Note: Descriptions are shown in the official language in which they were submitted.
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_2156176
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DNA POLYMERASES WITH ENHANCED TI~RMOSTABILITY AND ENHANCED
LENGTH AND EFFICIENCY OF PRIIVVIER EXTENSION
BACKGROUND OF THE INVENTION
The present invention is directed to DNA polymerises, and more particularly,
to a novel
mutation of Thermus aguaticus and Thermus flavus DNA polymerises exhibiting
enhanced
thermostability over any form of these enzymes now known. The invention is
also directed to
recombinant DNA sequences encoding such DNA polymerises, and vector plasmids
and host cells
suitable for the expression of these recombinant DNA sequences. The invention
is also directed to a
novel formulation of the DNA polymerises of the present invention and other
DNA polymerises,
which formulation of enzymes is capable of efficiently catalyzing the
amplification by PCR (the
polymerise chain reaction) of unusually long and faithful products.
DNA polymerise obtained from the hot springs bacterium Thermus aquaticus (Taq
DNA
polymerise) has been demonstrated to be quite useful in amplification of DNA,
in DNA sequencing,
and in related DNA primer extension techniques because it is thermostable.
Thermostable is defined
herein as having the ability to withstand temperatures up to 95°C for
many minutes without becoming
irreversibly denatured, and the ability to polymerize DNA at high temperatures
(60° to 75° C.). The
DNA and amino acid sequences described by Lawyer et al., J. Biol. Chem.
264:6427 (1989),
2 0 GenBank Accession No. J04639, define the gene encoding Thermus aguaticus
DNA polymerise and
the enzyme Thermus aquaticus DNA polymerise as those terms are used in this
application. The
highly similar DNA polymerise (Tfl DNA polymerise) expressed by the closely
related bacterium
Thermus flavus is defined by the DNA and amino acid sequences described by
Akhmetzjanov, A.A.,
and Vakhitov, V.A. (1992) Nucleic Acids Research 20:5839, GenBank Accession
No. X66105.
2 5 These enzymes are representative of a family of DNA polymerises, also
including Thermus
thermonhilus DNA polymerise, which are thermostable. These enzymes lack a 3'-
exonuclease
activity such as that which is effective for editing purposes in DNA
polymerises such as E. coli DNA
polymerise I, and phages T7, T3, and T4 DNA polymerises.
Gelfand et al., U.S. Patent 4,889,818 describe a wild-type (abbreviation used
here: WT),
3 0 native Thermus aguaticus DNA polymerise. Gelfand et al., U.S. Patent
5,079,352 describe a
recombinant DNA sequence which encodes a mutein of Thermus aguaticus DNA
polymerise from
which the N-terminal 289 amino acids of Thermus actuaticus DNA polymerise have
been deleted
(claim 3 of '352, commercial name Stoffel Fragment, abbreviation used here:
ST), and a
recombinant DNA sequence which encodes a mutein of Thermus a4uaticus DNA
polymerise from
3 5 which the N-terminal 3 amino acids of Thermus aguaticus DNA polymerise
have been deleted (claim
4 of '352, trade name AmpliTaq~, abbreviation used here: AT). Gelfand et al.
report their muteins
to be "fully active" in assays for DNA polymerise, but data as to their
maximum thermostability is
not presented.
The development of other enzymatically active mutein derivatives of Thermus
Av
WO 94126766 I 2 1 5 6 17 6 ~T10S94101867
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aduaticus DNA polymerise is hampered, however, by the unpredictability of the
impact of any
particular modification on the structural and functional characteristics of
the protein. Many factors,
including potential disruption of critical bonding and folding patterns, must
be considered in modifying
an enryme and the DNA for its expression. A significant problem associated
with the creation of N-
terminal deletion muteins of high-temperature Thermus aauaticus DNA polymerise
is the prospect that
the amino-terminus of the new protein may become wildly disordered in the
higher temperature
ranges, causing unfavorable interactions with the catalytic domains) of the
protein, and resulting in
denaturation. In fact, a few deletions have been constructed which appear to
leave the identifiable
domain for DNA polymerise intact, yet none of these deletions have
thermostability at temperatures as
high as 99° C. While Thermus aquaticus DNA polymerise has shown
remarkable thermostability at
much higher temperatures than that exhibited by other DNA polymerises, it
loses enzymatic activity
when exposed to temperatures above 95-97oC. Moreover, its fidelity at 72~C
(the recommended
temperature for DNA synthesis) is limited to an effective error rate of
approximately 1/9000 bp.
Gelfand et al.'s mutein ST of Thermus aquaticus DNA polymerise (with an N-
terminal 289 a.a.
deletion) is significantly more stable than AT, but ST exhibits significantly
decreased activity when
cycled to 98~C, and much less, if any, activity when cycled to 99~C, during
the denaturation phase of
PCR cycles.
Amplification of DNA spans by the polymerise chain reaction (PCR) has become
an
important and widespread tool of genetic analysis since the intro-duction of
thermostable Taq DNA
polymerise for its catalysis. Two limitations to prior art methods of PCR are
the fidelity of the final
product and the size of the product span that can be amplified. The fidelity
problem has been partially
addressed by the replacement of Taq DNA polymerise by Pfu (Pyrococcus
furiosus) DNA
polymerise, which exhibits an integral 3'-(editing) exonuclease that
apparently reduces the
mutations/bp/cycle from about 10° to about 10'S. However, experiments
suggest that this enzyme is
unable to amplify certain DNA sequences in the size range of 1.5 to 2 kb that
Klentaq-278 (also
known as Klentaql) (N-terminal deletion of Taq DNA polymerise; WMB
unpublished) or AmpliTaq
(full-length Taq DNA polymerise; ref. 3) can amplify handily, and that Pfu is
no more able (i.e. not
able) to amplify DNA product spans in excess of S-7 kb than is any form of Taq
DNA polymerise.
For full-length Taq DNA Polymerise and for N-terminally truncated variants
Klentaq-278, Klentaq5
and Stoffel Fragment, PCR amplification apparently rapidly becomes inefficient
or non-existent as the
length of the target span exceeds 5-6 kb. This was shown even when 30 minutes
was used during the
extension step of each cycle.
Although there are several reports of inefficient but detectable amplification
at 9-10
kb target length and one at 15 kb, most general applications are limited to 5
kb.
Kainze et al. (Analytical Biochem. 202:46-49(1992)) report a PCR amplification
of
over 10 kb: a 10.9 kb and a 15.6 kb product, utilizing an enryme of
unpublished biological source
(commercially available as "Hot 1~b" DNA polymerise). Kainze et al. report
achieving a barely
WO 94126766 PCTlUS94101867
215~'t76
visible band at 15.6 kb after 30 cycles, starting with 1 ng of ~ DNA template
per 100 ul of reaction
volume. The efficiency of this amplification was shown to be relatively low,
although a quantitative
calculation of the efficiency was not presented. Attempts by Kainze et al. to
make VJT Thermus
aquaticus DNA polymerise perform in the 10-15 kb size range were not
successful, nor have
successful results been reported by anyone else for any form of Thermus
aquaticus DNA polymerise
in this size range. There is no report of any longer DNA products amplifiable
by PCR.
A DNA polymerise which retains its thermostability at 98° or 99~C
would allow
more efficient and convenient DNA analysis in several situations including
"colony PCR" (see Figure
5), andlor allow thermal cycler overshoot without inactivation of the enzyme
activity. A thermostable
DNA polymerise or DNA polymerise formulation which exhibits improved fidelity
relative to AT or
WT Thermus aquaticus DNA polymerise at optimum temperatures for synthesis
would be highly
desirable for applications in which the target and product DNA is to be
expressed rather than merely
detected.
A DNA polymerise formulation capable of efficient amplification of DNA spans
in
excess of 6 kb would significantly expand the scope of applications of PCR.
For instance, whole
plasmids, and constructs the size of whole plasmids, could be prepared with
this method, which would
be especially valuable in cases in which a portion of the DNA in question is
toxic or incompatible with
plasmid replication when introduced into E. cola. If this thermostable DNA
polymerise preparation
simultaneously conferred increased fidelity to the PCR amplification, the
resulting large products
would be much more accurate, active and/or valuable in research and
applications, especially in
situations involving expression of the amplifed sequence. If the thermostable
DNA polymerise
preparation allowed, in addition, more highly concentrated yields of pure
product, this would enhance
the method of PCR to the point where it could be used more effectively to
replace plasmid replication
as a means to produce desired DNA fragments in quantity.
SUMMARY OF THE INVENTION
Among the several objects of the invention, therefore, may be noted the
provision of
a DNA polymerise which can survive meaningful repeated exposure to
temperatures of 99~C; the
provision of a highly thermostable DNA polymerise which exhibits greater
fidelity than Thermus
aguaticus DNA polymerise when utilized at standard Thermus aquaticus DNA
polymerise extension
reaction temperatures; the provision of such a DNA polymerise which is useful
for PCR amplification
techniques from DNA templates and from single colonies of E. cola, single-
stranded (linear)
amplification of DNA, nucleic acid sequencing, DNA restriction digest filling,
DNA labelling, in vivo
footprinting, and primer-directed mutagenesis. Further objects of the
invention include the provision
of recombinant DNA sequences, vectors and host cells which provide for the
expression of such DNA
polymerise.
Additional objects include the provision of a formulation of DNA polymerise
capable
WO 94/26766 PCTIUS94I01867
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of efficiently catalyzing primer extension products of greater length than
permitted by conventional
formulations, including lengths up to at least 35 kilobases, that reduces the
mutagenicity generated by
the PCR process, particularly in comparison with prior art DNA polymerises and
for any target
lengths, that maximizes the yield of PCR target fragments and, concomitantly,
enhances the intensity
aad sharpness of PCR product bands, without significant sacrifice in
flexiblity, specificity, and
efficiency; and the provision of an improved process for amplification by PCR
which can be utilized
to reliably synthesize nucleic acid sequences of greater length and which can
effectively utilize PCR
products as primers.
Briefly, therefore, the present invention is directed to a novel, recombinant
DNA
sequence encoding a DNA polymerise having an amino acid sequence comprising
substantially the
same amino acid sequence as the Thermus aauaticus or Thermus flavus DNA
polymerise, excluding
however the N-terminal 280 amino acid residues of WT Thermus aquaticus or the
N-terminal 279
amino acids of Thermus flavus DNA polymerise.
Additionally, the present invention is directed to a DNA polymerise having an
amino
acid sequence comprising substantially the same amino acid sequence of the
Thermus aguaticus or
Thermus flavus DNA polymerise, but lacking the N-terminal 280 amino acid
residues of Thermus
aquaticus DNA polymerise, or the N-terminal 279 amino acids of Thermus flavus
DNA polymerise.
In a further embodiment, the present invention is directed to a novel
formulation of
thermostable DNA polymerises, including a majority component comprised of at
least one
thermostable DNA polymerise lacking 3'-exonuclease activity and a minority
component comprised of
at least one thermostable DNA polymerise exhibiting a 3'-(editing) exonuclease
activity.
The present invention is also directed to a formulation of DNA polymerise
which
includes at least one DNA polymerise which in wild-type form exhibits 3'-
exonuclease activity and
which is capable of catalyzing a temperature cycle type polymerise chain
reaction, wherein the 3'-
exonuclease activity of said at least one DNA polymerise has been reduced to
between about 0.2
and about 7R6, of the 3'-exonuclease activity of the at least one DNA
polymerise in its wild-type
form.
In another aspect, a formulation of DNA polymerise comprising E1 and E2 is
provided. E1 is one or more DNA polymerises which lack any significant 3'-
exonuclease activity,
and E2 is one or more DNA polymerises which exhibit significant 3'-exonuclease
activity. 'Ihe
mixture provided has a relative DNA polymerise unit ratio of El to E2 of at
least about 4:1.
The invention is further directed to an improvement in a process for
amplification of
nucleic acid sequences by PCR wherein the improvement comprises formulating
DNA polymerise of
the types described above. The formulation thereby created is used to catalyze
primer extension
during the PCR process, thus extending the applicable size range for efficient
PCR amplification.
DNA polymerises such as those discussed in this application are commonly
composed of up to three identifiable and separable domains of enzymatic
activity, in the physical order
215617fi
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from N-terminal to C-terminal, of 5'-exonuclease, 3'-exonuclease, DNA
polymerise. Taq DNA
polymerise has never had a 3'-exonuclease, but the invention in the first pan
is directed to a deletion
of its 5'-exonuclease. Other DNA polymerises mentioned, such as Pfu DNA
polymerise, do not
have the 5'-exonuclease, but their 3'-exonuclease function is central to the
aspect of the invention
directed to mixtures of DNA polymerises E1 (lacking significant 3'-
exonuclease) and E2 (having 3'-
exonuclease). In these mixtures, the presence of 5'-exonuclease in either E1
or E2 has not been
shown to be essential to the primary advantages of the vresent invention.
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2156176
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In accordance with an aspect of the present invention is a recombinant DNA
sequence encoding a DNA polymerise having an amino acid sequence comprising
substantially the same amino acid sequence as Thermus a4uaticus DNA
polymerise, but
lacking the N-terminal 280 amino acid residues of said DNA polymerise.
In accordance with another aspect of the present invention is a DNA
polymerise having an amino acid sequence comprising substantially the same
amino acid
sequence as that of Thermus a4uaticus DNA polymerise, excluding the N-terminal
280 amino
acid residues of Thermus aquaticus DNA polymerise.
In accordance with yet another aspect of the present invention is a DNA
polymerise having an amino acid sequence comprised substantially of amino
acids 280-831
of the DNA polymerise of Thermus flavus.
In accordance with another aspect of the present invention is a formulation of
thermostable DNA polymerise comprising a thermostable DNA polymerise lacking
3'-
exonuclease activity and a thenmostable DNA polymerise exhibiting 3'-
exonuclease activity,
wherein the ratio of DNA polymerise units of the thermostable DNA polymerise
lacking 3'-
exonuclease activity to the thermostable DNA polymerise exhibiting 3'-
exonuclease activity
is four to one or greater.
In accordance with yet another aspect of the present invention is a method for
amplifying nucleic acid sequences by polymerise chain reaction, of the
repeating cycle type
wherein two complementary strands, for each different specific sequence being
amplified, are treated with a single, a pair, or a mixture of pairs of
oligonucleotide primers, and a DNA polymerise or a mixture of DNA
polymerises is used to catalyze synthesis of an extension product of
each primez which is complementary to each nucleic acid strand under
effective conditions for said synthesis, and wherein said primers are
selected so as to be sufficiently complementary to different strands of
each specific sequence to hybridize therewith such that the extension
product synthesized from one primer, when it is separated from its
complement, can serve as a template for synthesis of the complementary
strand of the extension product by extending the same or another
included primer, the primer extension products are separated from the
templates on which they were synthesized to produce single-stranded
molecules, and the single-stranded mo~ecules thus generated are treated
with the primers and a DNA polymerise or a mixture of DNA polymerises
is used to catalyze synthesis of an extension product of each primer
under effective conditions for the synthesis of a primer extension
product, wherein the improvement comprises formulating the DNA
p°l~erase as set forth in claim 6, and catalyzing primer extension
with said formulation of DNA polymerise.
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In accordance with yet another aspect of the present invention is a method for
amplifying nucleic acid sequences by polymerise chain reaction, of the
repeating
cycle type wherein two complementary strands, for each different specific
sequence
being amplified, are treated with a single, a pair, or a mixture of pairs of
oligonucleotide primers, and a DNA polymerise or a mixture of DNA polymerises
is
used to catalyze synthesis of an extension product of each primer which is
complementary to each nucleic acid strand under effective conditions for said
synthesis, and wherein said primers are selected so as to be sufficiently
complementary to different strands of each specific sequence to hybridized
therewith
such that the extension product synthesized form one primer, when it is
separated
from its complement, can serve as a template for synthesis of the
complementary
strand of the extension product by extending the same or another included
primer,
the primer extension products are separated from the templates on which they
were
synthesized to produce single-stranded templates on which they were
synthesized to
produce single-stranded molecules, and the single-stranded molecules thus
generated are treated with the primers and a DNA polymerise or a mixture of
DNA
polymerises is used to catalyze synthesis of an extension product of each
primer
under effective conditions for the synthesis of a primer extension product,
wherein the
improvement comprises formulating the DNA polymerise as set forth in claim 7,
and
catalyzing primer extension with said formulation of DNA polymerise.
In accordance with another aspect of the present invention is a formulation of
thermostable DNA polymerises comprising at least one thermostable DNA
polymerise lacking 3' - 5' exonuclease activity and at least one thermostable
DNA
polymerise exhibiting 3' - 5' exonuclease activity, wherein the ratio of DNA
polymerise units of the thermostable DNA polymerise lacking 3' - 5'
exonuclease
activity to the thermostable DNA polymerise lacking 3' - 5' exonuclease
activity is
four to one or greater.
In accordance with yet another aspect of the present invention a kit for the
synthesis of a polynucleotide, said kit comprising a first DNA polymerise,
wherein
said first polymerise is thermostable and possesses 3'- 5' exonuclease
activity, and
a second DNA polymerise, wherein said second polymerise is thermostable and
lacks 3' - 5' exonuclease activity whereby said second polymerise and said
first
polymerise are utilized in a ratio of 4 to 1 or greater.
In accordance with another aspect of the present invention is a kit for the
synthesis of a polynucleotide, said kit comprising:
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2156176
(a) a first DNA polymerise, wherein said first polymerise possesses 3' - 5'
exonuclease activity and is selected from the group consisting of Pyrococcus
furiosus
DNA polymerise, Theremotoga maritima DNA polymerise, Theremococcus litoralis
DNA polymerise, and Pyrococcus GB-D DNA polymerise, and
(b) a second DNA polymerise, wherein said second polymerise lacks 3' - 5'
exonuclease activity and is selected from the group consisting of Theremus
aouaticus
DNA polymerise, (exo-) Thermococcus literalis DNA polymerise, (exo-)
Pyrococcus
furiosus DNA polymerise, and (exo-) Pyrococcus GB-D DNA polymerise, wherein
the ratio of DNA polymerise units of the DNA polymerise lacking 3' - 5'
exonuclease
activity to the DNA polymerise possessing 3' - 5' exonuclease activity is four
to one
or greater.
In accordance with yet another aspect of the present invention is a method of
amplifying a polynucleotide sequence, said method comprising: the steps of
mixing a
composition with a synthesis primer, and a synthesis template, said
composition
comprising a first thermostable DNA polymerise possessing 3' - 5' exonuclease
activity, and a second thermostable DNA polymerise lacking 3' - 5' exonuclease
activity, wherein the ratio of DNA polymerise units of the thermostable DNA
polymerise lacking 3' - 5' exonuclease activity to the thermostable DNA
polymerise
exhibiting 3' - 5' exonuclease activity if four to one or greater.
In accordance with yet another aspect of the present invention is a method of
amplifying a polynucleotide sequence, said method comprising: the steps of
mixing a
composition with a synthesis primer, and a synthesis template, said
composition
comprising
(a) a first thermostable DNA polymerise possessing 3' - 5' exonuclease
activity
selected from the group consisting of Pyrococcus furiosus DNA polymerise,
Thermotoga maritima DNA polymerise, Thermococcus litoralis DNA polymerise,
and Pyrococcus GB-D DNA polymerise, and
(b) a second thermostable DNA polymerise, wherein said polymerise lacks 3' -
5' exonuclease activity and is selected from the group consisting of Thermus
aquaticus DNA polymerise, (exo-) Thermococcus litoralis DNA polymerise, (exo-)
Pvrococcus furiosus DNA polymerise, and (exo-) Pyrococcus GB-D DNA
polymerise, wherein the ratio of DNA polymerise units of the thermostable DNA
polymerise lacking 3' - 5' exonuclease activity to the thermostable DNA
polymerise
exhibiting 3' - 5' exonuclease activity if four to one or greater.
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Other objects and features will be in part apparent and in part pointed out
hereinafter.
SUMMARY OF ABBREVIATIONS
The listed abbreviations, as used herein, are defined as follows:
Abbreviations:
by = base pairs
kb = kilobase; 1000 base pairs
nt = nucleotides
BME = beta-mercaptoethanol
PP; = sodium pyrophosphate
p~ = pvr~~cus furiosus
Taq = Thermus aouaticus
Tfl = Thermus flavus
Tli = Thermococcus literalis
Klentaq-nnn = N-terminally deleted Thermus a4uaticus DNA polymerise
that starts with codon ttttn + 1, although that start codon and the next codon
may not match the WT
sequence because of alterations to the DNA sequence to produce a convenient
restriction site.
WT = wild-type (full length) or deletion of only 3 as
as = amino acids)
ST = Stoffel fragment, an N-terminal deletion of Thermus
a uaticus DNA polymerise that could be named Klentaq-288. -LA
= Long and Accurate; an unbalanced mixture of two DNA polymerises, at least
one lacking
significant 3'-exonuclease activity and at least one exhibiting significant 3'-
exonuclease activity.
PCR = (noun) 1. The Polymerise Chain Reaction
2. One such reactionlamplification experiment. 3.(verb) To amplify via the
polymerise chain reaction.
ul = microliter(s)
ATCC = American Type Culture Collection
Megaprimer = double-stranded DNA PCR product used as
primer in a subsequent PCR stage of a multi-step procedure.
Deep Vents = DNA polymerise from Pyrococcus species GB-D; purified
enzyme is available from New England Biolabs. .
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215617 6
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a 6
3' (editing)-exonuclease.
Deep Vent~ exo- = mutant form of Deep Vent~ DNA polymerase lacking
Vent~ = DNA polymerase from Thermococcus litoralis; purified enzyme
is available from New England Biolabs.
Vent~ exo- = mutant form of Vent~ DNA polymerase lacking 3'(editing)-
exonuclease.
Pfu = DNA polymerase from Pyrococcus furiosus; purified enzyme is
available from Stratagene Cloning Systems, Inc.
Pfu exo- = mutant form of Pfu DNA polymerase lacking 3' (editing)-
exonuclease; purified enzyme is available from Stratagene Cloning Systems,
Inc.
Sequenase~ = A chemically modified or a mutated form of phage T7 or
T3 DNA polymerase wherein the modification or mutation elinunates the 3'-
exonuclease activity.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts the nucleotide sequence of primers that can be used for
amplification of the gene for a preferred embodiment of the DNA polymerase of
this invention. The
bulk of the DNA sequence for the gene (between the primers) and the resultant
amino acid sequence
of the enzyme, is defined by the indicated GenBank entry.
Figure 2 depicts the nucleotide sequence of the same primers as in Figure
1, and shows that these same primers can be used for amplification of the
analogous gene from
2 0 Thermus flavus.
Figure 3 is a photograph of an agarose gel depicting a PCR amplification
reaction conducted using the prior art enzyme Thermus aquaticus DNA polymerase
(AmpliTaq~; AT)
and a preferred embodiment of the DNA polymerase of this invention, tested
with differing peak
denaturation temperatures lasting a full 2 min each for 20 cycles. Full
activity at 98° and partial but
2 5 useful activity at 99° is exhibited by the preferred embodiment of
this invention, whilst AT is unable
to withstand these temperatures. Figure 3 demonstrates that the enzyme of the
present invention is
more thermostable than AT in a practical test -- PCR amplification.
Figure 4 is a photograph of an agarose gel depicting a PCR amplification
reaction conducted using 4 enzymes: the prior art enzyme Thermus aQUaticus DNA
polymerase
3 0 (AmpliTaq~; AT); the DNA polymerase of this invention (KlenTaq-278); the
prior art enzyme
AmpliTaq~ Stoffel Fragment (ST); and KlenTaq-291. All were tested with PCR
denaturation steps
carried out at 95°C (control standard temperature), and at 98°C.
All were tested at two levels of
enzyme, the lower level being as close as practicable to the minimum necessary
to support the
reaction at the control temperature.
3 5 Note that both KlenTaq-291 and ST behave identically, losing most, but
not all, of their activity when used at 98° C, yet HIenTaq-278 is at
least twice as able to withstand
use of the
WO 94/26766 PCTlUS94101867
2~5fi17fi
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higher denaturation temperature. AT is seen to be drastically reduced in
effectiveness by exposure to
98 ° C. The behaviour of these enzymes is reproducible except for ST,
which is at its best in the
presented experiment, but performs more poorly when used in the amounts
recommended by the
manufacturer.
Figure 5 is a photograph of an agarose gel analysis of the products of colony
PCR
carried out at the standard peak denaturation temperature of 95° C
compared to the newly available
temperature of 98 ° C allowed by the enryme of the present invention.
Figure 5 demonstrates an
application advantage of the use of the newly available peak denaturation
temperature.
Figure 6(A-C) is three photographs, each of an agarose gel on which was loaded
a
portion of a test PCR experiment. Figure 6 demonstrates the large increase in
efficiency of large
DNA span PCR achieved by variations of a preferred embodiment of the enryme
formulation of the
invention. Although KlenTaq-278 or Pfu DNA polymerise, alone, are shown to
catalyze a low level
of 6.6 kb PCR product formation, various combinations of the two are seen to
be much more efficient.
Lower and lower amounts of Pfu in combination with Klentaq-278 are seen to be
effective, down to
the minimum presented, 1/640. Of those shown, only a combination of Klentaq-
278 and Pfu can
catalyze efficient amplification of 6.6 kb. Per 100 ul, the indicated level of
each enzyme (see
Methods, example 8, for unit concentrations) was used to catalyze PCR
reactions templated with 19 ng
~plac5 DNA and primers MBL and MBR. 20 cycles of 94° 2 min., 60°
2 min., 72° 10 min.
Figure 7 is a photograph of an agarose gel on which were analyzed the products
of
PCR experiments to test the performance of an embodiment of the invention in
catalyzing the
amplification of fragments even longer than 6.6 kb. Figure 7 demonstrates the
ability to amplify 8.4
kb, 12.5 kb, 15 kb, and 18 kb with high efficiency and large yield, utiliziing
the 1/640 ratio
embodiment of the enzyme formulation of the invention. Target product size is
indicated above each
lane as kb:. Level of template per 100 ul is indicated as ng a:. 20 or 30
cycles of PCR were each 2
sec. 94°, 11 min. 70°. These early amplifications were non-
optimal in several respects compared to
the current optimal procedure (see Methods, example 8): thick-walled tubes
were employed instead of
thin, catalysis was by 1 ul KlentaqLA-64 (63:1::Klentaq-278:Pfu) instead of
KlentaqLA-16, the 27mer
primers were used (see Table 1) instead of longer primers, the
extension/annealling temperature was
70° instead of 68°, and the Omnigene thermal cycler was used.
Figure 8 is a bar graph enumerating the differences in the number of mutations
introduced into a PCR product, the lacZ gene, by the near full-length prior
art -3 deletion of Thermus
aquaticus DNA polymerise, compared to the number of mutations introduced by
Klentaq-278.
Figure 9 is a photograph of an agarose gel depicting a PCR amplification
attempted
using a 384 by megaprimer (double-stranded PCR product) paired with a 43-mer
oligonucleotide
primer BtVS. Per 100 ul of reaction volume, the following enzymes (see Ex. 8,
Methods for unit
concentratioas) were used to catalyze amplifications: lane 1, 1 ul Pfu DNA
polymerise; lane 2, 1/16
ul Pfu; lane 3, 1 ul Klentaq-278; lane 4, both enzymes together (1 ul Klentaq-
278 + 1/16 ul Pfu).
ii
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WO 94126766 PCT/US94/01867
g
The 384 by band near the bottom of the gel is the megaprimer, which was
originally amplified using
Klentaq-278. ~H3 = lambda DNA digested with HindBI. The only successful
amplification resulted
from the combination of the two enrymes (lane 4). Vent DNA polymerase could
substitute for Pfu
with the same result (data not shown).
Figure 10 is a photograph of an agarose gel demonstrating that 33mers are
better
than 27mers. Per 100 ul of reaction volume, 2 ng (lanes 1-6) or 10 ng (lanes 7-
12) of lambda
transducing phage template were amplified using 27mer primers (lanes 1-3, 7-9)
or 33mer primers
(lanes 4-6, 10-12). Besides being longer, the 33mer lambda primer sequences
were situated 100 by to
the left of primer MBL and 200 by to the right of primer MBR on the lambda
genome. KlentaqLA-16
in the amounts of 1.2, 1.4, and 1.6 ul was used to catalyze the amplifications
of 12.5, 15, and 18 kb,
respectively. 15 ul aliquots (equivalent to 0.3 or 1.5 ag of ~ template) were
analyzed by 0.8 °6
agarose electrophoresis.
Figure 11 is a photograph of an agarose gel showing a CHEF pulse-field
analysis
(ref. 11, 4 sec. switching time) of large PCR products amplified by KlentaqLA-
16 (1.2 ul) under
conditions which were suboptimal with respect to pH (unmodified PC2 buffer was
used) and thermal
cycler (Omnigene). Starting template (see Table 1) was at 0.1 ng/ul and the
time at 68° in each cycle
was 21 min. for products over 20 kb, 13 min. for lanes 4 & 5, and 11 min. for
lanes 11-14. The
volumes of PCR reaction product loaded were adjusted to result in
approximately equal intensity; in
ul: 12,12,4,2; 10,10,10; 2,2,4,1. The standard size lanes (S) show full-length
~plac5 DNA (48645
bp) mixed with a Hind digest of ~ DNA. As for Table 1, the sizes in 5 figures
are in base pairs, as
predicted from the primer positions on the sequence of ~plac5 DNA, and sizes
with decimal points are
in kb, as determined from this gel.
Figure 12 is a photograph of an agarose gel depicting 28 kb and 35 kb products
without (lanes 2,3) and with (lanes 5,6) digestion by restriction enzyme Hind.
Before HindIll
digestion, the 28 kb product was amplified with 21 min. extension time per
cycle, and the 35 kb
product was cycled with 24 min. extension times, both in the RoboCycler at
optimum pH (see Ex. 8,
Methods). Lanes S (1,4,7) contain markers of undigested ~plac5 and Hind-
digested aplac5 DNA.
Figure 13 is a photograph of an agarose gel showing the results of a Pfu exo
mutant
test. PCR amplification of 8.4 kb by 30 units (0.? ug) of Klentaq-278 alone
(lanes 1,7) and in
combination with a very small admixture (1/16 ul or 1/64 ul, equivalent to 116
or 1/25 unit) of
archaebacterial Pfu wild type exo+ DNA polymerase (+; lanes 2,3) or a mutant
thereof lacking the 3'-
exonuclease activity (-; lanes 4,5). Lane 6 is the result if 1 ul (2.5 units)
of solely Pfu DNA
polymerase (wt, exo*) is employed.
DESCRIPTION OF THE PREFERRED EMBODIMENT
Referring now to Figure 1, the primers and logic for amplification by PCR of
the
recombinant DNA sequence encoding a preferred embodiment of the DNA polymerase
of the invention
WO 94/26766 PCTIUS94101867
,,..-. - _ . , ,, ..
9 21~ ~ ~ -1 ?' ~ '.
(referred to herein as Klentaq-278), are set forth. As depicted in Figure 1,
an initiator methionine and
a glycine residue occupy the first two N-terminal positions of Klentaq-278,
previously occupied by
residues 279 and 280 of WT Thermos aquaticus DNA polymerise, followed by the
amino acid
sequence of wild-type Thermos aquaticus DNA polymerise, beginning with the
amino acid residue at
position 281 as described by Lawyer et al. The codons encoding amino acid
residues 1 through 280 of
Thermos aquaticus DNA polymerise are therefore deleted, and the amino acids 1
thru 280 are not
present in the resulting gene product. Another preferred embodiment of the DNA
polymerise of the
invention is depicted in Figure 2. In this embodiment, the same deletion
mutation described above is
made to the highly analogous enzyme Thermos flavus DNA polymerise.
It will be appreciated that minor variations incorporated into the DNA
encoding for,
or the amino acid sequence as described herein, which retain substantially the
amino acid sequence as
set forth above, and which do not significantly affect the thermostability of
the polymerise are
included within the scope of the invention.
Surprisingly, the mutant DNA polymerise Klentaq-278 exhibits thermostability
at
temperatures above those reported for any previous variant of Thermos
aquaticus DNA polymerise
and has demonstrated a fidelity in final PCR products which is greater than
that of VJT Thermos
aquaticus DNA polymerise, when both are utilized at the 72 ° C
temperatures recommended for DNA
synthesis. Further, since Klentaq-278 does not have the 5'-exonuclease
activity associated with
Thermos aquaticus DNA polymerise (removed as a consequence of the N-terminal
deletion), it is
significantly superior to wild-type Thermos aJc uaticus DNA polymerise for DNA
sequencing.
Mutagenesis results, and mismatched primer testing, suggest that Klentaq-278
is less processive and is
less likely to extend a mispaired base than wild-type Thermos aguaticus DNA
polymerise.
Thermostability tests with Klentaq-278, Stoffel Fragment (ST, alternative
designation,
Klentaq-288) and Klentaq-291 have been carried out. The test used involves 20
PCR cycles with a
full 2 minutes each at the peak test temperature, such as 97° C,
98° C, or 99° C, and the intensity of
the resulting amplified bands is compared to 2 minutes at 97° C, or at
a lower control denaturation
temperature, such as 95° C (at which all of these variants are stable).
These data indicate that ST and
Klentaq-291 behave similarly, having thermolability at 98° C that is
similar to each other yet distinct
from Klentaq-278, which exhibits little detectable thermolability at
98° C in these tests. These data
suggest that the number of N-terminal amino acids is important to the enhanced
thermostability
exhibited by the DNA polymerise of the invention. Evidently deletions ST and
Klentaq-291 are in a
class which has removed too many amino acids (10 more and 13 more) for the
optimum stability
demonstrated by the invention Klentaq-278.
The DNA polymerise from the bacterium sometimes designated Thermos flavus (and
sometimes Thermos aquaticus -- see ATCC catalog) is highly homologous to the
WT Thermos
ac~uaticus DNA polymerise. In the region of the deletions being discussed
here, the enzymes and
genes are exactly homologous, and it is believed that the differences between
the pair ST, Klentaq-291
CA 02156176 2000-O1-25
10.
and the superior Klentaq-278 would remain if the analogous
deletions were construed. Indeed, the primers in Figure 2
could be used on Thermus flavus DNA to construct KlenTfl-277
in exactly the manner described here for the construction and
isolation of Klentaq-278. The Thermus flavus DNA polymerase-
277 enzyme and variations thereof which exhibit similar
thermostability are therefore also within the scope of this
invention.
The invention also features a vector which includes a
recombinant DNA sequence encoding a DNA polymerase comprising
the amino acid sequence of Thermus aquaticus or Thermus favus
DNA polymerase, except that it adds a methionine and glycine
residue at the N-terminal and excludes the N-Terminal 280
amino acids of wild-type Thermus aquaticus DNA polymerase
(see Lawyer et al., su ra).
In preferred embodiments, the vector is that nucleic
acid present as plasmid pWB254b (SEQ ID N0:5) deposited as
ATCC No. 69244 or a host cell containing such a vector.
In a related aspect, the invention features a purified
DNA polymerase having an amino acid sequence as discussed
above. As used herein, "purified" means that the polymerase
of the invention is isolated from a majority of host cell
proteins normally associated with it. Preferably, the
polymerase is at least 10~ (w/w) of the protein of a
preparation. Even more preferably, it is provided as a
homogeneous preparation, e.g., a homogeneous solution.
In general, the recombinant DNA sequence of the present
invention is amplified from a Thermus aquaticus genomic DNA
or from a clone of the portion of the Thermus aquaticus DNA
polymerase gene which is larger than the desired span, using
the polymerase chain reaction (PCR, Saiki et al., Science
239-487, 1988), employing primers such as those in Figure 2
into which appropriate restriction sites have been
incorporated for subsequent digestion.
The recombinant DNA sequence is then cloned into an
expression vector using procedures well know to those in this
art. Specific nucleotide sequences in the vector are cleaved
by sit-specific restriction enzymes such as NcoI and HindIII.
Then, after optional alkaline phosphatase treatment of the
CA 02156176 2000-O1-25
l0a
vector, the vector and target fragment are ligated together
with the resulting insertion of the target codons in place
adjacent to desired control and expression sequences. The
particular vector employed will depend in part on the type of
host cell chosen for use in gene expression. Typically, a
host-compatible plasmid will be used containing genes for
markers such as ampicillin or tetracycline resistance, and
also containing suitable promoter and terminator sequences.
In a preferred procedure, the recombinant DNA expression
sequence of the present invention is cloned into plasmid
pWB253 (expresses KlenTaq-235 deposited as ATCC No. 68431) or
pWB250 (expresses luciferase/NPTII fusion), the backbone of
which is pTAC2 (J. Majors, Washington University), a pBR322
derivative. The specific sequence of the resulting plasmid,
disgnated pWB254b is SEQ ID NO: 5.
Bacteria, e.g., various strains of E.coli, and yeast,
e.g., Baker's yeast, are most frequently used as host cells
for expression of DNA polymerase, although techniques for
using more
a ' 2156176
complex cells are known. See, e.g., procedures 1 i for using plant cells
described by Depicker, A.,
et al., J. Mol. Appl. Gen. (1982) 1:561. E. coli host strain X7029, wild-type
F-, having deletion
X74 covering the lac operon is utilized in a preferred embodiment of the
present invention.
A host cell is transformed using a protocol designed specifically for the
particular host cell. For E. coli, a calcium treatment, Cohen, S.N., Proc.
Natl. Acid. Sci. 69:2110
(1972), produces the transformation. Alternatively and more efficiently,
electroporation of salt-free
E. coli is performed after the method of Dower et al. (1988), Nucleic Acids
Research 16:6127-6145.
After transformation, the transformed hosts are selected from other bacteria
based on characteristics
acquired from the expression vector, such as ampicillin resistance, and then
the transformed colonies
of bacteria are further screened for the ability to give rise to high levels
of isopropylthiogalactoside
(IPTG)-induced thermostable DNA polymerise activity. Colonies of transformed
E. coli are then
grown in large quantity and expression of HIentaq-278 DNA polymerise is
induced for isolation and
purification.
Although a variety of purification techniques are known, all involve the
steps of disruption of the E. coli cells, inactivation and removal of native
proteins and precipitation of
nucleic acids. The DNA polymerise is separated by taking advantage of such
characteristics as its
weight (centrifugation), size (dialysis, gel-filtration chromatography), or
charge (ion-exchange
chromatography). Generally, combinations of these techniques are employed
together in the
purification process. In a preferred process for purifying Klentaq-278 the E.
coli cells are weakened
2 0 using lysozyme and the cells are lysed and nearly all native proteins are
denatured by heating the cell
suspension rapidly to 80°C and incubating at 80-81 ° C for 20
minutes. The suspension is then cooled
and centrifuged to precipitate the denatured proteins. The supernatant
(containing Klentaq-278) then
undergoes a high-salt polyethylene-imine treatment to precipitate nucleic
acids. Centrifugation of the
extract removes the nucleic acids. Chromatography, preferably on a heparin-
agarose column, results
2 5 in nearly pure enzyme. More detail of the isolation is set forth below in
Example 3.
The novel DNA polymerise of the present invention may be used in any
process for which such an enzyme may be advantageously employed. In
particular, this enzyme is
useful for PCR amplification techniques, nucleic acid sequencing, cycle
sequencing, DNA restriction
digest labelling and blunting, DNA labelling, in vivo DNA footprinting, and
primer-directed
3 0 mutagenesis.
Amplification
Polymerise chain reaction (PCR) is a method for rapidly amplifying
specific segments of DNA, in geometric progression, up to a million fold or
more. See, e.g., Mullis
U.S. Patent No. 4,683,202. The technique relies on repeated cycles of DNA
polymerise-catalyzed
3 5 extension from a pair of primers with homology to the 5' end and to the
complement of the 3' end of
the DNA segment to be amplified. A key step in the process is the heat
denaturing of the DNA
primer extension products from their templates to permit another round of
amplification. The
operable temperature range for the denaturing step generally ranges from
WO 94126766 PCTIUS94/01867
12
about 93oC to about 95oC, which irreversibly denatures most DNA polymerises,
necessitating the
addition of more polymerise after each denaturation cycle. However, no
additional DNA polymerise
needs to be added if thermo~ta~le DNA polymerises such as Thermus aquaticus
DNA polymerise are
used, since they are able to rexain their activity at temperatures which
denature double-stranded nucleic
acids. As described in Example 4, below, Klentaq-278 has demonstrated the
ability to survive
meaningful repeated exposure to temperatures of 99aC, higher than for any
previously known DNA
polymerise.
Klentaq-278 has also been demonstrated to have a higher fidelity than wild-
type
Thermus aquaticus DNA polymerise at 72~C, the recommended synthesis
temperature. The data for
this has been gathered by a method involving the PCR amplification of a lacZ
DNA gene flanked by
two selectable markers [Barnes, W.M. (1992) Gene 112, 29-25]. Representative
data comparing the
preferred embodiment of this invention Klentaq-278 to AT and another analogous
N-terminal deletion,
Klentaq-235, is shown in Figure 8, which demonstrates that different N-
terminal deletions reproducibly
exhibit differing fidelities as measured in the final PCR product.
Similar fidelity data for the enzyme ST is not available, since it is
difficult for the
commercial preparation of this enzyme to catalyze PCR of the long test
fragment (4.8 kb) used for this
assay. It is not yet known whether the difficu[ty with ST for these
experiments is caused merely by
formulation (its concentration is less, such that 10-15 times more volume is
necessary for a 2 kb PCR
amplification, and for these deletions more enzyme is needed for longer target
DNAs), or whether ST
may be intrinsically unable to catalyze such a long-target PCR amplification.
DNA Sequencins
Particular DNA sequences may be elucidated by the Singer Method (Singer, F.,
Nicklen, S. and Coulson, A.R., DNA sequencing with chain-terminating
inhibitors,
Proc.Nat.Acad.Sci.USA, 74 (1977) 5463-5467), using dideoxy analogs. DNA
polymerises are used
in these methods to catalyze the extension of the nucleic acid chains.
However, in its natural form,
Thermus arc uaticus DNA polymerise (like many other polymerises) includes a
domain for
5'-exonuclease activity. This associated exonuclease activity can, under
certain conditions including
the presence of a slight excess of enzyme or if excess incubation time is
employed, remove 1 to 3
nucleotides from the 5' end of the sequencing primer, causing each band in an
alpha-labelled
sequencing gel to appear more or less as a multiplet. If the label of the
sequencing gel is 5', the
exonuclease would not be able to cause multiplets, but it would instead reduce
the signal. As a result
of the deletion of the N-terminal 280 amino acid residues of Thermus aquaticus
DNA polymerise,
Klentaq-278 has no exonuclease activity and it avoids the sequencing hazards
caused by 5'-exonuclease
activity.
Klentaq-278 can be used effectively in thermostable DNA polymerise DNA sequen-
cing. There are basically two types of dideoxy DNA sequencing that Klentaq-278
is good for --
original dideoxy (Singer et al. supra; Innis et al., Proc. Natl. Acid. Sci.
USA 85:9436, 1988) and
2156 9~'6
>13
1988) and cycle sequencing.
Innis et al. (U.S. Patent No. 5,075,216) describe a good procedure for
dideoxy sequencing, but when the WT Thermus aQUaticus DNA polymerise is used
this procedure is
prone to doubled or tripled bands on the sequencing gel. Klentaq-278 is
effective in curing this
problem.
The procedure recommended for orignal-type (non-cycled, Innis et al.
dideoxy sequencing with Klentaq-278 is that in the USB Taquence~ 2.0 dideoxy
sequencing kit, the
title page of which is appended to this application (Appendix 1).
The procedure recommended for cycle sequencing is that in the USB Cycle
Sequencing Kit. The title page of this procedure is appended to this
application (Appendix 2).
Other Uses
Klentaq-278 has also been used successfully for primer-directed
mutagenesis, in vivo footprinting, DNA labelling and for preparation of non-
sticky Lambda DNA
fragment size standards. These procedures are discussed below.
Klentaq-278, especially in the formulation Klentaq-LA (discussed below),
can be used to extend site-specific mutagenic primers) which are annealled to
single-stranded
templates. It substitutes for Klenow enzyme (the large fragment of E. coli DNA
polymerise I) and
T7 DNA polymerise in this process, showing more primer selectivity at 60-
65°C than Klenow
enzyme at 37°C, and working to completion or sufficient incorporation
in 12 mins., as compared to
2 0 the one hour or more required for Klenow enzyme.
Klentaq-278 has also been shown to be useful (and superior to wild-type
Thermus aguaticus DNA polymerise) for the post-PCR labelling steps with the
third (second nested)
primer in ligase-mediated, PCR-assisted, in vivo footprinting. I am indebted
to I. Ghattas,
Washington University, St. Louis, MO for this information. These studies are
similar to those of
Garritty & Wold (Garrity, P.A., and Wold, B.J. (1992) Effects of different DNA
polymerises in
ligation-mediated PCR: enhanced genomic sequencing and in vivo footprinting.
Proc Natl Acad Sci
U S A 89, 1021-1025)
Klentaq-278 is also useful for DNA labelling. For random primers, a
length of at least 9 nt is recommended, and preferably the reaction is warmed
slowly (over 20-30
3 0 mins.) from 37 to 65°C. Most preferably, a programmable heat block,
using procedures well-known
to those in this art, is utilized for the DNA labelling.
Another use of Klentaq-278 is for the preparation of Lambda DNA
restriction digests that do not have the sticky ends partially stuck together.
As a result of including
Klentaq-278 and the four DNA dNTPs in with a HindIII digest performed at
55°C, bands 1 and 4 are
3 5 not partially attached
fr
WO 94126766 l ~ ~ ~. ~ ~ ~ ~ PCT/US94101867
14
to each other.
Deposit
Strain pWB254b/X7029 was deposited with the American Type Culture Collection,
Maryland, on February 18, 1993 and assigned the number ATCC 69244. Applicant
acknowledges his
responsibility to replace this culture should it die before the end of the
term of a patent issued hereon,
5 years after the last request for a culture, or 30 years, whichever is the
longer, and its responsibility
to notify the depository of the issuance of such a patent, at which time the
asits will be made
available to the public. Until that time the deposits will be made available
to the Commissioner of
Patents under the terms of 37 C.F.R. Section 1-14 nad 35 U.S.C. ~112.
In a further aspect of the invention a target length limitation to PCR
amplification of
DNA has been identified and addressed. Concomitantly, the base pair fidelity,
the ability to use PCR
products as primers, and the maximum yield of target fragment were increased.
These improvements
were achieved by the combination of a DNA polymerase lacking significant 3'-
exonuclease activity,
preferably, Klentaq-278, with a low level of a DNA polymerase exhibiting
significant 3'-exonuclease
activity (for example, Pfu, Vent, or Deep Vent). Surprisingly, target
fragments of at least 35 kb can
be amplified to high yields from, for example, 1 ag lambda DNA template with
this system.
Moreover, products in the range 6.6 to 8.4 kb can be efficiently amplified by
a
formulation of thermostable DNA polymerases consisting of a majority component
comprised of at
least one thermostable DNA polymerase lacking 3'-exonuclease activity and a
minority component
comprised of at least one thermostable DNA polymerase exhibiting 3'-
exonuclease activity.
The prior art technology only allowed relatively inefficient and sporadic
amplification
of fragments in this size range, resulting in only relatively faint product
bands or no detectable product
at all. In light of the current discovery, I believe I understand (without
limiting myself to any
particular theory) the reason for the inefficiency of the prior art. It is
believed that Thermus aquaticus
DNA polymerase and its variants are slow to extend a mismatched base pair
(which they cannot
remove since they lack any 3'-exonuclease). A couple of companies (New England
Biolabs and
Stratagene) have introduced thermostable enzymes which exhibit a 3'-(editing)
exonuclease which
should, one would think, allow the removal of mismatched bases to result in
both efficient extension
and more accurately copied products. In practice, these two enzymes (Vent and
Pfu DNA
polymerase) are unreliable and much less efficient than expected. One possible
explanation for the
unreliability of these enzymes for PCR is that the 3'-exonuclease often
apparently attacks and partially
degrades the primers so that little or no PCR is possible. This primer attack
problem is worse for
some primers than others. It has been reported (Anonymous, The NEB Transcript,
New England
Biolabs, (March, 1991) p. 4.) that the Vent DNA polymerase leaves the S' 15 nt
intact, so that if the
annealling conditions allow that 15 nt to prime, PCR could presumably proceed.
This would of course
only allow annealling at lower, non-selective temperatures, and the 5' 15 at
of the primers must be
exactly homologous to the template.
WO 94/26766 ~ 5 g ~ PCT/I1S94l01867
I have discovered that the beneficial effects of a 3'-exonuclease can be
obtained with
an unexpectedly minute presence of one or more DNA polymerises which exhibit
significant (defined
as being biochemically assayable) 3'-exonuclease activity (herein called "E2")
such as certain
Archaebacterial DNA polymerises, whilst efficient extension is being catalyzed
by a large amount of
5 one or more DNA polymerises which lack any significant 3'-exonuclease
activity, such as Klentaq-278
or AT (herein called "E1"). As a minority component of a formulation or
mixture of DNA
polymerises, the unreliability and inefficiency of the 3'-exonuclease DNA
polymerise, discussed
above, is substantially reduced or eliminated. Moreover, since it is believed
that the 3'-exonuclease is
removing mismatches to eliminate pausing at the mismatches, the resulting DNA
exhibits fewer base
10 pair changes, which is a valuable decrease in the mutagenicity of PCR
without sacrificing flexibility,
specificity, and efficiency. In fact, the combination, even for KlenTaq-
278/Pfu units ratios as high as
2000, exhibited greatly increased efficiency of amplification. For most
applications, the mixture of
DNA polymerises must be at a relative DNA polymerise unit ratio of E1 to E2 of
at least about 4:1,
before enhanced product length and yield can be achieved. When Pfu DNA
polymerise was used in
15 the formulation, the ratio preferably is in the range 80 to 1000 parts
KlenTaq-278 per part (unit) Pfu,
more preferably from about 150 to about 170:1, and most preferably, is about
160:1, depending
somewhat on the primer-template combination. Similar ratios are preferred for
mixtures of Pfu and
Klentaq-291.
If Deep Vent is substituted for Pfu for use in combination with Klentaq-278 or
-291,
the most preferred ratios for most applications increases to from about 450 to
about 500:1 E1 to E2; if
full-length (WT) Taq or Amplitaq is included as E1, the most preferred ratio
to Pfu or other E2
component is between about 10 and about 15:1 of E1 to E2.
E2 of the invention includes, but is not limited to, DNA polymerise encoded by
genes from Pfu, Vent, Deep Vent, T7 coliphage, Tma, or a combination thereof.
EI of the invention
includes, but is not limited to, a mutant, 3'-exonuclease negative form of an
E2 DNA polymerise, or
alternatively, a DNA polymerise which, in unmutated form, does not exhibit
significant 3'-
exonuclease activity, such as the DNA polymerises encoded by genes from Taq,
Tfl, or Tth, or a
combination thereof.
As discussed below, the formulation of DNA polymerises of the present
invention
also includes formulations of DNA polymerise wherein E1 comprises a reverse
transcriptase such as
Sequenase.
Additional examples of the formulations of the present invention include
mixtures
wherein E1 comprises or consists of a mutant or chemical modification of T7 or
T3 DNA polymerise
and E2 comprises or consists of a wild-type T7 or T3 DNA polymerise, or, in
another variation, E1
comprises or consists of a Vent DNA polymerise lacking any significant 3'-
exonuclease activity (sold
by New England Biolabs as Vent exo-) and E2 comprises or consists of Vent.
The principal here discovered, namely the use of low levels of 3' exonuclease
during
2156 976
primer extension by a DNA polymerase lacking ; 3' exonuclease, is applicable
to general DNA
polymerase primer extensions, including normal temperature incubations (i.e.
using non-thermostable
DNA polymerases) and including reverse transcriptase enzymes, which are known
to lack a 3'-
(editing) exonuclease (Battula & Loeb, 1976). An example of the former is the
use of Sequenase~
(exo-) as the majority enzyme, and wild-type T7 DNA polymerase (exo+) or
Klenow fragment as
the minority component. An example of the latter is AMV (Avian Myoblastosis
Virus) or MLV
(Murine Leukemia Virus) Reverse Transcriptase as the major component, and
Klenow fragment, T7
DNA polymerase, or a thermostable DNA polymerase such as Pfu or Deep Vent~ as
the minor
component. Because of the lower activity of thermostable DNA polymerases at
the temperatures of
37 degrees and 42 degrees used by these reverse transcriptases, higher levels
are likely to be required
than are used in PCR. Although HIenow fragment DNA polymerse is not a
preferred DNA
polymerase using RNA as a template, it does function to recognize this
template (Karkas, 1973;
Gulati, Kacian & Spiegelman, 1974), particularly in the presence of added Mn
ion. Added Mn ion is
routinely used to achieve reverse transtription by thermostable DNA polymerase
Tth, unfortunately
(in the prior art) without the benefit of an exo+ component. It must be
stressed that for the use of
the exo+ component for reverse transcriptase reactions, extra care must be
taken to ensure that the
exo+ component is entirely free of contaminating RNAse.
The following references describe methods known in the art for using
reverse transcriptases.
2 0 Battula N. Loeb LA. On the fidelity of DNA replication. Lack of
exodeoxyribonuclease activity and error-correcting function in avian
myeloblastosis virus DNA
polymerase. Journal of Biological Chemistry. 251(4):982-6, 1976 Feb 25.
Gulati SC. Kacian DL. Spiegelman S. Conditions for using DNA
polymerase I as an RNA-dependent DNA polymerase. Proceedings of the National
Academy of
2 5 Sciences of the United States of America. 71(4):1035-9, 1974 Apr.
Karkas JD. Reverse transcription by Escherichia coli DNA polymerase I.
Proc Natl Acad Sci U S A. 70(12):3834-8, 1973 Dec.
DNA Polymerase with no polymerase activity, only 3'-exonuclease activity:
3 0 While not limiting himself to a particular theory, applicant believes that
the
enzymatic activity of value in the minor (E2) component is the 3'-exonuclease
activity, not the DNA
polymerase activity. In fact, it is further believed that this DNA polymerase
activity is potentially
troublesome, leading to unwanted synthesis or less accurate synthesis under
conditions optimized for
the majority (E1) DNA polymerase component, not the minority one. As taught by
[Bernad, Blanco
35 and Salas (1990) Site-directed mutagenesis of the YCDTDS amino acid motif
of the phi 29 DNA
polymerase, Gene 94:45-51.] who mutated the "Region I" DNA conserved DNA
polymerase motif of
phi 29 DNA polymerase, either Region III or Region I of the Pfu DNA polymerase
gene are
mutated,
--. ~~ ~_ 215616
which has been sequenced by [Uemori,T., '; Ishino,Y., Toh,H., Asada,F. and
Kato,I.
Organization and nucleotide sequence of the DNA polymerase gene from the
archaeon Pyrococcus
furiosus, Nucleic Acids Res. 21, 259-265 (1993)].
In a further embodiment of the present invention, a formulation of DNA
polymerase is provided including or consisting of at least one DNA polymerase
which, in wild-type
form, exhibits 3'-exonuclease activity and which is capable of catalyzing a
temperature cycle type
polymerase chain reaction in which the 3'-exonuclease activity of the one or
more DNA polymerases
discussed above has been reduced substantially, but not eliminated. The
diminished 3'-exonuclease
activity is obtained by mutation, or by chemical or other means known to those
in this art. This
formulation of DNA polymerase may then be used to catalyze primer extension in
PCR
amplification. The Spanish researchers [Soengas, M.S., Esteban, J.A., Lazaro,
J.M. Bernad, S.,
Blasco, M. A.,.1 Salas, M., and Blanco, L., Embo Journal 11:4227-4237 (1992)],
studying a
distantly related DNA polymerase of the alpha class which includes Pfu, Vent~
and Deep Vent~
DNA polymerases, also identified and demonstrated mutation of the 3'-
exonuclease domain as well
separated and easily avoided while one is mutating the DNA polymerase
motif(s). In this same
report, they demonstrate that the exonuclease can be reduced to 4-7 % activity
by a change of the
conserved Tyr residue to a Phe or a Cys, whilst a reduction to only 0.1 %
activity is obtained by
replacing the conserved Asp with Ala. For long and accurate PCR, the optimum
exo- (3'-exonuclease
negative) mutation to create in a thermostable DNA polymerase such as Pfu,
Vent~ or Deep Vent~,
2 0 is one that reduces but does not eliminate the exonuclease, for instance a
reduction to 0.2-7 % ,
preferably, 0.5-7 % , and most preferably, 1-7 % of the 3'-exonuclease
activity of the wild-type DNA
polymerase.
As an alternative way to introduce the mutations (and as demonstrated for
the Bt CryV gene example above) Klentaq-LA, the current invention, is used to
introduce the
2 5 changes into a small PCR product spanning the DNA sequence coding for the
two homologies
REGION III and REGION I. The changes are chosen to change conserved amino
acids in REGION
III and/or REGION I, using as a guide the conserved motifs displayed below
with the aid of the
MACAW computer program.
I (data details not shown, but analogous to Example 1 above and readily
3 0 carried out by one skilled in the art) have PCR-amplified the ORF of the
Pfu DNA polymerase gene
from Pfu DNA, using the sequence published by Uemori et al (1993) (supra) as a
guide.
Analogously to Example 2 above, I have cloned this ORF into the same
expression vector as used for
expression of Klentaq-278, and I have shown that the DNA polymerase can be
purified by the same
procedure as described here for Klentaq-278 in Example 3 above.
3 5 As an alternative way (alternative to Soengas et. al, supra) to introduce
the
mutations (and as demonstrated for the Bt CryV gene example above) Klentaq-LA,
the current
invention, would be used to introduce the changes into a small PCR product
spanning the DNA
sequence coding for the two homologies REGION III and REGION I. The changes
would be chosen
to change conserved
. A
WO 94126766 E 215 61 ~ s PCTlUS94101867
.
18
amino acids in REGION III and/or REGION I, using as a guide the conserved
motifs displayed below
with the aid of the MACAW computer program.
The following is numbered output from the computer program MACAW,
demonstrating two of the DNA polymerise conserved motifs. A useful
presentation of these and other
S DNA polymerise motifs can also be found in Perler et al (1992) P.N.A.S. 89,
5577-5581.
SEQ ID N0:30 REGION III
Phi29 -------KLMLNSLYGkfasapdvtgkvpylkengalgfrlgeeetkdpvytpmgvfITA 435
Pfu dyrqkaiKLLANSFYGyygyakarwyckecaes------------------------VTA 516
SEQ ID N0:31
SEQ ID N0:32 REGION I
Phi29 WARyttitaaqacyd----RIIYCDTDSIHLTgteipdvikdivdpkklgywah------ 485
Pfu WGRkyielvwkeleekfgfKVLYIDTDGLYATipggeseeikkkalefvkyinsklpgll 576
SEQ ID N0:33
The following examples illustrate the invention.
EXAMPLE 1
Construction of an Expressible Gene for Klentag-278
In order to construct the Klentaq-278 DNA polymerise gene having a recombinant
DNA sequence shown as the nucleotide sequence of Figure 1, the following
procedure was followed.
The mutated gene was amplified from 0.25 ug of total Thermus aquaticus DNA
using
the polymerise chain reaction (PCR, Saiki et al., Science 239:487, 1988)
primed by the two synthetic
DNA primers of Figure I. Primer KTI, SEQ ID NO:1, has homology to the wild-
type DNA starting
at colon 280; this primer is designed to incorporate a NcoI site into the
product amplified DNA.
Primer Klentaq32, SEQ ID N0:3, a 33mer spanning the stop colon on the other
strand of the
wild-type gene encoding Thermus actuaticus DNA polymerise, and incorporating a
HindllI site and a
double stop colon into the product DNA.
The buffer for the PCR reaction was 20 mM Tris HCl pH 8.55, 2.5 mM MgCl2, 16
mM (NH,)zSO" 150 ug/ml BSA, and 200 uM each dNTP. The cycle parameters were 2'
950, 2'
650, 5' 72~.
In order to minimize the mutations introduced by PCR (Saiki et al., sub), only
16
WO 94/26766 21 5 fi 1 ~ 6 ~ 1'CZ'IUS94I01867
19 -
cycles of PCR were performed before phenol extraction, ethanol precipitation,
and digestion with the
restriction enzymes NcoI and HindI)l.
EXAMPLE 2
Preparation of an Expression Vector
The product NcoI and Hind)TI fragment was cloned into plasmid pWB254b which
had been digested with NcoI, HindBI, and calf intestine alkaline phosphatase.
The backbone of this
plasmid, previously designated pTAC2 and obtained from J. Majors, carries the
following elements in
counter-clockwise direction from the PvuII site of pBR322 (an apostrophe '
designates that the
direction of expression is clockwise instead of counter clockwise): a partial
IacZ' sequence, lacI',
lacPUVS (orientation not known), two copies of the tac promoter from PL
Biochemicals
Pharmacia-LKB; catalog no. 27-4883), the T7 gene 10 promoter and start codon
modified to consist of
a NcoI site, a HindBI site, the ~A terminator (PL no. 27884-01), an M13 origin
of replication, and
the ~R gene of pBR322. Expression of the cloned gene is expected to be induced
by 0.1 mM
IPTG.
Ampicillin-resistant colonies arising from the cloning were assayed by the
single
colony thermostable DNA polymerase assay of Sagner et al. (1991) [GENE 97:119-
23) and 4 strong
positives were sized by the toothpick assay (Barnes, Science 195:393, 1977).
One of these, number
254.7, was of the expected size except for a small proportion of double
insert. This plasmid was
further purified by electroporation into E. coli X7029 aad screened for size
by the toothpick assay,
and one plasmid of the expected size with no double insert contamination was
designated pWB254b.
This plasmid was used for the production of Klentaq-278 described herein.
EXAMPLE 3
Purification of Large Amounts of Klenta4-278
Plasmid pWB254 has a double (tandem repeat) tac promoter and the T7 gene 10
leader sequence, an ATG start codon, a glycine codon and then codons 280-832
of Thermus aquaticus
DNA polymerise, then a tandem pair of stop codons followed by the trp
transcription terminator. The
pBR322-based plasmid vector (pTac2 from John Majors) is ampicillin resistant.
The cells are grown
on very rich medium (see below). Bacterial host X7029 is wild-type H E. coli
except for deletion
X74 of the lac operon.
Medium: Per liter water, 100 mg ticarcillin (added when cool), 10 g Y.E., 25
g.
Tryptone, 10 g. glucose, 1XM9 salts with no NaCI (42 mM NazPO" 22 mM KHZPO" 19
mM NH,CI).
Do not autoclave the glucose and the lOXM9 together; instead, autoclave one of
them separately and
mix in Later. Adjust pH to 8 with 5 M NaOH (about 1 ml). Add IPTG to 0.1 mM at
ODS~ = 1 or 2, and shake well at 30° C. From OD = 2 up to 8 or 10,
every half hour or so do the
following:
2~~fi~~~
Read the pH with pH sticks 5-10. Adjust to pH 8.5 with 5 M NaOH
and swirling (2 to 5 ml per liter) whenever the pH falls below 8.
2. Read and record the ODSSO, usually as a 1/10 or 1/50 dilution.
3. This addition of glucose is optional and not necessarily of any value
(evaluation of this question is incomplete at this time.) Read the glucose
level with glucose sticks,
and add an additional 0. 5 % ( 10 ml of 50 % ) if the level falls below 0.2 %
.
If it is late, the cells can shake at 30° C all night after the last
pH ad-
justment. Alternatively, set them in the cold room if they have not grown much
in a few hours.
Concentrate the cells e.g. by centrifugation in a GS3 rotor for 8 minutes at
8 krpm. Pour off the supernatant and add culture to spin more down onto the
same pellets.
Lysis:
Resuspend the cells in milliliters of TMN buffer equal to twice the packed
cell weight in grams: (50 mM Tris-HCl pH 8.55, 10 mM MgCl2, 16 mM (NH4)zS04).
To each 300 ml of cell suspension add 60 mg lysozyme and incubate the
cells at 5-10° C. with occasional swirling for 15 minutes. Then add
Tergitol~ NP40 or Triton~ X100
to 0.1 %, and Tween 20 to 0.1 % , by adding 1/100 volume of a solution of 10%
in each. Then heat
the cell suspension rapidly to 80° C. by swirling it in a boiling water
bath, then maintain the cells
(fast becoming an extract) at 80-81° C. for 20 minutes. Use a clean
thermometer in the cells to
measure temperature. Be sure the flask and bath are covered, so that even the
lip of the flask gets
2 0 the full heat treatment. After this treatment, which is expected to have
inactivated all but a handful
of enzymes, cool the extract to 37° C. or lower in an ice bath and add
2 ml of protease inhibitor (100
mM PMSF in isopropanol). From this point forward, try not to contact the
preparation with any
flask, stir bar, or other object or solution that has not been autoclaved.
(Detergents and BME are not
autoclavable. The PEI and ammonium sulfate are also not autoclaved.) The
purpose of the
2 5 autoclaving is not only to avoid microbial contamination, but also to
avoid contamination with DNA
or nucleases.
Distribute into centrifuge bottles and centrifuge at 2° C. (for
instance, 30
minutes at 15 krpm in a Sorval SS-34 rotor or 14 h at 4 krpm in a GS3 rotor).
The supernatant is
designated fraction I, and can be assayed for DNA polymerase activity.
3 0 High-salt PEI precipitation
After rendering fraction I 0.25 M in NaCI (add 14.6 g per liter), add
five percent Polymin-P~ (PEI, polyethylene-imine, Sigma) dropwise with
stirring on ice to
precipitate nucleic acids. To determine that adequate Polymin-P~ has been
added, and to avoid
addition of more than the minimum amount necessary, test 1/2 ml of centrifuged
extract by adding a
3 5 drop of Polymin-P~, and only if more precipitate forms, add more Polymin-
P~ to the bulk extract,
mix and retest. Put the test aliquots of extract back into the bulk without
contaminating it.
To confirm that enough PEI has been added, centrifuge 3 ml and aliquot
the supernatant into 1/2 ml aliquots. Add 0, 2, 4, 6 or 10 ul of 5 % PEI.
Shake, let sit on ice, and
centrifuge in the cold. Load 15 ul of these aliquot supernatants onto an
agarose gel containing ethidi-
4 0 um bromide and electrophorese until the blue dye has travelled 2 cm.
Inspect the gel on a UV light
box for detectable DNA or RNA in the supernatant. For the bulk extract, use
about 1/100 volume
(i.e. 2-3 ml for a 300 ml extract) excess 5% PEI over the minimum necessary to
remove all DNA by
the agarose gel test.
r~..
21561~~
,, m
Stir in the cold for at least 15 minutes. Centrifugation of the extract then
removes most of the nucleic acids. Keep the supernatant, avoiding any trace of
the pellet.
Dilute the PEI supernatant with KTA buffer until the conductivity is
reduced to at or below the conductivity of KTA buffer with added 22 mM
ammonium sulfate. (Check
conductivity of 1/40 dilution compared to similar dilution of genuine 22 mM
A.S. in KTA.) Usually
this is about a 5-fold dilution.
Chromatography with Bio-Rex 70 (used by Joyce & Grindley) (Joyce,
C.M. & Grindley, N.D.E. (1983) Constrnction of a plasmid that overproduces the
large proteolytic
fragment (Klenow fragment) of DNA polymerase I of E. coli, Proc. Natl. Acad.
Sci. U.S.A. 80,
1830-1834) is unsuccessful (no binding), but unavoidable, since without it,
the next column (heparin
agarose) will not work efficiently. We believe that the important function of
the Bio-Rex 70 step is
to remove all excess PEI, although it is possible that some protein is removed
as well. CM-cellulose
does not substitute for Bio-Rex 70.
Pass the diluted PEI supernatant through equilibrated Bio-Rex 70 ( 10 ml
per 100 g. cells). The polymerase activity flows through. Rinse the column
with 2 column volumes
of 22 mM A.S. / KTA. Our procedure is to set up the following heparin agarose
column so that the
effluent from the Bio-Rex 70 column flows directly onto it.
Heparin Agarose Chromatography (room temperature, but put fractions
on ice as they come off.)
2 0 Load the Bio-Rex flow-through slowly onto heparin agarose (Sigma; 10 ml
per 100 grams of cells [this could be too little heparin agarose].) Wash with
several column volumes
of KTA + 22 mM A.S., then three column volumes of KTA + 63% glycerol + 11 mM
A.S., then
elute the pure enzyme with KTA + 63 % glycerol + 222 mM A.S. + 0.5 % Thesit~
(this is more
Thesit~ for the final eluate.)
2 5 Pool the peak of polymerase activity or ODzBO/(starts about at 213 of one
column volume after 222 mM starts, and is about 2 column volumes wide). Store
pool at -20° C.
The storage buffer is a hybrid of, and a slight variation of, AmpliTaq~
storage buffer as recommended by Perkin-Elmer Cetus and Taq storage buffer
used by Boehringer-
Mannheim: 50% glycerol (vlv; 63% wlv), 222 mM ammonium sulfate (diluted to
about 50 mM for
3 0 bench-strength samples), 20 mM Tris-HCl pH 8.55,0.1 mM EDTA, 10 mM
mercaptoethanol, 0.5%
Thesit~)
The Thesit~ causes some thickening and cloudiness below -10° C.
This
seems to cause no harm, but we suggest you warm the enzyme to 0° C. on
ice before aliquoting for
use.
A~
WO 94126766 2 1 5 6 1 ~ ~ PCT/LTS94/01867
;. , r ,
s
22
Thesit replaces the combination of 0.5 f6 Triton-X 100, 0. 5 °6 Tween
20, which you may want to
consider as an alternative.
We have had sporadic reports that freezing can inactivate the enzyme.
Exercise caution in this regard. This question is under current investigation.
Storage at -80°
(after quick-cooling with liquid nitrogen) is being tested and looks
promising, but more than
one freeze-thaw cycle has been deleterious to the enzyme preparation on some
occasions.
Our final yield of enzyme from 7 liters (100 g cells) was once 28 ml at a
concentration of 120,000 units per ml (4 x bench-strength).
1/4 ul of bench-strength enzyme will support the PCR of a 2 kb span of
DNA in a 100 ul reaction. Template is 5-10 ng of plasmid DNA. Each cycle
consists of 1
min 98° C, 1 min 65° C, 6 min 72° C. Cycle number is 16-
20. Less enryme is needed for
smaller-sized products (1/8 ul for 500 bp) and more enzyme is needed for
larger products (1
ul for 5 kb).
KTA Buffer per liter
_____-___________ _________________
mM Tris 8.55 10 ml of 2
M
10 mM BME 0.7 ml neat
109b w/v Glycerol 100 g.
0.1 mM EDTA 0.2 ml of .5
M
20 0.1 b w/v Thesit 10 ml of 10~n
Rough Incorporation Assay
1 X PC2 Buffer (20 mM Tris-HC1 pH 8.55, 2.5 mM MgCl2,
16 mM (NH~ZSO,, 100 uglml BSA)
200-250 ug/ml activated salmon sperm DNA
40 uM each dNTP + 10-50 uCi a '2P-dATP per ml
To 25 ul assay mix on ice add 0.2 ul of enzyme fraction, undiluted, or
diluted in 8 ul of 1XPC2 buffer (or a 1/5 or 1/25 dilution thereof.) Prepare
standard Klentaq
or Amplitaq, zero enzyme and total input samples, also. Incubate 10 min. at
72° C., then
chill. Spot 5 or 8 ul onto filter paper and wash twice for S - 10 min. with 5
9b TCA, 1 °6 PP;.
If pieces of paper were used, count each using Cerenkov radiation or hand
monitor. If a
single piece of 3 MM paper was used, autoradiograph for 60'.
PCR Assay to give 2 kb product.
Make up 1 ml of PCR reaction containing 50 ng of plasmid pLc (a clone of
an R color control cDNA from maize. PNAS 86:7092; Science 247:449), 200 pmoles
each of
WO 94/26766 ~': y t x ~3 ~ t,~ PCTIUS94101867
,~.. 21 5~ 17 fi
23
primers Lc5 (SEQ ID NO:11) and Lc3 (SEQ ID N0:12), PC2 buffer and 200 uM
dNTPs,
but no enryme.
Distribute 100 ul into tube one, and 50 ul into the rest of 8-10 tubes: Add 1
ul of final pool of KlenTaq to tube one and mix. Then remove 50 ul to tube two
and mix
that, and so on down the series, which will then contain decreasing amounts of
enryme in
two-fold steps. Cover each 50 ul reaction with a drop of mineral oil, spin,
and PCR 16
cycles at 2' 95 ° C, 2' 65 ° C, 5' 72 ° C.
Final Bench-Strength KlenTaq-278 Enzyme
Using 63 9b glycerol / KTA (.5 9b Thesit) buffer with 222 mM ammonium
sulfate, dilute the pool conservatively so that 1/4 ul should easily catalyze
the amplification
the 2 kb span by PCR. Do not decrease the ammonium sulfate concentration below
50 mM.
Store at -20° C.
EXAMPLE 4
DNA Amplification
As reported in Figure 3, a PCR amplification assay to produce 2 kb of DNA
product was conducted using Thermus aquaticus DNA polymerase (AmpliTaq) (prior
art
DNA polymerase) and Klentaq-278. To test polymerase thermostability at
elevated
temperatures, the DNA denaturation step of the PCR amplification reactions
were conducted
for 2 min. at 97~C, 98~C and 99~C, respectively, using graduated
concentrations of DNA
polymerase.
The amplification procedures used followed approximately the protocol for
amplifying nucleic acid sequences outlined by Saiki et al., Science 239:487,
1988. A 1 ml
reaction mixture was prepared containing 100 ng of plasmid pLC, 200 pmoles
each of
primers Lc5 (SEQ ID NO:11) and Lc3 (SEQ ID N0:12), reaction buffer (20 mM Tris-
HCl
pH 8.55, 16 mM ammonium sulfate, 2.5 mM MgCl2 and 150 uglml BSA), 200 uM
dNTPs,
but no enzyme. 100 ul of the reaction mixture was placed into tubes. Aliquots
of AmpliTaq
and Klentaq-278 were then added and 20 cycles of PCR were undertaken.
Figure 3 shows the results of the experiment to compare the practical
thermostability limits. The only change between the 3 panels shown is the
temperature of the
2 min. denaturation step: 97 ° C, 98 ° C, or 99° C. A
range of enzyme concentrations was
used in order to be able to detect small effects on the effective PCR
catalysis activity. The
template was 10 ag of pLc (a clone of an R color control cDNA from maize. PNAS
86:7092,
Science 247:449). The primers were L,cS (SEQ ID NO:11) and Lc3 (SEQ ID N0:12).
Other
details of the reactions are given in the assay section of Example 3.
It can be seen in this experiment that 98° C was not detectably
detrimental
to KlenTaq-278, yet AT was nearly completely inactivated by this temperature.
WO 94126766 ~' ~ 1 5 6 1 ~ 6 ~T/US94I01867
i
24
IIrt the experiment shown in Figure 4, each of four enrymes (AT, KlenTaq-
278', S'Z', and KlenTaq-291) was tested for thermostability at 98° C.
Each was tested in pairs
of two concentrations differing by a factor of Z. The volumes of actual enryme
preparation
are indicated above each lane in ul. The amount used was adjusted from
previous titrations
(conducted as described for the 2 kb PCR assay in Example 3 and the legend to
Figure 3) so
that a 2-fold drop-off in activity would be detectable. Note the large amount
of ST necessary
to function at the 95 ° C control PCR. A previous attempt at this
experiment (data not shown)
used only 1/4 these volumes of ST (which would have been equivalent standard
DNA
polymerase incorporation units compared to KT-291 and KT-278), and no product
was
obtained.
EXAMPLE 5
Single Colony PCR
The analysis of single E. coli colonies by PCR is a convenient screen for the
presence and/or orientation of desired DNA fragments during a cloning or
recloning
procedure. In the prior art, the bacteria may not be simply added to a
complete PCR
reaction, since they evidently do not lyre efficiently enough to release the
plasmid DNA that
is to be the template for the PCR. Instead, and cumbersomely, since it
requires a complete
extra set of labelled test tubes, bacteria must first be suspended in water,
not buffer, in the
optional but recommended (Riggs et al.) presence of chelating resin, and
heated to 100° C for
several (such as 10) minutes. Then 1-10 ul of the heated bacterial suspension
is added to an
otherwise complete PCR reaction, which is then cycled and analyzed normally.
The improvement here is that, since Klentaq-278 can withstand 98-
99° C.
during the denaturation step of each PCR cycle, the bacteria can be added
directly and
conveniently to a complete (including Klentaq-278 enryme) PCR reaction and
then the PCR
cycling can begin without further pretreatment. The only difference from a
normal PCR
cycling is that the full 98° C (2 min.) or 99° C (1 min.)
temperature is used during each
denaturation step (or at least the first 5-10 steps) of the PCR. The
experiment in Figure 5
used 2 min. at 98° C for all 25 cycles, and demonstrates that this
method gives rise to a more
intense and reliably distinguished product band even than the prior art method
which utilizes a
10' 100° C separate treatment. This improvement is not possible with AT
enzyme, since AT
enryme is inactivated at 98° C (as shown in figures 3 and 4).
Figure 5 is a photograph of an agarose gel of a demonstration of the
advantage of a 98 ° C denaturation step in colony PCR, compared to the
standard 95 ° C
temperature. Lanes 1 and 3 employed the prior art pre-treatment of the
bacteria in distilled
water at 100° C for 10 minutes before addition to the PCR reaction.
Lanes 2 and 4
conveniently dispensed with this step and the same amount of bacterial
suspension (about 2 to
i
WO 94/26766 ~ ~ ~ ~ ~"~ ~ ~~ y PCTILTS94101867
25 ,
4 X 106 cells, but the identical volume of the same bacterial suspension) was
simply
introduced into the complete PCR reaction (including buffer, triphosphates,
primers and
enryme KlenTaq-278. ) Lanes 1 and 2 employed the standard 95 ° C, and
lanes 3 and 4
employed the newly possible 98 ° C denaturation/cell-disruption
temperature. The cycle
conditions were 2 min. at 98 ° C or 95 ° C, 2 min. at 65
° C, and 5' at 72 ° C., for 25 cycles.
The primers used were KT2 (37mer GAG CCA TGG CCA ACC TGT GGG GGA GGC TTG
AGG GGG A) and KIenTaq32 (see Figure 1). The bacterial cells were X7029
containing
plasmid pWB319, a broad-host range plasmid containing the coding region of the
gene for
KlenTaq-278.
Lane 4 is the most convenient and the most effective method, and it takes
advantage of the new stability of KlenTaq-278.
EXAMPLE 6
Efficent and Accurate PCR Amplification of
Long DNA Tareets: (Part A)
A preferred embodiment of the above formulation (designated KIenTaq-LA):
Starting with the purified enrymes in storage buffer, mix 1 ul of Pfu DNA
polymerase at 2.5
u./ul with 64 ul of KlenTaq-278 at 25 u./ul. Store at -20° C.
Larger amounts of Pfu are detrimental to some PCR amplifications, perform
equally for some, and are beneficial for some. For testing of the optimum
level of Pfu,
several reactions complete with KlenTaq-278 are aliquoted in the amount left
to right of 75 ul,
ul, 25 ul, and as many additional 25 ul aliquots as desired. Then 3/8 ul of
Pfu (equivalent
to 0.5 ul per 100 ul -- this is about the most that one would ever want) is
added to the
leftmost, 75 ul reaction and mixed. Serial, two-fold dilutions are then made
as 25 ul + 25 ul
left to right along the row of tubes, adding no Pfu to the last one, as a
control of KlenTaq-
25 278 alone. A reaction of 1/2 or 1 ul (per 100 ul) of Pfu alone should also
be run.
Reaction buffer is PC2 as above, supplemented with 200 uM of each dNTP
and 800 uM of MgClz (total Mg++ 3.3 mM), and per 100 ul of reaction volume, 20
pmoles
of each primer MBL (SEQ ID N0:7) and MBR (SEQ ID N0:8), and 30 ng of ~plac5
intact
phage. Per 100 ul of reaction volume, 1 or 1/2 ul of KTLA are effective levels
of enryme.
Suitable PCR cycling conditions are two-temperature: 20 seconds at 94°
C, 11 minutes at
70° C, for 20 cycles. Alternate cycling conditions include two-
temperature PCR with 1
minute at 98° C and 10 minutes at 65° C. 10 to 16 ul are loaded
onto an agarose gel for
product analysis by staining with ethidium bromide. See Figure 6 for other
details and
variations. The template was ~plac5, which carries a portion of the lac operon
region of the
E. coli genome. Thirty ng of phage DNA were included in each 100 ul of
reaction volume,
WO 94126766 f ~'~ 5 6~~ ~ ~ PCTIUS94101867
26
introduced as intact phage particles. The primers are homologous to wild-type
lambda DNA
and amplify ~ DNA, not the lac DNA. Primer MBL No. 8757 (5' nuclei~ti(de
matches:,~a~e
vf, q. :, .~: .. ..
pair 27914 of ~ DNA) is GCT TAT CTG CTT CTC ATA GAG TCT TGC (SEQ YD X10:7).
Primer IVIBR No. 8835 (5' nucleotide matches by 34570 of ~ DNA) is ATA ACS FTC
~-A'FA .;t,
TAC ATG GTT CTC TCC (SEQ ID N0:8). The size of the amplified product is
therefore
predicted to be 6657 bp.
As shown in Figure 6A and 6B, each DNA polymerase enzyme (KlenTaq-
278 or Pfu) alone gives rise to a faint product band (except for some
reactions, when Pfu
alone does not work at all), but the combinations all give rise to product
bands that are 20 to
50 times more intense than either enzyme can catalyze on its own.
Figure 6C, second lane from the right, shows the surprising result of adding
as little as 1/64 ul of Pfu to 1 ul of KlenTaq-278 (a units ratio of 1/640).
Not shown are data
that as little as 1/200 ul (1/2000 in units) of Pfu contributed a noticeable
improvement to the
efficiency of this test amplification.
Vent DNA polymerase required 10-fold higher amounts (yet still minority
amounts) for similar functionality.
An additional, beneficial, and unexpected attribute to the PCR reactions
catalyzed by KlenTaq-LA was a phenomenal, never previously observed intensity
and
sharpness to the PCR product bands. In part, this increased yield is
manifested by a dark
area in the middle of the bands as photographed. This darker area in the
ethidium
flourescence is believed to be due to UV absorbance by the outside portions of
the band,
reducing the potential UV-activated flourescence. The system apparently
allowed a much
greater yield of product then did the prior art, which tended to create a
broad smear of
product, and increasing amounts of side product, when amplification was
allowed to proceed
to this extent.
EXAMPLE 7
Efficent and Accurate PCR An~lification of
Lone DNA Targets: (Part B)
Efficient amplification of 8.4 kb, 12.5 kb, 15 kb, and 18 kb was
demonstrated by the experiment depicted in Figure 7. This experiment extended
the
demonstrated performance of the a preferred embodiment of the invention, 1/640
KlenTaq-
LA, even further. The amplification was highly successful for the size range
8.4 to 15 kb,
delectably successful for 18 kb, but not successful for an attempted 19.7 kb.
Eight different PCR reactions were run in this experiment, differing from
each other in the template or amount of template or in the primer pair
employed, as shown in
the legend on Figure 7. Each reaction was divided 3 ways and cycled
differently in parts A,
WO 94/26766 PCTIL1S94101867
2,
B, and C. Between parts A and B, this experiment compared 20 cycles to 30
cycles at 94 °
denaturatioa phase. In parts B and C, this experiment compared 94° to
93° for 30 cycles.
This experiment utilized 1.3 ul of Klentaq-LA (at a Klentaq-278/Pfu ratio of
640) per 100 ul
of reaction. This may have been a little too much enzyme, since high enzyme
has been
associated in previous experiments with the catastrophic synthesis of product
which cannot
enter the gel, as occurred here for the reaction products in channels 2B and
6C. At the
current stage of development of long PCR using the invention, this poor
outcome occurs
about 10°k of the time.
Comparing conditions B and C, it is apparent that somewhat lower denaturation
temperature is desirable. This is consistent with similar experiments
comparing time at 94° C., in
which yield of long PCR products was found to be decreased as the denaturation
time increased in the
order 2, 20, 60, and 180 seconds at 94° C for the denaturation step of
each cycle. These data indicate
that there was at least one weak link, i.e. least thermostable component, in
the reactions which is
subject to inactivation at 94°. Since 94° is below the
temperature known to damage the DNA
polymerise activity and the DNA, it is believed that it is not the
thermolabile element. In an
alternative embodiment of this aspect of the invention Pfu DNA polymerise is
replaced as the minority
component with a more thermostable 3'-exonuclease of a DNA polymerise such as,
but not limited to,
that from the Archaebacterium strain ES4, which can grow at temperatures up to
114° C [Pledger,
R.J. and Baross, J.A., J. Gen. Microbiol. 137 (1991)], which maximum growth
temperature exceeds
that of the source of the Pfu DNA polymerise (103° C.; Blumentals, LI.
et al. (1990) Annals of the
N.Y. Acid. Sci. 589:301-314.)
In the experiment in Figure 7 the final intensity of the 15 kb band matched in
only
20 cycles the yield obtained by Kainze et al.su~ra in 30 cycles for a band of
similar size and from
similar DNA template amounts. This was a measure of the improved efficiency
provided by the
invention, and the further result was that the yield catalyzed by the
invention in 30 cycles greatly
exceeded the yield reported by these authors for 30 cycles. Accurate
quantitation has not yet been
carried out to measure the efficiency of the two methods, but inspection of
Figure 7 compared to the
figure published by Kainze et al. shows a yield for the 15 kb fragment that is
estimated to be some
100 times higher. This corresponds approximately to a doubled efficiency of
PCR extension.
EXAMPLE 8
Efficent and Accurate PCR Aa~lification of
Long DNA Tareets: fPart C)
Materials and Methods
DNA Polymereses. DNA polymerises Vent and Deep Vent were supplied by New
England Biolabs. Pfu DNA polymerise and its exo' mutant were supplied by
Stratagene at 2.5
WO 94126766 PCTlUS94101867
158176
28
units/ul. Klentaq-278 is an N-terminal deletion variant of Taq DNA polymerase
(WMB, unpublished).
The deletion endpoint is between that of Klentaq5 (10) and Stoffel Fragment
(3). Purified Klentaq-278
was as supplied by Ab Peptides, St. Louis, MO, USA at 25-35 units/ul (a
protein concentration of
about 0.7 ug/ul). One unit of DNA polymerase activity incorporates 10 amoles
of nucleotide in 30
min. at 72° C., utilizing activated (partially degraded) calf thymus
DNA as template. Since activated
calf thymus DNA is a somewhat undefined substrate and is structurally
different from PCR reaction
substrate, this assay was routinely eschewed in favor of a PCR-based assay to
set the above stock
concentration of Klentaq-278: the concentration of Klentaq-278 stock was
adjusted so that 0.25 ul
effectively (but .12 ul less effectively) c:~ ~zes the amplification of a 2 kb
target span from 10 ng of
plasmid substrate with cycling conditions including 7 min. of annealing /
extension at 65°. The
mixture of 15/16 ul Klentaq-278 + 1/16 ul Pfu DNA polymerases is designated
KlentaqLA-16.
Agarnse gel electrophoresis employed 0.7 k to 1 °b agarose in 1XGGB
(TEA) buffer
[40 mM Tris acetate pH 8.3, ZO mM sodium acetate, 0.2 mM EDTA] at 2-3 v/cm,
with 3 ~ ficoll
instead of glycerol in the loading dye. Figure 11 employed 1~ agarose pulsed-
field CHEF (11) with a
switching time of 4 sec. Standard DNA fragment sizes in every figure are, in
kilobases (kb): 23.1,
9.4, 6.6, 4.4, 2.3, 2.0, and 0.56. Figure 11 and 12 also have a full-length
~plac5 standard band,
48645 bp.
All agarose gels were run or stained in ethidium bromide at 0.5 ug/ml and
photographed (35 mm ASA 400 black and white film) or videographed (Alpha
Innotech or Stratagene
Eagle Eye) under UV illumination. While printing the gel photographs, the left
halves of Figure 7 and
10 were exposed 50°.b less than the right halves.
DNA primers are listed in Table 1 and in the Sequence Listing.
Lambda DNA templates. wacA, a gift from S. Phadnis, is a ~EMBL4-vectored
clone of the cytotoxin gene region of Helicobacter pylori DNA. This DNA was
extracted and stored
frozen. The other phage template DNAs ~plac5 (12) and ~IC138 (13) were added
as intact phage
particles that had been purified by CsCI equlibrium centrifugation, dialyzed,
and diluted in 1X PC2
buffer.
Long and Accurate PCR. PC2 Reaction buffer (10) consisted of 20 mM Tris-HCI
pH 8.55 at 25°, 150 ug/ml BSA, 16 mM (NH,~ZSO,. 3.5 mM MgCl2, 250 uM
each dNTP. For
success above 28 kb (at 35 kb), 1.5 ul of 2 M Tris base was added to each
reaction, corresponding to
pH 9.1 measured for the Tris-HCl component only at 20 mM in water at
25° C. Contact with a pH
probe was detrimental to the reactions, so pH was only measured on separate
aliquots, and found to be
8.76 in the final reaction at 25° C. Each 100 ul of reaction volume
contained 20 pmoles of each
primer, and 0.1 to 10 ng of phage DNA template. 0.8 or 1.2 ul of KlentaqLA-16
was appropriate for
under 20 kb and over 20 kb, respectively. Reaction volumes per tube were 33-50
ul, under 40 ul of
mineral oil in thin-walled (PGC or Stratagene) plastic test tubes.
PCR reactions utilizing the primers at the ends of ~ required a preincubation
of S
WO 94/26766 ~ PCTIUS94101867
.1581 . ..
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min. at 68°-72° to disrupt the phage particles and to allow fill-
in of the ~ sticky ends to complete the
primer homology. Optimal cycling conditions were in a multiple-block
instrument (Robo Cycler,
Stratagene) programmed per cycle to 30 sec. 99°, 30 sec. 67°,
and 11 to 24 min. at 68°, depending
on target length over the range shown in Table 1. The second-best cycler was
the Omnigene
(HybAid), programmed under tube control per cycle to 2 sec. at 95 °,
then 68 ° for similar anneal
ing/extension times. Unless otherwise stated, all of the experiments reported
here used 24 cycles.
For reported results of comparison of conditions such as cycling temperatures
and
times, thermal cycler machines, thick and thin-walled tubes, etc., reactions
were made up as 100 ul
complete and then split into identical aliquots of 33 ul before subjecting to
PCR cycling.
Table 1. Primer and template combinations.
Product Left Right Template
Size Primer Primer DNA
SEQ ID SEQ ID
,
5.8 25 MBL101 28 MSA1933 ~IC138
6657 7 MBL 8 MBR ~plac5
8386 9 MBL-1.7 8 MBR AplacS
8.7 26 MBR001 29 ~R36 ~IC138
12.1 22 lacZ333 27 MBR202 AK138
12.5 7 MBL 27mer 8 MBR 27mer wacAI
or
25 or MBL101 27 MBR202 33mer
33mer
15560 10 MSA19 28mer 27 MBR202 ~plac5
28 or MSA1933
33mer
18.0 25 MBL101 27 MBR202 ~IC138
19.8 20 ~I36 24 MBL002 ~IC138
20707 25 MBL101 29 ~R36 36mer ~plac5
19584 20 ~I36 22 lacZ333 ~plac5
13971 26 MBR00133mer 29 aR36 ~plac5
22.0 20 ~L36 21 lacZ'S33 aIC138
24.6 20 ~I36 28 MSA1933 AK138
22495 20 ~L36 23 IacZ536 aplac5
26194 21 lac2'S33 29 ~R36 aplac5
28083 20 ~L36 24 MBL002 ~plac5
34968 20 aI36 27 MBR202 aplac5
Legend to Table 1.
Product sizes in integer base pairs are as predicted from the sequence and
structure of ~ and J~plac5 as
documented in Gcnbank accession no. J02459 and ref. (21). Product sins with
decimal points in kb
were determined by comparison with these products and with the ~+HindBI size
standards labelled
~H3. The sequence of the primers is given in the Sequence Listing.
Megaprimer consisted of gel-purified 384 by PCR product DNA homologous to the
region between the BamHl site and EcoRI site of the gene coding for the CryV
ICP of Bacillus
WO 94126766 PCTIUS94101867
l ~ 15 6 ~ ~_ :~ ~. . ' ..
thurin i~ (14), and primer-modified to remove these restriction sites. The PCR
reactions in Figure
10 each employed megaprimer (300 ng) , primer BtVS and 20 ng of genomic DNA
from Bacillus
thuringiensis strain NRD12 (15), and enzyme as indicated in the descripiton
above of Figure 9.
Cycling conditions were 30 sec. 95°, 7 min. 60°, for ZO cycles.
5 HindI)Q digestion. Unfractionated, total PCR reactions for 28 and 35 kb
targets
were supplemented with 1/10 volume of IOXNaTMS (1X = 50 mM NaCI, 10 mM Tris-
HCI pH 7.7,
10 mM MgCl2, 10 mM mercaptoethanol) and 2 ul (10 units) of restriction enzyme
Hind)TI, and
incubated at 55 ° C. for 90 min.
Test of exo- Pfu. Each 100 ul of reaction (incubated as 33 ul under 40 ul of
oil)
10 contained 2 ng aplac5 DNA as purified phage particles, 20 pmoles each of
primers MBL-1.7 and
MBR , reaction buffer PC2 and 1 ul of Klentaq-278 (0.7 ug), except for
reaction 6, which contained I
ul Pfu DNA polymerise (2.5 u.) alone. Other details are in the description of
Fig. 12. Thermal
conditions were 24 cycles of 2 sec.at 94°, 11 min. at 70°.
The discovery leading to the DNA polymerise mixture of the present invention
was
15 made during attempts to utilize in PCR a primer with a mismatched A-A base-
pair at its 3' end. In
fact the primer was itself a PCR product "megaprimer" of 384 bp, and the
mismatched A had been
added by Klentaq-278 using non-templated terminal transferase activity common
to DNA polymerises
(16). Neither Klentaq-278 (Figure 9, lane 3) nor Pfu DNA polymerise (Figure 9,
lanes 1 & 2 and
other levels of enzyme not shown) could catalyze amplification of the 1500 by
target that lay between
20 the PCR-product megaprimer and a 42mer oligonucleotide primer. The
combination of the two
enzymes, however, was well able to catalyze amplification of the desired
target fragment (Fig 9, lane
4). Evidently, the Pfu DNA polymerise removed the presumed 3' A-A mismatch,
allowing Klentaq-
278 catalysis to proceed efficiently for each step of the PCR. The same result
was obtained with Vent
DNA polymerise substituted for Pfu (data not shown).
25 I hypothesized that mismatched 3'-ends are a general cause of inefficient
primer
extension during PCR of targets larger than a few kb. As a test system I
employed a 6.6 kb lambda
DNA target which was amplified detectably but poorly by AmpliTaq, Klentaq-278
or Pfu DNA
polymerise in a variety of standard conditions. Per 100 ul reaction volume, 1
ul of Klentaq-278 was
combined with various amounts of Pfu DNA polymerise, from 1/2 ul down to as
little as 1/200 ul of
30 Pfu. Since the Pfu stock (2.5 units/ul) was at least 10 times less
concentrated than the Klentaq-278
stock (25-30 units/ul), the actual ratios tested were 1/20 to 1/2000 in DNA
polymerise units.
Representative results of these tests are shown in Figure 6B. A high yield of
target band was
observed for all tested combinations of the two enzymes, yet several levels of
each enryme on its own
failed to catalyze more than faintly detectable amplification. The lowest
level of Pfu tested, 1/200 ul,
exhibited only a slight beneficial effect. The apparent broad optimum ratio of
Klentaq-278:Pfu1 was
16 or 64 by volume, which is about 160 or 640 on the basis of DNA polymerise
incorporation units.
When tested at 6-8 kb (data not shown), other combinations of 3'-exo and 3'-
exo+ thermostable DNA
- .T
WO 94/26766 ~" '~ PCT/US94101867
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31
polymerases also showed the effect, including Amplitaq/Pfu, Klentaq-278/Vent,
Klentaq5 (DeltaTaq,
USB) I Pfu, Stoffel Fragment/Pfu, Klentaq-278/Deep Vent (our second choice),
and Pfu exo- I Pfu
exo+. Although comparatively few trials and optimizations were carried out, no
other combination
tried was as effective as Klentaq-278/Pfu.
A very short heat step is preferred. I next attempted to amplify DNA in the
size
range 8.4 to 18 kb from lambda transducing phage template. Our early cycling
protocol employed a
denaturation step of 1 or 2 minutes at 95° or 98° C, but no
useful product in excess of 8.4 kb was
obtained until the parameters of this heat step were reduced to 2 sec. or 20
sec. at 93° or 94° C. In
an experiment with the denaturation step at 94° for 20, 60, or 180 sec,
the 8.4 kb product exhibited
decreasing yield with increased length of this heat step (data not shown).
Apparently, a component of
the reaction is at its margin of thermostability. Figure 7 shows that, using
the short 2 sec.
denaturation step, target fragment was obtained for some reactions at all
sizes in the range 8.4 to 18
kb, with very high product yields up to 15 kb if 30 PCR cycles were employed.
Figure 7 also shows
some failed reactions which I cannot explain. The failure mode that gives rise
to massive ethidium
tl 15 staining in the sample well (30-cycle lane 2) was particularly common,
especially at high enryme
levels.
Longer Primers. A change in primer length from 27 to 33 greatly reduced the
frequency of failed reactions. Figure 10 demonstrates improved reliability for
amplification of 12.5,
and 18 kb with the longer 33mer primers, under conditions of otherwise
optimally high enzyme
levels in which the 27mer primers failed to give rise to desirable target
product. This result does not
represent an extensive survey of primer length, and it has not yet been
repeated with the improvements
below. Therefore the optimum primer length for long PCR remains to be
determined. Some of the
amplifications analyzed in Figure 11 utilized 36mer primers from the very ends
of ~. A 2-S min.
preincubation at 68-72° (22) was necessary to release the template DNA
from the phage particles and
to fill in the sticky ends of lambda to complete the template homology with
primers J~I36 and ~R36.
Rapid cycling. A change to thin-walled tubes, which have lower heat capacity
and
conduct heat more efficiently, further improved the reactions. Figure 1 I
shows a CHEF pulse-field
agarose gel analysis of successful amplifications of DNA spans 6-26 kb in
size. The target of 28 kb
was not amplifiable in the Omnigene thermal cycler (data not shown), but did
appear (Figure 12, lane
2) when the RoboCycler was employed.
Several models of thermal cycler have been employed, and although not all have
been optimized, some are preferable to others for long PCR. As may be
concluded from the
advantaggl'of thin-walled tubes noted above, success seems to be positively
correlated with a high
speed of temperature change made possible by the design of the thermal cycler.
The RoboCycler
a'~hieves rapid temperature change by moving tubes from block to block, and
observations with a
thermistor temperature probe indicate that it raises the reactions to 93-95
° for only 5 sec. under the
denaturation conditions employed (30 sec. in the 99° block), before
rapidly (within 30 sec) returning
WO 94/26766 " PCTIUS94101867
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32
the reaction to 68°.
Higher pH. The current record 35 kb (Figure 12, lane 3) was only amplifiable
if
the pH was increased. A preliminary scan of higher pH was carried out (data
not shown), and this
resulted in the appearance of the 35 kb band at pH 8.8 to 9.2, with the
optimum at 9.1 as described in
Methods (above). Further improvement to a high yield of the 35 kb product was
achieved by
lengthening the extension time to 24 min. Other than the higher pH, the long
PCR procedure has not
yet realized any potential benefits from changes in buffer conditions from
those optimized for 8.4 kb.
For Targets over 20 kbs extension times exceeding 20 min. are preferred and
the extension
temperature is preferably below 69° C.
Identity of long PCR products. It can be seen in Figures 7, 10 and 11 that
llae
mobilities of the successful large DNA products agree with those predicted in
Table 1 from the known
map positions of the primers used.
HindllI restriction enzyme digestion of the unpurified 28 and 35 kb products
(Figure
12, lanes 6 and 7) resulted in the expected left arm of lambda (23 kb) and 2.3
kb band from both, and
the predictable bands terminated by the right PCR primer: 447 by (barely
visible) from the 28 kb
product and 7331 by from the 35 kb product.
Exonuclease mutant. The available mutant of Pfu DNA polymerise (8) which is
defective in the 3'-exonuclease activity was tested. Figure 13 shows that the
3'-exo mutant of Pfu
DNA polymerise fails to promote efficient amplification of a long DNA target.
This supports our
hypothesis that the 3'-exonuclease activity is important for the efficiency of
PCR amplification in this
size range.
Fdelity test. Since the biological purpose of 3'-exonuclease is to edit base
pair
mismatches for high replication fidelity, we tested the fidelity of the PCR
product using an assay
involving the amplification and molecular cloning of an entire IacZ (~-
galactosidase) gene flanked by
two selectable markers (10). Heretofore the highest reported fidelity of PCR
amplification is that
catalyzed by Pfu DNA polymerise (2). Table 2 shows that the fidelity of the
product amplified by the
640:1 mixture of Klentaq-278 and Pfu DNA polymerise at least matches that
obtained for Pfu DNA
polymerise, alone, when each are used for 16 cycles of PCR. Our designation of
the enryme mixture
as Klentaq-LA (KlenTaq Long and Accurate) reflects this high fidelity
performance.
,\ \..~ , ~J
WO 94/26766 P~'fI~JS9,4(01867
33 2 ~~5 6 1
Table 2. Non-silent mutations introduced into the lacZ gene by 16 cycles of
PCR (10).
LacZ+ LacZ- % Effective Errors Fold Improve-
Enryme Blue Light Blue mutant cycle no. per 105 meat over
or White (c) by (b) full-length Taq
KTLA-64 571 34 5.6 12 1.05 12.7
Pfu 528 37 6.5 8 1.9 6.9
Klentaq5' 442 85 16.1 8 5.1 2.6
Klentaql3225 985 26.4 8 9.0 1.5
Amplitaq 525 301 36.4 8 13.4 1.0
(a) Klentaq' is the N-terminal deletion of Taq DNA polymerase described
in ref. 10. (b) Equation 1 of reference 10 was rearranged to be as
follows to solve for errors per bp: X = - (ln(2F~'~~'~ - 1 ) ) /1000,
where X is the errors per by incorporated, 1000 is the effective
target size in the lacZ gene (10), F is the fraction of blue colonies,
and m is the effective cycle number. (c) As in ref. 10, the effective
cycle number was estimated at less than the machine cycles to reflect
the actual efficiency of the reaction, yet higher than the minimum
calculated from the fold-amplification. Strand loss due to incomplete
synthesis of product strands is a probable cause of lower than ideal
amplification efficiency. Therefore successful (not lost) product
molecules are judged to have undergone more than the calculated
minimum number of replications. KThA-64 (Klentaq-278:Pfu::64:1 by
volume) was assigned a higher effective cycle number since its
reactions started with 10 times less DNA (1.5 ng vs. 15 ng plasmid
pWB305) to result in comparable levels of product.
DISCUSSION
The previous length limitation for PCR amplification is
postulated to have been caused by low efficiency of extension at the
sites of incorporation of mismatched base pairs. Although it would
have seemed that the cure for these mismatches would be to employ
enzymes With 3'-(editing)-exonucleases, I believe that when Pfu and
Vent DNA polymerase are used to catalyze our amplifications on their
own, their failure is due to degradation of the PCR primers by their
3'-exonucleases, especially during the required long synthesis times
and at optimally high DNA polymerase levels. Evidently, low levels of
3'-exonuclease are sufficient and optimal for removal of the mismatches
to allow the Klentaq-278 and amplification to proceed. It has been
demonstrated that the optimally low level of 3'-exonuclease can be set
"3
WO 94126766 ,::
r;, ~~ ~; PCTIC1S94101867
. ~ . ~' 34
effectively, conveniently, and flexibly by mixing and dilution. ,
Preferably the ratio of exo-/exo+ enzyme is high. If'equal
levels of the two types of enzymes are used (or where the E2 component
is in excess), or if the ratio of exo-/exo+ is 4 or less, the
effectiveness of the long PCR, even under optimal cycling conditions
discussed below, is non-existent or much reduced.
It is preferred, and for certain applications, important
that the length and temperature of the heat denaturation step of the
PCR be kept to a minimum. Further, the improvement obtained by
increasing the pH slightly may correspond to a decrease in template
depurination. If so, further improvements may result if depurination
can be reduced, or if a majority DNA polymerase component can be found
which is able to bypass depurination sites.
The short denaturation time found to be optimal, preferably
less than 20 sec., and most preferably, 5 sec. or less in the reaction
itself at 95°, is surprisingly effective for the amplification of 35
kb, whereas it might have been expected that longer PCR targets would
need longer denaturation time to become completely denatured. If
complete denaturation is required for PCR, and if longer DNA requires
more time to unwind at 95°, the required unwinding time may eventually
become significantly more than 5 seconds. This could limit the size of
amplifiable product because of the increased depurination caused by
longer denaturation times.
These amplifications were successful with several different
target sequences, with several primer combinations, and with product
sizes up to nearly twice the maximum size of inserts cloned into
Whole viruses and plasmids up to 35 kb in length should now be
amplifiable with this system. Should this method prove applicable to
DNA of higher complexity than 1, it could prove a boon to genomic
mapping and sequencing applications, since in vitro amplification is
convenient and avoids the DNA rearrangement and gene toxicity pitfalls
of in vivo cloning.
In view of the above, it will be seen that the several
objects of the invention are achieved and other advantageous results
attained.
As various changes could be made in the above methods and
products Without departing from the scope of the invention, it is
intended that all matter contained in the above description shall be
interpreted as illustrative and not in a limiting sense.
WO 94126766 PCTIUS94101867
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References
1. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R.,
Horn, G.T., Mullis, K.B., and Erlich, H.A. (1988) Science 239, 487-491.
2. Lundberg, K.S., Shoemaker, D.D., Adams, M.W.W., Short, J.M. Sorge,
5 J.A., and Mathur, E.J. (1991) Gene 108, 1-6.
3. Lawyer, F.C., Stoffel, S., Saiki, R.K., Chang, S-Y., Landre, P.A.,
Abramson, R.D., and Gelfand, D.H. (1993) PCR Methods and ADnlications
2, 275-287.
4. Jeffreys, A.J., Wilson, V., Neumann, R., and Keyte, J. (1988)
10 Nucleic Acids Res. 16. 10953-10971.
5. Krishnan, B.R., Kersulyte, D., Brikun, I., Berg, C.M. & Berg, D.E.
(1991) Nucleic Acids Res. 19. 6177-6182.
6. Maga, E.A., & Richardson, T. (1991) BioTechnicrues 11: 185-186.
7. Ohler, L.D. & Rose, E. A. (1992) PCR Methods and A~~lications 2, 51-
15 59 .
8. Rychlik, W., Spencer, W.J., and Rhoads, R.E. (1990) Nucleic Acids
Res. 18: 6409.
9. Kainz, P. Schmiedlechner, A., & Strack, H.B. (1992) Analytical
Biochem. 202, 46-49.
20 10. Barnes, W.M. (1992) Gene 112, 29-35.
11. Chu, G.D., Vollrath, D. and Davis, R.W. (1986) fence 234, 1582-.
12. Ippen, K., Shapiro, J.A., and Beckwith, J.R. (1971) J.Bact. 108, 5-
9.
13. Kohara, Y. (1990) pp 29-42 in The Bacterial Chromosome, eds Drlica,
25 K. & Riley, M., ASM Washington D.C.
14. Tailor, R., Tippett, J., Gibb, G., Pells, S., Pike, D., Jordan, L.,
Ely, S. (1992) Mol. Microbiol. 6, 1211-1217.
15. Dubois, N.R. Reardon, R.C., Kolodny-Hirsh, D.M. J.Econ.Entomol.
81, 1672 (1988)],18.
30 16. Clark, J.M. (1988) Nucleic Acids Res. 16, 9677-.
17. Brewer, A.C., Marsh, P.J and Patient, R.K. (1990) Nucleic Acids
Res. 18~ 5574.
18. Lindahl, T. (1993) Nature 362: 709-715.
19. Lindahl, T. & Nyberg, B. (1972) Biochemistnr 11: 3611-3618.
35 20. Sigma Chemical Co. Technical Bulletin No. 106B.
21. Shpakovski, G.V., Akhrem, A.A., and Berlin, Y.A. (1988) Nucleic
Acids Res 16, 10199 (1988).
WO 94/26766 ~ , PCT/US94101867
21 ~ ~ ~:,,
36
Table of Contents of Sequence Listing
SEQ IDNO:1: PCR primer KT1.
SEQ IDN0:2: N-terminus
of
Klentaq-278.
SEQ IDN0:3: PCR primer Klentaq32.
SEQ IDN0:4: C-terminus
of
Klentaq-278
and
Taq
DNA
polymerase.
SEQ IDN0:5: pWB254b
plasmid
expression
vector.
SEQ IDN0:6: Amino
acid
sequence
of
Klentaq-278,
complete.
SEQ IDN0:7: PCR primer I~L on left side of lambda E1~L4
inserts.
SEQ IDN0:8: PCR primer NIBR on right side of lambda ENIBL4
inserts.
SEQ IDN0:9: PCR primer I48L-1.7, 1729 by to left of 1~L.
SEQ IDNO:10:PCR primer NlSAl9, at C-terminus of lacZ.
SEQ IDN0:11:PCR primer Lc5
SEQ IDN0:12:PCR primer Lc3
SEQ IDN0:13:PCR primer KT2
SEQ IDN0:14:Taq DNA polymerase gene, KT1 end
SEQ IDN0:15:Taq DNA polymerase gene, 3' end
SEQ IDN0:16:Tfl DNA polymerase gene, KT1 end
SEQ IDN0:17:Tfl DNA polymerase gene, 3' end
SEQ IDN0:18:PCR primer BtV3
SEQ IDN0:19:PCR primer BtVS
SEQ IDN0:20:PCR primer ~.L36
SEQ IDN0:21:PCR primer lacZ'S33
SEQ IDN0:22:PCR primer lacZ333
SEQ IDN0:23:PCR primer lacZ536
SEQ IDN0:24:PCR primer I~L002
SEQ IDN0:25:PCR primer I~L101
SEQ IDN0:26:PCR primer I~R001
SEQ IDN0:27:PCR primer I~R202
SEQ IDN0:28:PCR primer MSA1933
SEQ IDN0:29:PCR primer ~1R36
SEQ IDN0:30:Phi 29 fragment 1, Region III
SEQ IDN0:31:Pfu fragment 1, Region III
SEQ IDN0:32:Phi 29 fragment 2, Region I
SEQ IDN0:33:Pfu fragement 2, Region I
WO 94126766 ~ '~ PCTlUS94l01867
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37
SEQBSNCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Barnes Ph.D., Wayne M
(ii) TITLE OF INVENTION: DNA polymerases with
enhanced thermostability and enhanced length and
efficiency of primer extension
'~.ii) NUMBER OF SEQUENCES: 33
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Senniger, Powers, Leavitt & Roedel
(B) STREET: One Metropolitan Square
(C) CITY: St. Louis
(D) STATE: Missouri
(E) COUNTRY: USA
(F) ZIP: 63102
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: 22-FEB-1994
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Blosser, G.Harley
(B) REGISTRATION NUMBER: 33,650
(C) REFERENCE/DOCKET NUMBER: WN84903
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (314) 231-5400
(B) TELEFAX: (314) 231-4342
(C) TELEX: 6502697583 MCI
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Thermus aquaticus
(B) STRAIN: YT1
(vii) IMMEDIATE SOURCE:
(A) LIBRARY: synthetic
(B) CLONE: KT1
WO 94/26766 ~ ~ ~ PCTIUS94101867
A t. ~
v
38
( ix) FEATURE : ' i'
(A) NAME/KEY: CDS
(B) LOCATION: 6..35
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
GAGCC ATG GGC CTC CTC CAC GAG TTC GGC CTT CTG G 36
Met Gly Leu Leu His Glu Phe Gly Leu Leu
1 5 10
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Gly Leu Leu His Glu Phe Gly Leu Leu
1 5 10
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(v) FRAGMENT TYPE: C-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Thermus aquaticus
(B) STRAIN: YT1
(vii) IMMEDIATE SOURCE:
(A) LIBRARY: synthetic
(B) CLONE: Klentaq32
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: complement (8..34)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
GCGAAGCTTA CTACTCCTTG GCGGAGAGCC AGTCC 35
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
WO 94!26766 , ~ ; PCT/US94I01867
r
:161?fi
2
39
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Asp Trp Leu Ser Ala Lys Glu
1 5
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6714 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Expression vector
(vii) IMN~DIATE SOURCE:
(B) CLONE: pWB254b
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1665
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
ATGGGC CTCCTCCAC GAGTTC GGCCTTCTG GAAAGCCCC AAGGCCCTG 48
MetGly LeuLeuHis GluPhe GlyLeuLeu GluSerPro LysAlaLeu
1 5 10 15
GAGGAG GCCCCCTGG CCCCCG CCGGAAGGG GCCTTCGTG GGCTTTGTG 96
GluGlu AlaProTzp ProPro ProGluGly AlaPheVal GlyPheVal
20 25 30
CTTTCC CGCAAGGAG CCCATG TGGGCCGAT CTTCTGGCC CTGGCCGCC 144
LeuSer ArgLysGlu ProMet TrpAlaAsp LeuLeuAla LeuAlaAla
35 40 45
GCCAGG GGGGGCCGG GTCCAC CGGGCCCCC GAGCCTTAT AAAGCCCTC 192
AlaArg GlyGlyArg ValHis ArgAlaPro GluProTyr LysAlaLeu
50 55 60
AGGGAC CTGAAGGAG GCGCGG GGGCTTCTC GCCAAAGAC CTGAGCGTT 240
ArgAsp LeuLysGlu AlaArg GlyLeuLeu AlaLysAsp LeuSerVal
65 70 75 80
CTGGCC CTGAGGGAA GGCCTT GGCCTCCCG CCCGGCGAC GACCCCATG 288
LeuAla LeuArgGlu GlyLeu GlyLeuPro ProGlyAsp AspProMet
85 90 95
CTCCTC GCCTACCTC CTGGAC CCTTCCAAC ACCACCCCC GAGGGGGTG 336
LeuLeu AlaTyrLeu LeuAsp ProSerAsn ThrThrPro GluGlyVal
100 105 110
GCCCGG CGCTACGGC GGGGAG TGGACGGAG GAGGCGGGG GAGCGGGCC 384
AlaArg ArgTyrGly GlyGlu TrpThrGlu GluAlaGly GluArgAla
115 120 125
CA 02156176 2000-O1-25
4Q
GCC CTT TCC GAG AGG CTC TTC GCC AAC CTG TGG GGG AGG CTT GAG GGG 432
Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu Trp Gly Arg Leu Glu Gly
130 135 140
GAG GAG AGG CTC CTT TGG CTT TAC CGG GAG GTG GAG AGG CCC CTT TCC 480
Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu Val Glu Arg Pro Leu Ser
145 150 155 160
GCT GTC CTG GCC CAC ATG GAG GCC ACG GGG GTG CGC CTG GAC GTG GCC 528
Ala Val Leu Ala His Met Glu Ala Thr Gly Val Arg Leu Asp Val Ala
165 170 175
TAT CTC AGG GCC TTG TCC CTG GAG GTG GCC GAG GAG ATC GCC CGC CTC 576
Tyr Leu Arg Ala Leu Ser Leu Glu Val Ala Glu Glu Ile Ala Arg Leu
180 185 190
GAG GCC GAG GTC TTC CGC CTG GCC GGC CAC CCC TTC AAC CTC AAC TCC 624
Glu Ala Glu Val Phe Arg Leu Ala Gly His Pro Phe Asn Leu Asn Ser
195 200 205
CGG GAC CAG CTG GAA AGG GTC CTC TTT GAC GAG CTA GGG CTT CCC GCC 672
Arg Asp Gln Leu Glu Arg Val Leu Phe Asp Glu Leu Gly Leu Pro Ala
210 215 220
ATC GGC AAG ACG GAG AAG ACC GGC AAG CGC TCC ACC AGC GCC GCC GTC 720
Ile Gly Lys Thr Glu Lys Thr Gly Lys Arg Ser Thr Ser Ala Ala Val
225 230 235 240
CTG GAG GCC CTC CGC GAG GCC CAC CCC ATC GTG GAG AAG ATC CTG CAG 768
Leu Glu Ala Leu Arg Glu Ala His Pro Ile Val Glu Lys Ile Leu Gln
245 250 255
TAC CGG GAG CTC ACC AAG CTG AAG AGC ACC TAC ATT GAC CCC TTG CCG 816
Tyr Arg Glu Leu Thr Lys Leu Lys Ser Thr Tyr Ile Asp Pro Leu Pro
260 265 270
GAC CTC ATC CAC CCC AGG ACG GGC CGC CTC CAC ACC CGC TTC AAC CAG 864
Asp Leu Ile His Pro Arg Thr Gly Arg Leu His Thr Arg Phe Asn Gln
275 280 285
ACG GCC ACG GCC ACG GGC AGG CTA AGT AGC TCC GAT CCC AAC CTC CAG 912
Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser Ser Asp Pro Asn Leu Gln
290 295 300
AAC ATC CCC GTC CGC ACC CCG CTT GGG CAG AGG ATC CGC CGG GCC TTC 960
Asn Ile Pro Val Arg Thr Pro Leu Gly Gln Arg Ile Arg Arg Ala Phe
305 310 315 320
ATC GCC GAG GAG GGG TGG CTA TTG GTG GCC CTG GAC TAT AGC CAG ATA 1008
Ile Ala Glu Glu Gly Trp Leu Leu Val Ala Leu Asp Tyr Ser Gln Ile
325 330 335
GAG CTC AGG GTG CTG GCC CAC CTC TCC GGC GAC GAG AAC CTG ATC CGG 1056
Glu Leu Arg Val Leu Ala His Leu Ser Gly Asp Glu Asn Leu Ile Arg
340 345 350
i
CA 02156176 2000-O1-25
' 4Qa
GTC TTC CAG GAG GGG CGG GAC ATC CAC ACG GAG ACC GCC AGC TGG ATG 1104
Val Phe Gln Glu Gly Arg Asp Ile His Thr Glu Thr Ala Ser Trp Met
355 360 365
TTC GGC GTC CCC CGG GAG GCC GTG GAC CCC CTG ATG CGC CGG GCG GCC 1152
Phe Gly Val Pro Arg Glu Ala Val Asp Pro Leu Met Arg Arg Ala Ala
370 375 380
AAG ACC ATC AAC TTC GGG GTC CTC TAC GGC ATG TCG GCC CAC CGC CTC 1200
Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly Met Ser Ala His Arg Leu
285 390 395 400
WO 94126766 2 1 ~ t~ ~1 ~ ~ ~~ , ~ ~ PCTlUS94101867
41
TCC CAG CTA GCC ATC CCT TAC GAG CAG GCC ATT GAG 1248
GAG GAG GCC TTC
Ser Gln Leu Ala Ile Pro Tyr Glu Gln Ala Ile Glu
Glu Glu Ala Phe
405 410 415
CGC TAC CAG AGC TTC CCC AAG GTG TGG ATT AAG ACC 1296
TTT CGG GCC GAG
Arg Tyr Gln Ser Phe Pro Lys Val Trp Ile Lys Thr
Phe Arg Ala Glu
420 425 430
CTG GAG GGC AGG AGG CGG GGG TAC ACC CTC GGC CGC 1344
GAG GTG GAG TTC
Leu Glu Gly Arg Arg Arg Gly Tyr Thr Leu Gly Arg
Glu Val Glu Phe
435 440 445
CGC CGC GTG CCA GAC CTA GAG GCC AAG AGC CGG GAG 1392
TAC CGG GTG GTG
Arg Arg Val Pro Asp Leu Glu Ala Lys Ser Arg Glu
Tyr Arg Val Val
450 455 460
GCG GCC CGC ATG GCC TTC AAC ATG CAG GGC GCC GCC 1440
GAG CCC GTC ACC
Ala Ala Arg Met Ala Phe Asn Met Gln Gly Ala Ala
Glu Pro Val Thr
465 470 475 480
GAC CTC AAG CTG GCT ATG GTG AAG CCC AGG GAG GAA 1488
ATG CTC TTC CTG
Asp Leu Lys Leu Ala Met Val Lys Pro Arg Glu Glu
Met Leu Phe Leu
485 490 495
ATG GGG AGG ATG CTC CTT CAG GTC GAG CTG CTC GAG 1536
GCC CAC GAC GTC
Met Gly Arg Met Leu Leu Gln Val Glu Leu Leu Glu
Ala His Asp Val
500 505 510
GCC CCA GAG AGG GCG GAG GCC GTG CTG GCC GAG GTC 1584
AAA GCC CGG AAG
Ala Pro Glu Arg Ala Glu Ala Val Leu Ala Glu Val
Lys Ala Arg Lys
515 520 525
ATG GAG GTG TAT CCC CTG GCC GTG GAG GTG GTG GGG 1632
GGG CCC CTG GAG
Met Glu Val Tyr Pro Leu Ala Val Glu Val Val Gly
Gly Pro Leu Glu
530 535 540
ATA GGG GAC TGG CTC TCC GCC AAG 1682
GAG GAG TAGTAAGCTT ATCGATGATA
Ile Gly Asp Trp Leu Ser Ala Lys
Glu Glu'
545 550 555
AGCTGTCAAACATGAGAATT AGCCCGCCTA ATGAGCGGGCTTTTTTrTAATTCTTGAAGA1742
CGAAAGGGCCTCGTGATACG CCTATTTTTA TAGGTTAATGTCATGATAATAATGGTITCT1802
TAGCGTCAAAGCAACCATAG TACGCGCCCT GTAGCGGCGCATTAAGCGCGCCGGGTGTGG1862
TGGTTACGCGCAGCGTGACC GCTACACTTG CCAGCGCCCTAGCGCCCGCTCCTITCGCTT1922
TCTTCCCTTCCTTTCTCGCC ACGTTCGCCG GCTITCCCCGTCAAGCTCTAAATCGGGGGC1982
TCCCTITAGGGTTCCGATTT AGTGC'ITTAC GGCACCTCGACCCCAAAAAACTTGATTTGG2042
GTGATGGTTCACGTAGTGGG CCATCGCCCT GATAGACGGTTTTTCGCCCTTTGACGTTGG2102
AGTCCACGTTCTTTAATAGT GGACTCTTGT TCCAAACTTGRACAACACTCAACCCTATCT2162
CGGGCTATTCTTTTGATITA TAAGGGATTT TGCCGATTTCGGCCTATTGGTTAAAAAATG2222
AGCTGATITAACAAAAATTT AACGCGAATT TTAACAAAATATTAACGTTTACAATTTCAG2
2
8
2
GTGGCACTTTTCGGGGAAAT GTGCGCGGAA CCCCTATTTGTTTATTITTCTAAATACATT2342
CAAATATGTATCCGCTCATG AGACAATAAC CCTGATAAATGCTTCAATAATATTGAAAAA2402
GGAAGAGTATGAGTATTCAA CATITCCGTG TCGCCCTTATTCCCTTTTTTGCGGCATTTT2462
GCCTTCCTGT TTTTGCTCAC CCAGAAACGC TGGTGAAAGT AAAAGATGCT GAAGATCAGT 2522
r, h
WO 94126766 , '~ 5 ~ '~ ~ ~ "~ ' ~; PCTIUS94101867
42
TGGGTGCACG AGTGGGTTACATCGAACTGG ATCTCAACAG CGGTAAGATC CTTGAGAGTT2582
TTCGCCCCGA AGAACGTTTTCCAATGATGA GCACTTTTAA AGTTCTGCTA TGTGGCGCGG2642
TATTATCCCG TGTTGACGCCGGGCAAGAGC AACTCGGTCG CCGCATACAC TATTCTCAGA2702
ATGACTTGGT TGAGTACTCACCAGTCACAG AAAAGCATCT TACGGATGGC ATGACAGTAA2762
GAGAATTATG CAGTGCTGCCATAACCATGA GTGATAACAC TGCGGCCAAC TTACTTCTGA2822
CAACGATCGG AGGACCGAAGGAGCTAACCG CTTTTTTGCA CAACATGGGG GATCATGTAA2882
CTCG~CTTGA TCGTTGGGAACCGGAGCTGA ATGAAGCCAT ACCAAACGAC GAGCGTGACA2942
CCACGATGCC TGCAGCAATGGCAACAACGT TGCGCAAACT ATTAACTGGC GAACTACTTA3002
CTCTAGCTTC CCGGCAACAATTAATAGACT GGATGGAGGC GGATAAAGTT GCAGGACCAC3062
TTCTGCGCTC GGCCCTTCCGGCTGGCTGGT TTATTGCTGA TAAATCTGGA GCCGGTGAGC3122
GTGGGTCTCG CGGTATCATTGCAGCACTGG GGCCAGATGG TAAGCCCTCC CGTATCGTAG3182
TTATCTACAC GACGGGGAGTCAGGCAACTA TGGATGAACG AAATAGACAG ATCGCTGAGA3242
TAGGTGCCTC ACTGATTAAGCATTGGTAAC TGTCAGACCA AGTTTACTCA TATATACTTT3302
AGATTGATTT AAAACTTCATTTTTAATTTA AAAGGATCTA GGTGAAGATC CTTTTTGATA3 3
6
2
ATCTCATGAC CAAAATCCCTTAACGTGAGT TTTCGTTCCA CTGAGCGTCA GACCCCGTAG3422
AAAAGATCAA AGGATCTTCTTGAGATCCTT TTTTTCTGCG CGTAATCTGC TGCTTGCAAA3482
CAAAAAAACC ACCGCTACCAGCGGTGGTTT GTTTGCCGGA TCAAGAGCTA CCAACTCTTT3542
TTCCGAAGGT AACTGGCTTCAGCAGAGCGC AGATACCAAA TACTGTCCTT CTAGTGTAGC3602
CGTAGTTAGG CCACCACTTCAAGAACTCTG TAGCACCGCC TACATACCTC GCTCTGCTAA3662
TCCTGTTACC AGTGGCTGCTGCCAGTGGCG ATAAGTCGTG TCTTACCGGG TTGGACTCAA3722
GACGATAGTT ACCGGATAAGGCGCAGCGGT CGGGCTGAAC GGGGGGTTCG TGCACACAGC3782
CCAGCTTGGA GCGAACGACCTACACCGAAC TGAGATACCT ACAGCGTGAG CTATGAGAAA3842
GCGCCACGCT TCCCGAAGGGAGAAAGGCGG ACAGGTATCC GGTAAGCGGC AGGGTCGGAA3902
CAGGAGAGCG CACGAGGGAGCTTCCAGGGG GAAACGCCTG GTATCTTTAT AGTCCTGTCG3962
GGTTTCGCCA CCTCTGACTTGAGCGTCGAT TTTTGTGATG CTCGTCAGGG GGGCGGAGCC4022
TATGGAAAAA CGCCAGCAACGCGGCCTTTT TACGGTTCCT GGCCTTTTGC TGGCCTTTTG4082
CTCACATGTT CTTTCCTGCGTTATCCCCTG ATTCTGTGGA TAACCGTATT ACCGCCTTTG4142
AGTGAGCTGA TACCGCTCGCCGCAGCCGAA CGACCGAGCG CAGCGAGTCA GTGAGCGAGG4202
AAGCGGAAGA GCGCCTGATGCGGTATTTTC TCCTTACGCA TCTGTGCGGT ATTTCACACC4262
GCATATGGTG CACTCTCAGTACAATCTGCT CTGATGCCGC ATAGTTAAGC CAGTATACAC4322
TCCGCTATCG CTACGTGACTGGGTCATGGC TGCGCCCCGA CACCCGCCAA CACCCGCTGA4382
CGCGCCCTGA CGGGCTTGTCTGCTCCCGGC ATCCGCTTAC AGACAAGCTG TGACCGTCTC4442
CGGGAGCTGC ATGTGTCAGAGGTTTTCACC GTCATCACCG AAACGCGCGA GGCAGAACGC4502
CATCAAAAAT AATTCGCGTCTGGCCTTCCT GTAGCCAGCT TTCATCAACA TTAAATGTGA4562
WO 94126766 ' ~ 5 ~ ~ , ~ ~~ y - ~~ -~ - ~ PCTlUS94101867
,,.,
43
GCGAGTAACA ACCCGTCGGA GAACAAACGGCGGATTGACCGTAATGGGAT4622
TTCTCCGTGG
AGGTTACGTT GGTGTAGATGGGCGCATCGTAACCGTGCATCTGCCAGTTTGAGGGGACGA4682
CGACAGTATC GGCCTCAGGAAGATCGCACTCCAGCCAGCTTTCCGGCACCGCTTCTGGTG4742
CCGGAAACCA GGCAAAGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGAT4802
CGGTGCGGGC CTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGAT4862
TAAGTTGGGT AACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAAT4922
CCGTAATCAT GGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACAC4982
AACATACGAG CCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTC5042
ACATTAATTG CGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTG5102
CATTAATGAA TCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGT5162
TTTTCTTTTC ACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGA5222
GAGTTGCAGC AAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGT5282
GGTTGACGGC GGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGAT5342
ATCCGCACCA ACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTG5402
ATCGTTGGCA ACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTG5462
TTGAAAACCG GACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATT5522
GCGAGTGAGA TATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGG5582
GCCCGCTAAC AGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCG5642
CGTACCGTCT TCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAG5702
AAATAACGCC GGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAG5762
CGGATAGTTA ATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTT5822
ACAGGCTTCG ACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATC5882
GGCGCGAGAT TTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGT5942
GGCAACGCCA ATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAAT6002
GTAATTCAGC TCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCT6062
GGCCTGGTTC ACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATC6122
GTATAACGTT ACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCA6182
TGCCATACCG CGAAAGGTTTTGCGCCATTCGATGGTGTCCCAGTGAATCCGTAATCATGG6242
TCATAGCTGT TTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCC6302
GGAAGCATAA AGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCG6362
TTGCGCTCAC TGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATC6422
GGAGCTTACT CCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAAT6482
TGTGAGCGGA TAACAATTTCACACAGGAAACAGGATCGATCCAGCTTACTCCCCATCCCC6542
CTGTTGACAA TTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTC6602
WO 94/26766 ~ ~ 7 fi ~ ~ ~'i C~ ;' y PCTILTS94101867
t
44
ACACAGGAAA CAGGATCTGG GCCCTTCGAA ATTAATACGA CTCACTATAG GGAGACCACA 6662
ACGGTTTCCC TCTAGAAATA ATTTTGTTTA ACTTTAAGAA GGAGATATAT CC 6714
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 554 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Met Gly Leu Leu His Glu Phe Gly Leu Leu Glu Ser Pro Lys Ala Leu
1 5 10 15
Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly Ala Phe Val Gly Phe Val
20 25 30
Leu Ser Arg Lys Glu Pro Met Trp Ala Asp Leu Leu Ala Leu Ala Ala
35 40 45
Ala Arg Gly Gly Arg Val His Arg Ala Pro Glu Pro Tyr Lys Ala Leu
50 55 60
Arg Asp Leu Lys Glu Ala Arg Gly Leu Leu Ala Lys Asp Leu Ser Val
65 70 75 80
Leu Ala Leu Arg Glu Gly Leu Gly Leu Pro Pro Gly Asp Asp Pro Met
85 90 95
Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn Thr Thr Pro Glu Gly Val
100 105 110
Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu Glu Ala Gly Glu Arg Ala
115 120 125
Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu Trp Gly Arg Leu Glu Gly
130 135 140
Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu Val Glu Arg Pro Leu Ser
145 150 155 160
Ala Val Leu Ala His Met Glu Ala Thr Gly Val Arg Leu Asp Val Ala
165 170 175
Tyr Leu Arg Ala Leu Ser Leu Glu Val Ala Glu Glu Ile Ala Arg Leu
180 185 190
Glu Ala Glu Val Phe Arg Leu Ala Gly His Pro Phe Asn Leu Asn Ser
195 200 205
Arg Asp Gln Leu Glu Arg Val Leu Phe Asp Glu Leu Gly Leu Pro Ala
210 215 220
Ile Gly Lys Thr Glu Lys Thr Gly Lys Arg Ser Thr Ser Ala Ala Val
225 230 235 240
Leu Glu Ala Leu Arg Glu Ala His Pro Ile Val Glu Lys Ile Leu Gln
245 250 255
Tyr Arg Glu Leu Thr Lys Leu Lye Ser Thr Tyr Ile Asp Pro Leu Pro
260 265 270
WO 94!26766 ~ 7 fi PCTIL1S94101867
.,.~.~ 2 t~ ~ p ~ ..
Asp Leu Ile His Pro Arg Thr Gly Arg Leu His Thr Arg Phe Asn Gln
275 280 285
Thr Ala Thr Ala Thz Gly Arg Leu Ser Ser Ser Asp Pro Aen Leu Gln
290 295 300
Asn Ile Pro Val Arg Thr Pro Leu Gly Gln Arg Ile Arg Arg Ala Phe
305 310 315 320
Ile Ala Glu Glu Gly Trp Leu Leu Val Ala Leu Asp Tyr Ser Gln Ile
325 330 335
Glu Leu Arg Val Leu Ala His Leu Ser Gly Asp Glu Asn Leu Ile Arg
340 345 350
Val Phe Gln Glu Gly Arg Asp Ile His Thr Glu Thr Ala Ser Trp Met
355 360 365
Phe Gly Val Pro Arg Glu Ala Val Asp Pro Leu Met Arg Arg Ala Ala
370 375 380
Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly Met Ser Ala His Arg Leu
385 390 395 400
Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu Ala Gln Ala Phe Ile Glu
405 410 415
Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg Ala Trp Ile Glu Lys Thr
420 425 430
Leu Glu Glu Gly Arg Arg Arg Gly Tyr Val Glu Thr Leu Phe Gly Arg
435 440 445
Arg Arg Tyr val Pro Asp Leu Glu Ala Arg Val Lys Ser Val Arg Glu
450 455 460
Ala Ala Glu Arg Met Ala Phe Asn Met Pro Val Gln Gly Thr Ala Ala
465 470 475 480
Asp Leu Met Lys Leu Ala Met Val Lys Leu Phe Pro Arg Leu Glu Glu
485 490 495
Met Gly Ala Arg Met Leu Leu Gln Val His Asp Glu Leu Val Leu Glu
500 505 510
Ala Pro Lys Glu Arg Ala Glu Ala Val Ala Arg Leu Ala Lys Glu Val
515 520 525
Met Glu Gly Val Tyr Pro Leu Ala Val Pro Leu Glu Val Glu Val Gly
530 535 540
Ile Gly Glu Asp Txp Leu Ser Ala Lys Glu
545 550
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: bacteriophage lambda
WO 94126766 1 ~~ '~ ~ ~ ~ PCTILTS94101867
215
46
(B) STRAIN: Papa
(vii) IMMEDIATE SOURCE:
(B) CLONE: MBL
(viii) POSITION IN GENOME:
(B) MAP POSITION: 27940
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
GCTT~TCTGC TTCTCATAGA GTCTTGC 27
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: bacteriphage lambda
(B) STRAIN: Papa
(vii) IMMEDIATE SOURCE:
(B) CLONE: MBR
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
ATAACGATCA TATACATGGT TCTCTCC 27
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: bacteriophage lambda
( vi i ) IMMEDIATE SOURCE
(B) CLONE: MBL-1.7
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
TTTTGCTGGG TCAGGTTGTT CTTTAGG 27
(2) INFORMATION FOR SEQ ID N0:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(v) FRAGMENT TYPE: C-terminal
WO 94!26766 . , PCTlUS94l01867
~r~
215616
(vi) ORIGINAL SOURCE:
(A) ORGANISM: E.coli
(B) STRAIN: K12
(vii) IMMEDIATE SOURCE:
(B) CLONE: MSA19
(viii) POSITION IN GENOME:
(B) MAP POSITION: lacZ
~xi) SEQUENCE DESCRIPTION: SEQ ID N0:10:
GGAAGCTTAT TTTTGACACC AGACCAAC 28
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Zea maize
(vii) IMMEDIATE SOURCE:
(B) CLONE: Lc5
(viii) POSITION IN GENOME:
(B) MAP POSITION: 5' end of color control gene Lc
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
GTGATGGATC CTTCAGCTTC CCGAGTTCAG CAGGCGG 3 7
(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iv) ANTI-SENSE: YES
(v) FRAGMENT TYPE: C-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Zea maize
(vi i ) IMMEDIATE SOURCE
(B) CLONE: Lc3
(viii) POSITION IN GENOME:
(B) MAP POSITION: 3' end of color control gene Lc
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
WO 94/~~~'
6 ~ ~ 6 PCTIUS94/01867
8~ ~ ~_s 48
GGTCTCGAGC GAAGCTTCCC TATAGCTTTG CGAAGAG 37
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(v) FRAGMENT TYPE : internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Thertnus aquaticus
(B) STRAIN: YTl
(vii) IMMEDIATE SOURCE:
(B) CLONE: KT2
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
GAGCCATGGC CAACCTGTGG GGGAGGCTTG AGGGGGA 37
(2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Thertnus aquaticus
(B) STRAIN: YTl
(vii) IMMEDIATE SOURCE:
(B) CLONE: Genbank Accession no. J04639
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT: DNA polymerase gene
(B) MAP POSITION: 950
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
AGTTTGGCAG CCTCCTCCAC GAGTTCGGCC TTCTGG 36
(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Thermus aquaticus
(B) STRAIN: YT1
(vii) IMMEDIATE SOURCE:
WO 94/26766 r. ., PCTIUS94/01867
49
(B) CLONE: Genbank Accession No. J04639
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT: DNA polymerase gene
(B) MAP POSITION: 2595
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
GGACTGGCTC TCCGCCAAGG AGTGATACCA CC 32
(2) TNFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Thermus flavis
(vii) IMMEDIATE SOURCE:
(B) CLONE: Genbank Accession No. X66105
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT: DNA polymerase
(B) MAP POSITION: 1378
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
AGTTTGGAAG CCTCCTCCAC GAGTTCGGCC TCCTGG 36
(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Thermus flavis
(vii) IMMEDIATE SOURCE:
(B) CLONE: Genbank Accession No. X66105
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT: DNA polymerase gene
(B) MAP POSITION: 3023
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
GGACTGGCTC TCCGCCAAGG AGTAGGGGGG TCCTG 35
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
PCTlUS94/01867
WO 94/267~~
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iv) ANTI-SENSE: YES
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bacillus thuringiensis
(B) STRAIN: CryV
(C) INDIVIDUAL ISOLATE: NRD12
(vii) IMMEDIATE SOURCE:
(B) CLONE: BtV3
(xi) SEQUENCE DESCRIPTION: SfiQ ID N0:18:
GCGAAGCTTC TCGAGTTACG CTCAATATGG AGTTGCTTC 39
(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bacillus thuringiensis
(B) STRAIN: NRD12
(vii) IMMEDIATE SOURCE:
(B) CLONE: BtVS
(xi) SfiQUENCE DESCRIPTION: SEQ ID N0:19:
CCGAGATCTC CATGGATCCA AAGAATCAAG ATAAGCATCA AAG 43
(2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCfi CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: bacteriophage lambda
(B) STRAIN: Papa
(vii) IMMEDIATE SOURCE:
(B) CLONE: L36
(viii) POSITION IN GENOME:
(B) MAP POSITION: left end
WO 94/26766 . 1 1 5 6 1 '~ f ~ ~ y PCTIUS94/01867
51
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
GGGCGGCGAC CTCGCGGGTT TTCGCTATTT ATGAAA 36
(2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iv) ANTI-SENSE: YES
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: E.coli
(B) STRAIN: K12
(vii) IIrdHEDIATE SOURCE:
(B) CLONE: lacZ~533
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
CGACGGCCAG TGAATCCGTA ATCATGGTCA TAG 33
(2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(v) FRAGMENT TYPE: C-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: E.coli
(B) STRAIN: K12
( vi i ) IN~IEDIATE SOURCE
(B) CLONE: lacZ333
(viii) POSITION IN GENOME:
(B) MAP POSITION: lacZ
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
ACCAGCCATC GCCATCTGCT GCACGCGGAA GAA 33
(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
WO 94/26766 1 5 fi 1 '~. ~ . ~~~ k''!' ~ ~T~S94101567
2 -.,,
52
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: E.coli
(B) STRAIN: K12
(vii) IMMEDIATE SOURCE:
(B) CLONE: lacZ536
(v~ii) POSITION IN GENOME:
(B) MAP POSITION: lacZ
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
CTATGACCAT GATTACGGAT TCACTGGCCG TCGTTT 36
(2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: bacteriophage lambda
(B) STRAIN: Papa
(vii ) IMMEDIATE SOURCE
(B) CLONE: MBL002
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
GCAAGACTCT ATGAGAAGCA GATAAGCGAT AAG 33
(2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: bacteriophage lambda
(B) STRAIN: Papa
( vi i ) IMMEDIATE SOURCE
(B) CLONE: MBL101
(viii) POSITION IN GENOME:
(B) MAP POSITION: 27840
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
ATCATTATTT GATTTCAATT TTGTCCCACT CCC 33
(2) INFORMATION FOR SEQ ID N0:26:
WO 94!26766 ~ ~ PCTlUS94101867
:~2 ~1~5.6~1
...
53
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: bacteriophage lambda
(B) STRAIN: Papa
(vii) IMMEDIATE SOURCE:
(B) CLONE: MBR001
(viii) POSITION IN GENOME:
(B) MAP POSITION: 34576
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:
GGAGAGAACC ATGTATATGA TCGTTATCTG GGT 33
(2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: bacteriophage lambda
(B) STRAIN: Papa
(vii) IMMEDIATE SOURCE:
(B) CLONE: MBR202
(viii) POSITION IN GENOME:
(B) MAP POSITION: 34793
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
GCGCACAAAA CCATAGATTG CTCTTCTGTA AGG 33
(2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(v) FRAGMENT TYPE: C-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: E.coli
(B) STRAIN: K12
( vi i ) IMMEDIATE SOURCE
(B) CLONE: MSA1933
WO 94/26766 ~ ~ ~ ,~ ~~~''~ ~~ 4~ PCTlUS94101867
256
54
(viii) POSITION IN GENOME:
(B) MAP POSITION: lacZ
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
CCCGGTTATT ATTATTTTTG ACACCAGACC AAC 33
(2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: bacteriophage lambda
(B) STRAIN: Papa
(vii) IMMEDIATE SOURCE:
(B) CLONE: R36
(viii) POSITION IN GENOME:
(B) MAP POSITION: right end
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
AGGTCGCCGC CCCGTAACCT GTCGGATCAC CGGAAA 36
(2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 53 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE : internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: phi 29
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT: DNA polymerase
(B) MAP POSITION: ending at 435
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
Lys Leu Met Leu Asn Ser Leu Tyr Gly Lys Phe Ala Ser Asn Pro Asp
1 5 10 15
Val Thr Gly Lys Val Pro Tyr Leu Lys Glu Asn Gly Ala Leu Gly Phe
20 25 30
Arg Leu Gly Glu Glu Glu Thr Lys Asp Pro Val Tyr Thr Pro Met Gly
35 40 45
Val Phe Ile Thr Ala
(2) INFORMATION FOR SEQ ID N0:31:
WO 94126766 ~ ~ ~ ~ 7~ ~ PCT/L1S94/01867
,"",
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE : internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Pyrococcus furiosus
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT: DNA polymerase
(B) MAP POSITION: ending at 516
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:
Asp Tyr Arg Gln Lys Ala Ile Lys Leu Leu Ala Asn Ser Phe Tyr Gly
1 5 10 15
Tyr Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu
20 25 30
Ser Val Thr Ala
(2) INFORMATION FOR SEQ ID N0:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE : internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: phi 29
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT: DNA polymerase
(B) MAP POSITION: ending at 485
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:
Txp Ala Arg Tyr Thr Thr Ile Thr Ala Ala Gln Ala Cys Tyr Asp Arg
1 5 10 15
Ile Ile Tyr Cys Asp Thr Asp Ser Ile His Leu Thr Gly Thr Glu Ile
20 25 30
Pro Asp Val Ile Lys Asp Ile Val Asp Pro Lys Lys Leu Gly Tyr Trp
35 40 45
Ala His
(2) INFORMATION FOR SEQ ID N0:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
WO 94126766 PCTlUS94101867
~~58~7s
56
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE : internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Pyrococcus furiosus
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT: DNA polymerase
(B) MAP POSITION: ending at 576
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:33:
Trp Gly Arg Lys Tyr Ile Glu Leu Val Trp Lys Glu Leu Glu Glu Lys
1 5 10 15
Phe Gly Phe Lys Val Leu Tyr Ile Asp Thr Asp Gly Leu Tyr Ala Thr
20 25 30
Ile Pro Gly Gly Glu Ser Glu Glu Ile Lys Lys Lys Ala Leu Glu Phe
35 40 45
Val Lys Tyr Ile Asn Ser Lys Leu Pro Gly Leu Leu
50 55 60
WO 94/26766 ~ s : $T "~ ";~ ~ '~ PCT/L1S94101867
21561
s~
TAQuence~
Version 2.0
TaCr DNA Polymerase Sequencing System
Step-By-Step Protocols
For DNA Sequencing With
TAQuence~ Version 2.0
t st Edition
USB~
APPENDIX 1
Revision: 314191
PCTlLTS94101867
WO 94!26766
H
~Taq.,
Cycle-Sequencing
Kit
Cycle Sequencingt System
Fea~urin.g 0 Taq'~*
Version 2.0 DNA Polymerase
and the Cycled Labeling Step Protocol
Step-By-Step Protocols
For Cycle Sequencing
APPENDIX 2
United States Biochemical
