Note: Descriptions are shown in the official language in which they were submitted.
1 335962
1 607~4-2094E
This application is a divisional application of
application No. 556,390 filed on January 13th, 1988.
This invention relate~s to a method of inactivating
exonuclease activity in a DNA polymerase solution produced by
recombinant DNA techniques, comprising: incubating said solution
in a vessel containing oxygen, a reducing agent and a transition
metal.
The invention relates to DNA polymerases suitable for
DNA sequencing.
DNA sequencing involves the generation of four
populations of single stranded DNA fragments having one defined
terminus and one variable terminus. The variable terminus always
terminates at a specific given nucleotide base (either guanine
(G), adenine (A), thymine (T), or cytosine (C)). The four
different sets of fragments are each separated on the basis of
their length, on a high resolution polyacrylamide gel; each band
on the gel corresponds ~olinearly to a specific nucleotide in the
DNA sequence, thus identifying the positions in the sequence of
the given nucleotide base.
Generally there are two methods of DNA sequencing. One
method (Maxam and Gilbert sequencing) involves the chemical
degradation of isolated DNA fragments, each labeled with a single
radiolabel at its defined terminus, each reaction yielding a
limited cleavage specifically at one or more of the four bases (G,
A, T or C). The other method (dideoxy sequencing) involves the
enzymatic synthesis of a DNA strand. Four
1 335962
60724-209
-- 2
separate synthe~es are run, each reaction being caused
to terminate at a specific base (G, A, T or C) via
incorporation of the appropriate chain terminating
dideoxynucleotide. The latter method is prQferrQd since
the DNA fragment~ are uniformly labelled (instead of end
labelled) and thus the larger DNA fragments contain
incrQasinqly more radioactivity. Further,
35S-labelled nuclQotides can be used in place of
32P-labelled nuclQotides, resulting in sharper
definition; and tho reaction products are simple to
interpret since each lane corrasponds only to either G,
A, T or C. The enzyme used for most dideoxy sequencing
is the Escherichia coli DNA-polymerase I large fragment
~"Klenow"). Another ~olymerase used is AMV rever~Q
lS transcriptase.
Sum~ary of the Invention
The invention of the parent application features a method
for determining the nucleotide base sequence of a DNA
molecule, compri~ing annealing tha DNA molecule with a
primer molecule able to hybridize to the DNA molecule:
incubating separate portions of the annealed mixture in
at least four ve~els with four different
deoxynucl~osid~ triphosphates, a procQssive DNA
polymerase wherein the polymerase remains bound to a DNA
molocule for at l~aat ~00 bases before dissociating in
an environm-nt-~ condition normally us~d in the
extension r-action of a DNA seguencing reaction, the
polymera~- having less than S00 units of sYo~clease
acti~ty p~r mg of polymeras-, and on- of four DNA
synthesi~ tQrminating agent~ which terminate DNA
synthesis a~t a ~pecific nucleotide base. The agont
terminater at a different specific nucleotidQ basa in
each of the four vessels. The DNA products of the
incubating reaction are separated according to thoir
size so that at least a part of the nuclQotide base
sQguence of the DNA molecule can be determined.
~ 33~
-- 3 --
In preferred embodiments the polymerase remains
bound to the DNA molecule for at least.lOOo bases before
dissociating; the polymerase is substantially the same
as one in cQlls infected with a T7-type phage (i. Q.,
phage in which the DNA polymerase requires host
thioredoxin as a subunit; for example, the T7-type phage
is T7, T3, ~ II, H, W31, gh-l, Y, A1122, or SP6,
Studier, 9S Virology 70, 1979); thQ polymerase is
non-discriminating for dideoxy nucleotide analogs; the
lo polymerase i8 modified to have less than 50 units of
exonuclease activity per mg of polymerase, more
prefQrably less than 1 unit, even more prQfQrably 1QSS
than 0.1 unit, and most preferably has no detectable
~Yonl~clQase activity; the polymQrase i8 able to utilize
primers of a~ short as 10 bases or preferably as short
as 4 bases; the primer comprises four to forty
nucleotide basQs, and is single stranded DNA or RNA; the
annealing step comprises heating the DNA molecule and
the primer to above 6SC, preferably from 65C to 100C,
and allowing the heated mixture to cool to below 65C,
preferably to 0C to 30C; the incubating step comprises
a pulse and a chase step, wherein the pulse step
comprises mixing the annealed mixture with all four
d~fferent deox~,-ucleoside triphosphates and a processive
2s DNA polymerase, wherein at least one of the
deoxynucleoside t~iphosphates is labelled; most
preferably the pulse step performed under conditions in
which the polymerase does not exhibit its processivity
and is for 30 secon~ to 20 minutes at 0C to 20C or
where at least one of the nucleotide triphosphates is
limiting; and the chase step comprises adding one of the
chain terminating agents to four separate aliquots of
the mixture after the pulse step; preferably the chase
60724-2094E
1 3359b2
-- 4
step i~ for 1 to 60 minut~s at 30C to 50C; the
terminating agent is a dideoxynuclaotide, or a limiting
l~vel of ona deoxynucleoside triphosphate one of the
four deoxynuclQotide~ is dITP or deazaguanosine;
labelled primers are used so that no pulsQ step is
required, preferably the label i~ radioactiva or
fluorescent; and the polymerasQ is unable to exhibit its
processivity in a ~econd Qnvironmental condition
normally u~ed in the pulse reaction of a DNA sequencing
lo reaction
Inventions of divisional appl~cations feature a) a
method for producing blunt ended double-stranded DNA
molecule~ from a linear DNA molecule having no 3'
protruding termini, using a proce~ive DNA polymerase
free from e~o~clea~e activity; b) a method of
amplification of a ~NA ~equence compri~ing annealing a
irst and second primer to opposite ~trand~ of a double
stranded DNA seq~ence and incubating the annealed
mixtur~ with a processive DNA polymerase having le~s
than S00 units o eYon~clease activity per mg of
polymerasQ, pref~rably le~a than l unit, wherein the
first and second primers anneal to oppo~ite strands of
tho DNA sequence; in prefQrred embodiment~ the primers
have th-ir 3' enda dir-cted toward each other and th-
method further cempri~e-, after the incubation ~tep,
denaturing th- roaulting DNA, annealing the first and
~econd primer- to th~ re~ulting DNA and incubating the
anr~ d Q~xtur- with th- polymera~e preferably the
cycl- of ds~aturing, annealing and incubating i~
reFeat d from lO to ~0 time~ c) a method for in vitro
mutagene~ of cloned DNA fragment~, com~ri~ing
providing a clonQd fragm~nt and ~yntherizing a DNA
~trand w ing a proce~aive DNA polymera~- having le~
than l unit of eYen~clQa~- activity per mg of
polymera~e d) a method of producing active T7-type DNA
~olymera~e from cloned DNA fraqm~nt~ under the control
~ 1 3359~2
of non-leaky promoters (see below) in the same cell
comprising inducing expression of the genes only when
the cells are in logarithmic growth phase, or stationary
phase, and isolating the polymerase from the cell:
preferably the cloned fragments are under the control of
a promoter requiring T7 RNA polymerase for expressiOn;
e) a gene encoding a T7-type DNA polymera~e, the gene
being genetically modified to reducQ the activity of
naturally occurring exonuclease activity: most
preferably a histidine (His) residue i5 modified, even
more preferably His-123 of gene 5; f) the product of the
gene encoding genetically modified polymerase: g) a
method of purifying T7 DNA polymera~e from cells
comprising a vector from which the polymerase i8
expressed, compri~ing the steps of lysing the cells, and
passing the polymerase over an ion-exchange column, over
a DES2 DEAE column, a phosphocellulose column, and a
hydroxyapatite column; preferably prior to the passing
step the method comprises precipitatinq the polymerase
with ammonium sulfate; the method further comprises the
step of passing the polymerase over a Sephadex*DEAE A50
column; and the ion-exchange column is a DES2 DEAE
column; h) a method of inactivating exonuclease activity
in a DNA polymerase solution comprising incubating the
solution in a vessel containing oxygen, a reducing agent
and a transition metal: i) a kit for DNA sequencing,
comprising a proceusive DNA polymerase, defined as
above, having le~s than 500 unit~ of exonuclease
activity per mg of polymerase, wherein the polymerase is
able to exhibit it~ processivity in a first
environmental condition, and preferably unable to
exhibit its proces~ivity in a ~econd environmental
condition, and a reagent necQs~ary for the sequencing,
*Trademark
-
- 6 - 1 3 3 5 9 6 ~ 60724-209~
select~d from a chain terminating a~ent, and dITP; j) a
method for labelling the 3' end of a DNA fragment
comprising incubating the DNA fragment with a processive
DNA polymerase having less than SOO units of exonuclease
activity per mg of polymerase, and a labelled
deoxynucleotide; k) a method for in vitro mutagenesis of
a cloned DNA fragment comprising providing a primer and
a template, the primer and the template having a
specific mismatched base, and extending the primer with
lo a proce~ive DNA polymerase; and 1) a method for in
vitro mutagenesis of a cloned DNA fragment compri~ing
providing the cloned ragment and ~ynthesizing a DNA
~trand using a proces~ive DNA polymerase, having less
than SO unit~ of exonuclease activity, under conditions
lS which cause misincorporation of a nucleotide base.
The inventions provide a DNA polymerase which
i~ proces~ive, non-discriminating, and can utilize short
primer~. Further, the polymerase has no associated
Qxonuclease activity. The~e are ideal propertie~ for
the above dQscribed methods, and in particular for DNA
seguencing reaction~, ~ince the background level of
radioactivity in the polyacylamide gels i~ negligible,
there are few or no artifactual band~, and the bands are
sharp -- making th- DNA sequence ea~y to read. Further,
such a polymera~ allow~ novel mathod~ of sQquencing
long DNA fragm~ntr, a~ i~ d~scribQd in detail below.
Oth-r featurea and advantage- of the invention~
will b- apparent from the following de~cription of the
. preferr-d ~mbodi~-nt~ thereof and from th~ claim~.
1 3 3 5 9 ~ 2 60724-209~
Descriptlon of the Preferred Embodiments
The drawings will first briefly be described.
Drawinqs
Figs. 1-3 are d~agrammatic representations of
the vectors pTrx-2, mGPl-l, and pGPS-5 respectively;
Fig. ~ i8 a graphical represQntation of the
selective oxidation of T7 DNA polymerase;
Fig. S is a graphical representation of the
ability of modified T7 polymerase to synthesize DNA in
the presence of etheno-dATP: and
Fig. 6 i8 a diagrammatic representation of the
enzymatic amplification of genomic DNA using mcdified T7
DNA polymerase.
Fig. 7, 8 and 9 are the nucleotide sequences of
pTrx-2, a part of pGPS-5 and mGPl-2 resFectively.
lS Fig. 10 i~ a diagrammatic representation of
pGP5-6.
DNA PolYmerase
In general the DNA polymerase
is processi~Q, ha~ no associated Qxonucleaso activity,
does not discriminate against nucleotide analog
incorporation, and can utilize small oligonucleotides
(such as tetramers, heYamers _nd octamers) as s~ecific
primers. The~- ~ro~erties will now be discussQd in
detail.
2s Proce-aivity
By ~roc~-~ivity i~ meant that th~ DNA
polymora~ abl- to continuously incorporate many
nucleotid-~ u-in~ the ~ame primer-template without
dissociating fro~ th- template, under conditions
normally u&sd for DNA sequencing exten~ion reactions.
The degree of proce~ivity ~arie~ with different
~olymera~ some incorporata only a few bas~- before
dissociating (e.g. Rlenow (about 15 base-), T~ DNA
~ ~3~
-- 8
polymerase (abou~ 10 bases), T5 DNA polymerase (about
180 bases) and reVQrSQ transcriptasQ (about 200 bases)
(Das et al. J. Biol. Chem. 2S4:1227 1979; Bambara et
al., J. Biol. Chem 2S3:413, 1978) while others, such as
those of the present invention, will remain bound for at
least 500 base~ and preferably at least 1,000 bases
und~r suitable environmental eonditions. Sueh
environmental conditions inelude having adequatQ
supplie~ of all four deoxynueleoside triphosphate~ and
an ineubation temperature from lO~C-S0C. Proeessivity
is greatly enhaneed in the presenee of E. eoli single
stranded bi~ing (ssb), protein.
With processi~e enzymes termination of a
seguencing reaetion will oeeur only at those basQs which
have ineorporated a ehain terminating agent, sueh as a
dideoxynueleotid~. If the DNA polymerase is
non-proeessive, ~hen artifaetual bands will arise during
sequeneing reaetions, at positions corresponding to the
nucleotide where the polymerase dissoeiated. Frequent
dissoeiation ereates a baekground of bands at ineorreet
positions and ob w ures the true DNA sequence. This
problem i8 partially corrected by incubating the
reaction mixtura for a long time (30-60 min) with a high
coneentration of substratQs, whieh "chase" the
artifaetual band~ up to a high moleeular weight at the
top of the gel, away from the region where the DNA
seguenee is read. This i~ not an ideal solution sinc~e a
non-proee~sive D~A polymerase ha~ a high probability of
dissoeiating fro~ the t~mplate at regions of compact
secondary strueturQ, or hairpins. Reinitiation of
primer elongation at these site~ i8 inefficient and the
usual result i~ the formation of band~ at the same
position for all four nuclQotides, thus obseuring the
DNA sequQnee.
~ 33~9~2
9 60724-2094E
Analoq discrimination
The DNA polymerases do not discriminate significantly
between dideoxy-nucleotide analogs and normal nucleotides. That
is the chance of incorporation of an analog is approximately the
same as that of a normal nucleotide or at least incorporates the
analog with at least 1/10 the efficiency that of a normal analog.
The polymerases also do not discriminate significantly against
some other analogs. This is important since, in addition to the
four normal deoxynucleotide triphosphates (dGTP, dATP, dTTP and
dCTP), sequencing reactions require the incorporation of other
types of nucleotide derivatives such as: radioactively-or
fluorescently-labelled nucleotide triphosphates, usually for
labeling the synthesize~ strands with 35S, 32p, or other chemical
agents. When a DNA polymerase does not discriminate against
analogs the same probability will exist for the incorporation of
an analog as for a normal nucleotide. For labelled nucleotide
triphosphates this is important in order to efficiently label the
synthesized DNA strands using a minimum of radioactivity.
Further, lower levels of analogs are required with such enzymes,
making the sequencing reaction cheaper than with a discriminating
enzyme.
Discriminating polymerases show a different extent of
discrimination when they are polymerizing in a processive mode
versus when stalled, struggling to synthesize through a secondary
structure impediment. At such impediments there will be
variability in the intensity of different radioactive bands on the
gel, which may ob.scure t.he sequence.
9a l 3 3 5 9 6 2 60724-20g4E
Exonuclease Activity
The DNA polymerase has less than 50%, preferably less
than 1%, and most preferably less than 0.1%, of the normal or
naturally associated level of exonuclease activity (amount of
activity per polymerase
1 335962
-- 10 --
molecule). By normal or naturally associated level is
meant the eYo~uclease activity of unmodified T7-type
polymerase. Normally the associated activity is about
S,000 units of exonuclease activity per mg of polymerase,
measured as described below by a modification of the
procedure of Cha~e et al. (2~9 J. Biol. Chem. 4S4S,
197~). FYonueleases inerease the fidelity of DNA
synthesis by exelsing any newly synthQsized bases which
are ineorreetly basepaired to the template. Sueh
lo assoeiated sYonuclease activities are detrimental to the
quality of DNA sequencing reactions. They rais~ the
minimal required concentration of nueleotide preeursors
which must be added to the reaction sinee, when the
nueleotide concentration falls, the polymerase activity
slows to a rate comparable with the e~onuelease~activity,
resulting in no net DNA synthesis, or even degradation of
the synthesized DNA.
More importantly, associated eYo~uclease activity
will cause a DNA polymerase to idle at regions in the
template with secon~ary structure ~mps~ime~ts~ When a
polymerase approaches such a structure its rate of
synthQsis decreases as it struggle~ to pass. An
associated exonuclease will excise the newly synthesized
DNA when the polymerase stalls. As a consequenee numerous
cyeles of synthesi~ and exeision will occur. This may
result in ~he polymerase eventually synthesizing past the
- hairpin (with no detriment to the guality of~the
sequeneing reaetion); or the polymerase may dissoeiate
from the synthe~izQd strand (resulting in an artifaetual
band at the same po~ition in all four sequencing
reactions); or, a chain terminating agent may be
incorporated at a high freguency and produce a wide
variability in the intensity of different fragments in a
sequencing gel. This happens beeause the frequency of
11 60724-2094E
incorporation of a chain terminating agent at any given site
increases with the number of opportunities the polymerase has to
incorporate the chain terminating nucleotide, and so the DNA
polymerase will incorporate a chain-terminating agent at a much
higher frequency at sites of idling than at other sites.
An ideal sequencing reaction will produce bands of
uniform intensity throughout the gel. This is essential for
obtaining the optimal exposure of the X-ray film for every
radioactive fragment. If there is variable intensity of
radioactive bands, then fainter bands have a chance of going
undetected. To obtain uniform radioactive intensity of all
fragments, the DNA po~ymerase should spend the same interval of
time at each position on the DNA, showing no preference for either
the addition or removal of nucleotides at any given site. This
occurs if the DNA polymerase lacks any associated exonuclease, so
that it will have only one opportunity to incorporate a chain
terminating nucleotide at each position along the template.
Short Primers
The DNA polymerase is able to utilize primers of 10
bases or less, as well as longer ones, most preferably of ~-20
bases. The ability to utilize short primers offers a number of
important advantages to DNA sequencing. The shorter primers are
cheaper to buy and easier to synthesize than the usual 15-20-mer
primérs. They also anneal faster to complementary sites on a DNA
template, thus making the sequencing reaction faster. Further,
the ability to utilize small (e.g., six or seven base)
oligonucleotide primers for DNA sequencing permits strategies not
otherwise possible for sequencing long DNA fragments. For
12 ~ 60724-209
example, a kit containing 80 random hexamers could be generated,
none of which are complementary to any sites in the cloning
vector. Statistically, one of the 80 hexamers sequences will
occur an average of every 50 bases along the DNA fragment to be
sequenced. The determination of a sequence of 3000 bases would
require only five sequencing cycles. First, a "universal`' primer
le.g., New England Biolabs #1211 , sequence 5' GTAAAACGACGGCCAGT
3') would be used to sequence about 600 bases at one end of the
insert. Using the results from this sequencing reaction, a new
primer would be picked from the kit homologous to a region near
the end of the determined sequence. In the second cycle, the
sequence of the next 6~0 base~ would ~e determined using this
primer. Repetition of this process five times would determine the
complete sequence of the 3000 bases, without necessitating any
subcloning, and without the chemical synthesis of any new
oligonucleotide primers. The use of such short primers may be
enhanced by including gene 2.5 and 4 protein of T7 in the
sequencing reaction.
DNA polymerases having the above properties include
modified T7-type polymerases. That is the DNA polymerases
requires host thioredoxin as a sub-unit, and they are
substantially identical to a modified T7 DNA polymerase or to
equivalent enzymes isolated from related phage, such as T3, ~I,
~II, H, W31, gh-1, Y, A1122 and SP6. Each of these enzymes can be
modified to have properties similar to those of the modified T7
enzyme. It is possible to isolate the enzyme from phage infected
cells directly, but preferably the enzyme is isolated from cells
*Trademark
13 l 335962 60724-2094E
which overproduce it. By substantially identical is meant that
the enzyme may have amino acid substitutions which do not affect
the overall properties of the enzyme. One example of a
particularly desirable amino acid substitution is one in which the
natural enzyme is modified to remove any exonuclease activity.
This modification may be performed at the genetic or chemical
level (see below).
Cloning T7 Polymerase
We shall describe the cloning, overproduction,
purification, modification and use of T7 DNA polymerase. This
processive enzyme consists of two polypeptides tightly complexed
in a one to one stoichiometry. One is the phage T7-encoded gene 5
protein of 84,000 daltons (Modrich et al. 150 J. Biol. Chem. 5515,
1975), the other is the E. coli encoded thioredoxin, of 12,000
daltons (Tabor et al., J. Biol. Chem. 262:16, 216, 1987). The
thioredoxin is an accessory protein and attaches the gene 5
protein lthe non-processive actual DNA polymerase~ to the primer
template. The natural DNA polymerase has a very active 3' to 5'
exonuclease associated with it. This activity makes the
polymerase useless for DNA sequencing and must be inactivated or
modified before the polymerase can be used. This is readily
performed, as ~escribed below, either chemically, by local
oxidation of the exonuclease domain, or genetically, by modifying
the coding region of the polymerase gene encoding this activity.
~Trx-2
In order to clone the trxA (thioredoxin) gene of E. coli
wild type E. coli DNA was partially cleaved with Sau3A and the
13a 1 3 3 5 9 6 2 60724-2094E
fragments liyated to BamHI-cleaved T7 DNA isolated from .strain T7
ST9 (Tabor et al., in Thioredoxin and Glutaredoxin Systems:
Structure and
-
- 14 -
Funetion (Holmgren et al., eds) pp. 285-300, Raven
Press, NY; and Tabor et al., suPra). ThQ ligated DNA
was transfected into E. eoli trxA cells, the mixture
plated onto trxA cells, and the resulting T7 plaques
picked. Since T7 eannot grow without an active E. eoli
trxA gene only those phages containing the trxA gene
eould form plagues. ThQ cloned trxA genes were located
on a ~70 base pair HincIl fragment.
In order to overproduce thioreoAoY~n a plasmid,
lo pTrx-2, wa~ as construeted. Briefly, the ~70 base pair
HincII fragment eontaining the trxA gene wa~ isolated by
standard proeedure (Maniatis et al., Cloning: A
Laboratory Manual, Cold Spring Harbor Lab~., Cold Spring -
Harbor, N.Y.), and ligated to a derivative of pBR322
eontaining a Ptae promoter (pta~c-12, Amann et al., 2S
Gene 167, 1983). Referring to Fig. 2, ptae-12,
containing ~-laetamase and Col El origin, wa~ cut with
PvuII, to yield a fraqment of 2290 bp, which was then
ligated to two tandem eopies of trxA (HineII fraqment)
using commereially available linkers (SmaI-3amHI
polylin~er), to form pTrx-2. The eomplete nueleotide
sequence of pTrx-2 is shown in Figure ~. Thioredoxin
produetion is now under the eontrol of the tae promoter,
and thus ean be spaeifieally indueed, e.g. by IPTG
(isopropyl B-D-thiogalaetoside).
DGps-s and mGPl-2
Some gene produets ~of T7 are lethal when
expres~ed in E. eoli. An expression system was
developQd to faeilitate eloning and expre~-ion of,
lethal gene~, bas~d on the indueible expression of T7
RNA polymerase. Gene 5 protein i8 lethal in some E.
coli strains and an example of sueh a system is
deseribed by Tabor et al. 82 Proe. Nat. Aead. Sei. 1074
1 335962
- 15 -
(198S) where T7 gene 5 was placed under the control of
the ~10 promcter, and is only expressed when T7 RNA
polymeraso i~ present in the cQll.
Briefly, pGPS-5 (Fig. 3) wa~ constructed by
standard proeedures using synthetie 8amHI linkers to
join T7 fragment from 1~306 (Nde~) to 16869 (AhaIIr),
containing gene 5, to the S60 bp fragment of T7 from
S667 (HincrI) to 6166 (Fnu~Hl) eontaining both the
~l.lA and ~l.lB ~romoters, whieh are recognized by
T7 ~NA polymerasQ, and the 3~ BamHI-HincII fragment of
pACYC177 (Chang et al., 13~ J. Bacteriol. 11~1, 1978).
The nueleotide sequence of the T7 insert~ and linkers in
shown in Fig. 8. In thi~ pla~mid gene 5 i~ only
expre~sQd when T7 RNA polymeraso i8 provided in the cQll.
Referring to Fig. -3, T7 RNA polymerase i~
provided on phage veetor mGPl-2. This i~ similar to
pGPl-2 (Tabor et al., id.) exeept that the fragmen~ of
T7 from 3133 (HaeIII) to sa~o (HinfI), eontaining T7 RNA
polymerase was ligated, u~ing linker~ (B~lII and SalI
respectively), to BamHI-SalI eut M13 mp8, plaeing the
polymerase gQne under eontrol of tho lae promoter. The
complete nueleotide seguQnee of mGPl-2 is shown in
Fig. 9.
Sinee pGP5-5 and pTrx-2 have different origin~
of replieation (r-~e_~ively a P15A and a ColEl origin)
they ean b- tranformed into one eell simultaneously.
pTrx-2 expre-~e- larg- ~uantitie~ of thiore~Yin in the
presenee of rPTG. mGPl-2 ean eoexist in the same cell
as the-- two pla~id~ and be used to r~gulate exprQ~sion
of T7-DNA polymera~e from pGP5-S, simply by eau~ing
production of T7-RNA polymerase by indueing the lae
promoter with, e.g., IPTG.
_==
1 335962
- 16 -
OverProduction of T7 DNA polymerase
There are several potential strategies for
overproducing and reconstituting the two gene products
of trxA and gene S. The same cell strains and plasmids
can be utilized for all the strategies. In the
S preferred strategy the two genes are co-overexpressed in
the same cell. (This i~ because gene S is susceptible
to protease~ until thioredoxin is bound to it.) As
deseribed in detail below, one proeedure is to place the
two genes separatQly on each of two compatible plasmids
in the same cell. Alternatively, the two genes could be
placed in tandem on the same plasmid. It is important
that the T7-genQ S i8 plaeed under the control of a
non-leaky inducible promoter, such as ~l.lA, ~l.lB
and ~10 of T7, as the ~ynthesi~ of even small
quantities of the two polypeptides together is toxic in
most E. eoli eell~. By non-leaky is meant that less
than 500 moleeule~ of the gene product are produced, per
cell generation time, from the gene when the promoter,
controlling the gQne's expression, is not activated.
Preferably the T7 RNA polymeraæe expression system is
used although other expression systems which utilize
inducible promoter~ could also be used. A leaky
promoter, e.g., plae, allows more than 500 moleeules of
protein to be sy~thesiz~d, even when not induced, thus
cells eontaining lethal genes under the control of such
a promoter gro~ ~ao * y and are not suitable in thi~
invention. It i~ of eourse possible to produce these
produet~ in eell~ where they are not lethal, for
example, the plae promoter is suitable in such cells.
In a sseond strategy each ~ene can be cloned
and overexpressed sQparately. Using this strategy, the
cells containing the individually overproduced
polypeptides are combined prior to preparing the
~ - 17 - 1 3 3 5 9 6 2
extracts, at which point the two polypeptides form an
active T7 DNA polymerase.
Example 1: Production of T7 DNA Polymerase
E. coli strain 71.18 ~Messing et al., Proc.
Nat. Acad. Sci. 7~:36~2, 1977) is used for preparing
stocks of mGPl-2. 71.18 i8 stored in 50% glycerol at
-80~C. and i8 streaked on a standard minimal media agar
plate. A single colony is grown overnight in 2S ml
standard M9 media at 37-C, and a single plague of mGPl-2
i9 obtained by titering the stock using freshly prepared
71.18 cells. The plaque is used to inoculate 10 ml 2X
L3 ~2% 8acto-Tryptone,*1% yeast extract, 0.5% NaCl, ~mM
NaOH) containing JM103 grown to an A590~0.S. This
culture will provide the phage stock for preparing a
lS large culture of m~Pl-2. After 3-12 hours, the 10 ml
culture i8 centrifuged, and the supernatant used to
infect the large (2L) culture. For the large culture, 4
X 500 ml 2X L3 i8 inoculated with ~ X 5 ml 71.1~ cells
grown in M9, and is shaken at 37C. When the large
culture of cells has grown to an A590-1.0
(approximately three hours), they are inoculated with 10
ml of supernatant containing the starter lysate of
mGPl-2. The infected cell~ are then grown overnight at
37-C. The next day, the cells are r~...oved by
centrifugation, and the supernatant is ready to use for
induction of K38~paP5-S/pTrx-2 (see below). The
supernatant can ~be stored at ~C for approximately six
months, at a titer ~5 X 1011 ~/ml. At thi3 titer,
1 L of phage will infect 12 liter~ of cells at an
A590-S with a multiplicity of infection of 15. If the
titer is low, the maPl-2 phage can be concentrated from
the supernatant by dissolving NaCl t60 gm/liter) and
PE~-6000 (6S gm~liter) in the ~upernatant, allowing the
*Trademark
- 1 3359~2
- 18 -
mixture to settle at 0C for 1-72 hours, and then
centrifuging (7000 rpm for 20 min). The preeipitate,
which contains the mGPl-2 phage, is resuspended in
approximately l/20th of the original volume of M9
media.
K3~/pGPS-5/pTrx-2 i~ the E. coli strain
(genotype HfrC (~)) eontaining the two compatible
plasmids pGPS-5 and pTrx-2. pGPS-S plasmid has a PlsA
origin of replieation and expresse~ the kanamyein (Km)
resistanee qene. pTrx-2 has a ColEI origin of
replieation and expre8ses the ampieillin (Ap) resistanee
gene. The plasmids arQ intro~lee~ into K38 by standard
proeedure~, sQleeting KmR and ApR res~eetively. The
cells K38/pGPS-5/pTrx-2 are stored in S0% glycerol at
-80C. Prior ~o use they are streaked on a plate
containing 50~g/ml ampicillin and kanamyein, grown at
37C overnight, and a single eolony grown in 10 ml LB
media containing 50~g/ml ampicillin and kanamycin, at
37C for ~-6 hour3. The 10 ml cell eulture is used to
inoeulate 500 ml of ~B media eontaining 50~g/ml
ampieillin and ~anamyein and shaken at 37C overnight.
The following day, the S00 ml culture i~ used to
inoculate 12 liters of 2X LB-KPO~ media (2%
Baeto-Tryptone, 1~ yQast extraet, O.S% NaCl, 20 mM
KPO4, 0.2% dextrose, and 0.2% easamino acids, pH 7.4),
and grown w~t~ aeration in a fermentor at 37C. When
cells retaeh an Asgo~S~0 (i.e. logarithmie or
stationary phase eells), they are infeeted with mGPl-2
at a multiplieity of infeetion of 10, and IPTG is added
(final eoneentration O.SmM). The IPTG induee~
produetion of thioredoYin and the T7 RNA polymerase in
mGPl-2, and thenee induces produetion of the eloned DNA
1 33~9~2
-- 19 --
polymerase. The cells are grown for an additional 2.5
hours with stirring and aeration, and then har~ested.
The cell pellet i8 resuspended in l.S L 10% sucrose/20
mM Tris-HCl, pH ~.0/25 mM EDTA and re-spun. Finally,
the cell pellet is resuspended in 200 ml 10% sucrose/20
mM Tris-HCl, pH 8/1.0 mM EDTA, and frozen in liquid
N2. From 12 liter~ of induced cells 70 gm of cell
paste are obtained containing a~proximately 700 mg gene
S protein and 100 mg thioredoxin.
lo K38/pTr~-2 (R38 containing pTrx-2 alone)
ovQrproducQc thiore~oYi~, and it is added as a "booster"
to extracts of K3~/pGP5-5/pTrx-2 to insure that
thiors~oYin is in excess over gQne S protein at the
outset of the purification. The K38/pTrx-2 cells are
stored in S0% glycerol at -80-C. Prior to use they are
streaked on a plate containing 50 ~g/ml ampicillin,
grown at 37-C for 2~ hours, and a single colony grown at
37C overnight in 25 ml LB media containing S0 ~g/ml
ampicillin. The 25 ml culture i~ used to inoculate 2 L
of 2X L8 media and sha~en at 37-C. When the cells reach
an A590-3.0, the ptac promotQr~ and thus thioredoxin
production, i8 induced by the addition of IPTa (final
concentration O.S mM). The cells ar~ grown with shaking
for an additional 12-16 hours at 37C, har~e~ted,
resu~An~nd in 600 ml 10% sucrose/20 mM Tri~-HCl, pH
8.0/2S ~M EDTA, a~d re-~pun. Finally, the cell~ are
re~u~p ndQd in ~0 ml 10% sucro~e/20 mM Tris-HCl, pH
8/0.5 m~ EDTA, and frozen in liquid N2. From 2L of
cells 16 gm of cell past~ are obtained containing 150 mg
of thiore~o~in.
A~ay- for the polymerase involve the use of
~ingle-strand~d calf thymus DNA (6mM) a~ a substrate.
Th$s is prepared 1mms~iately prior to use by
1 3 3 ~
- 20 -
denaturation of double-stranded calf thymus DNA with 50
mM NaOH at 20C for 15 min., followed by neutralization
with HCl. Any purified DNA can be used as a template
for the polymerase assay, although preferably it will
have a length greater than 1,000 bases.
The standard T7 DNA polymQrasQ assay used is a
modification of the procedure described by Grippo et al.
(246 J. 3iol. Chem. 6867, 1971). The standard reaction
mix (200 ~1 final volume) contain~ ~0 mM Tri~/HCl pH
7.S, 10 mM MgC12, S mM dithiothreitol, 100 nmol
alkali-denatured calf thymus DNA, 0.3 mM dGTP, dATP,
dCTP and ~3H~dTTP (20 cpm/pm), 50 ~g/ml BSA, and
varying amount- of T7 DNA polymera~e. Incubation i~ at
37~C (10-C-~SC) for 30 min (S min-60 min). The
reaction i~ ~topped by the addition of 3 ml of cold
(0C) 1 N HCl-0.1 M pyrophosphate. Acid-insoluble
radioactivity i8 determined by the procedure of Hinkle
et al. (2S0 J. Biol. Chem. 5S23, 197~). The DNA is
precipitated on ice for lS min (S min-12 hr), then
precipitated onto glass-fiber filter~ by filtration.
The filters are wash~d five time~ with ~ ml of cold
(0C) 0.1M HCl-0.lM pyrophosphate, and twice with cold
(0C) 90% ethanol. After drying, th~ radioactivity on
the filter~ ir count~d using a non-agueous ~cintillation
2s fluor.
~ One unit of polymerase activity catalyzes the
inco~rporation of 10 n~ol of total nucleotide into an
aci~-~oluble form in 30 min at 37-C, under the
condition- given above. Native T7 DNA polymerasQ and
modified T7 DNA polymerase (see below) have the same
specific po~ymera~e activity + 20%, which ranges between
S,000-20,000 unit~/mg for native and S,000-50,000
units/mg for modifiQd polymera~e) dep6~tng upon the
preparation, u~ing the standard assay conditions stated
above.
1 335962
- 21 -
T7 DNA polymerase is purified from the above
extracts by precipltation and chromatography
tQchniques. An example of such a purification follows.
An extract of frozen cells (200 ml
K38/pGPS-5/pTrx-2 and ~0 ml ~38/p~rx-2) are thawed at
ooc overnight. The cells are combined, and 5 ml of
lysozyme (lS mg/ml) and 10 ml of NaCl (5M) are added.
After 45 min at 0C, the cells are placed in a 37C
water bath until their tempQrature reache~ 20nC. The
cells are then frozen in liquid N2. An additional 50
lo ml of NaCl (5M) is added, and the cell~ are thawed in a
37C water bath. After thawing, the cells are gently
mixed at O~C for 60 min. The lysate i8 centrifuged for
one hr at 3S,000 rpm in a Beckman ~5Ti rotor. The
supernatant (250 ml) is fraction I. It contains
lS approximately 700 mg gene 5 protein and 2S0 mg o
thioredoxin (a 2:1 ratio ~hioredoxin to gene S protein).
so gm of ammonium sulphate is dissolved in
fraction I (250 ml) and stirred for 60 min. The
suspension i8 allowed to sit for 60 min, and the
resulting precipitate collected by centrifugation at
8000 rpm for 60 min. The precipitate i8 redissolved in
300 ml of 20 mM Trls-HCl pH 7.5/5 mM
2-mercaptoethanol/0.1 mM EDTA/10~ glycerol (8uffer A).
This i~ raction II.
, A column of Whatman*DES2 DEAE (12.6 cm2 x 18
cm) i~ prepared and wa~hed with Buffer A. Fraction II
is dialyzed overnight against two changes of 1 L of
~uffer A each until the conductivity of Fraction II has
a conductivity equal to that of Buffer A containing 100
mM NaCl. Dialyzed Fraction II is applied to the column
at a flow rate of 100 ml/hr, and washed with ~00 ml of
8uffer A containing 100 mM NaCl. Proteins are eluted
*Trademark
1 335962
- 22 -
with a 3.5 L gradient from 100 to 400 mM NaCl in Bufer
A at a 1OW rate of 60 ml/hr. Fractions containing T7
DNA polymerase, which elute~ at 200 mM NaCl, are
pooled. This is fractlon lII (190 ml).
A column of Whatman Pll~phosphocellulose (12.6
cm2 x 12 cm) is prepared and washed with 20 mM KPO4
pH 7.~/5 mM 2-mercaptoethanol/0.1 mM EDTA/10 % glycerol
(~uffer ~). Fraction III is diluted 2-fold (380 ml)
with ~uffer B, then applied to the column at a flow rate
lo of 60 ml/hr, and washed with 200 ml of Buffer B
containing lOOmM KCl. protQins are elutQd with a 1.8 L
gradient from 100 to ~00 mM KCl in Buffer B at a flow
rate of 60 ml/hr. Fraction5 containing T7 DNA
polymerase, which elutes at 300 mM KCl, are pooled.
This is fraction IV (370 ml).
A column of DEAE-Sephade ~A-50 (~.9 cm2 x 15
cm) is prepared and washed with 20 mM Tris-HCl 7.0/0.1
mM dithiothreitol/0.1 mM EDTA/10~ glycerol (Buffer C).
Fraction IV is dialyzed against two changes of 1 L
Buffer C to a final conductivity equal to that of Buffer
C contalning 100 mM NaCl. Dialyzed fraction IV is
applied to the column at a flow rate of ~0 ml/hr, and
washed with 150 ml of Buffer C containing 100 mM NaCl.
Protein~ are eluted with a 1 ~ gradient from 100 to 300
mM NaCl in ~uffer C at a flow rate of ~0 ml~hr.
Fraction~ containing T7 DNA polymerase, which elutes at
210 mM NaCl, are pooled. This is fraction V (120 ml).
A column of BioRad HTP hydroxylapatite (~.9
cm2 x 15 cm) is prepared and washed with 20 mM KPO~,
pH 7.~/10 mM 2-mercaptoethanol/2 mM Na citrate/10%
glycerol (Buffer D). Fraction V iB dialyzed against two
changes of S00 ml Buffer D each. Dialyzed fraction V is
applled to the column at a flow rate of 30 ml/hr, and
*Trademark
1 335962
- 23 -
washed with 100 ml of Buffer D. Proteins are eluted
with a 900 ml gradi~nt from 0 to 180 mM KPO4, pH 7 . 4
in Bufer D at a flow rate of 30 ml/hr. Fractions
containing T7 DNA polymerase, which elutes at 50 mM
KPO~, are pooled. This is fraction VI (130 ml). It
contain~ 270 mq of homogeneous T7 DNA polymerase.
Fraction VI is dialyzed versus 20 mM KPO4 pH
7.4/0.1 mM dithiothreitol/0.1 mM EDTA/S0% glycerol.
This is concentrated fraction VI ~-6S ml, 4 mg/ml),
and is ~tored at -20-C.
The isolatQd T7 polymQrase has exonuclease
activity associated with it. As stated above this must
be inactivated. An example of inactivation by chemical
modification follows.
-15 Concentrated fraction VI is dialyzed overnight
against 20 mM KPO4 pH 7.4/0.1 mM dithiothreitol/10%
glycerol to remove the EDTA present in the storage
buffer. AftQr dialysis, the concentration i~ adjusted
to 2 mg/ml with 20 mM KPO4 pH 7.4/0.1 mM
dithiothreitol/10% glycerol, and 30 ml (2mg/ml) aliguots
are placed in S0 ml polypropylene tubes. (At 2 mg/ml,
the molar co~centration of T7 DNA polymerase i~
22 ~M.)
Dithiothreitol (DTT) and ferrous ammonium
sulfate (Fe(NH~)2(SO~)26H2O) are prepared
~ fresh immediately before use, and added to a 30 ml
; . aliquot of T7 DN~ polymerasQ, to concentrations of 5 mM
DTT (0.6 ml of a 2S0 mM stock) and 20~M
Fe(NH4)2(SO~)26H2O (0.6 ml of a 1 mM stock).
During modification the molar concentrations of T7 DNA
polymerase and iron are each approximately 20 ~M,
while DTT i8 in 2SOX molar excess.
- 24 -
The modification is carried out at 0C under a
saturated G~yen atmosphere as follows. The reaction
mixture is plaeed on ice within a dessicator, the
dessicator is purged of air by evaeuation and
subsequently filled with 100% oxygen. This cycle is
repeated three times. The reaetion can be performed in
air (20% oaygen)~ but oceurs at one third the rate.
The time course of 10s8 of exonuelease activity
i8 shown in Fig. ~. 3H-labeled double-~tranded DNA (6
lo epm/pmol) wa8 prepared from baeteriophage T7 a8
deseribed by Riehardson (lS J. Molee. 8iol. 49, 1966).
3H-labeled single-strandQd T7 DNA wa~ prepared
immediately prior to use by denaturation of
double-stranded 3H-labeled T7 DNA with SO mM NaOH at
20-C for 15 min, followed by neutralization with HCl.
The standard exonuelease assay used is a modifieation of
the proee~re deseribed by Chase et al. (su~ra). The
standard reaetion mixture (100 ~1 final volume)
contained ~0 mM Tris/HCl pH 7.5, 10 mM MgC12, 10 mM
dithiothreitol, 60 nmol 3H-labeled single-stranded T7
DNA (6 epm/pm), and varying amounts of T7 DNA
polymerase. 3H-labelQd double-stranded T7 DNA can
also be used as a substrate. Also, any uniformly
radioaetively labeled DNA, single- or double-stranded,
ean be used for the assay. Also, 3' end labeled single-
or double-~trandod DNA ean be used for the assay. After
incubation at 37^C for 15 min, the reaetion i8 stopped
by the addition of 30 ~1 of BSA (lOmg/ml) and 2S ~1
of TCA (100~ w/v~. The assay ean be run at 10C-~SC
for 1-60 min. The DNA is preeipitated on iee for 15 min
(1 min - 12 hr), then eantrifuged at 12,000 g for 30 min
(5 min - 3 hr). 100 ~1 of the supernatant i~ used to
determine the aeid-~oluble radioaetivity by adding it to
1 335962
400 ~1 water and 5 ml of aqueous scintillation
cocktail.
One unit of eYonl~elease aetivity eatalyzes the
acid solubilization of 10 nmol of total nucleotide in 30
min under the conditions of the assay. Native T7 DNA
polymera~e has a speeific exonuelease aetivity of 5000
units/mg, us$ng the standard a~say eonditions stated
above. The speeifie eYon~clease aetivity of the
modified T7 DNA polymerase depends upon the extent of
ehemical modifieation, but ideally is at least
10-100-fold lower than that of native T7 DNA polymerase,
or S00 to 50 or less unit~/mg using thQ standard assay
condition~ stated above. When double stranded substrate -
is used th~ eYonuelease aetivity is about 7-fold higher.
Under the eonditions outlined, the eYo~uelease
aetivity deeay~ Qxponentially~ with a half-life of deeay
of eight hours. Onee per day the reaction vessel is
mixed to di~tribute the soluble o~yyen, otherwise the
reaction will proceed more rapidly at the surface where
the concentratio~ of oAygen i~ higher. Once per day 2.5
mM DTT (O.3 ml of a fresh 250 mM stock to a 30 ml
reaction) is added to replenish the oxidized DTT.
After eight hours, the eYo~uelease activity of
T7 DNA polymeras6 has been reduced S0%, with negligible
loss of polymerase aetivity. The 50% 10~8 may be the
result of the eomplete inactivation of eYo~elease
activity of half the polymerase moleeules, rather than a
general reduetion of the rate of exonuelease activity in
all the moleeule~. Thus, after an eight hour reaction
all the moleeules have normal polymerase aetivity, half
the moleeules have normal sYon~elease aetivity, while
the other half have <0.1% of their-original eYonl)elease
aetivity.
1 335962
- 26 -
When 50% of the molecules are modified (an eight
hour reaction), the enzyme is suitable, although
suboptimal, for DNA sequencing. For more optimum quality
of DNA seguencing, th~ reaction is allowed to proceed to
greater than 99% modification (having less than 50 units
of eYonttclease activity), which rQquirQs four day~.
After four days, thQ reaction mixture is dialyzed
against 2 ch~nl~s of 2S0 ml of 20 mM KPOg pH 7.4/0.1 mM
dithiothreitol/0.1 mM EDTA/S0% glycerol to remove the
iron. The modified T7 DNA polymerase (-~ mg/ml) is
stor~d at -20-C.
The reaction mechanism for chemical modification
of T7 D~A polymerasQ dep~nds upon reactive o~y~e.. species
generated by the presencQ of raduced transition metals
such as Fe2 and oxygen. A possible reaction mechanism
for the gen~ration of hydroxyl radicals is outlined below:
(1) Fe2~ + 2 ~ Fe3+ + i
~2) 2 2 + 2 H ~ H2O~ + 2
(3) Fe2 + H2O2 ~ Fe3 + OH' + OH
In eguation 1, oxidation of the reduced metal
ion yields superoxide radical, 2 The superoxidQ
radical can unde~go a dismutation r~action, producing
hydrogen peroxide (~guation 2). Finally, hydrogen
peroxide can r-act with reduced metal ions to form
hydroxyl radical~, OH' (the Fenton reaction, equation
~ 33~
3). The oxidized metal ion is recycled to the reduced
form by redueing agents such as dithiothreitol (DTT).
These reaetive oxygen speeies probably
inactivate proteins by irreversibly chemically altering
specific amino aeid residues. Such damage is observed
in SDS-PAGE of fragments of gene S produced by CNBr or
trypsin. Some fragments disappear, high molecular
weight cross linlcing occurs, and some fragments are
broken into two smaller fragments.
lo As previou~ly mentioned, oxyqen, a reducing
agent (e.g. DTT, 2-mercaptoethanol) and a transition
metal (Q . g. iron) are essential elemQnts of thQ
modification reaetion. The reaetion oceur~ in air, but
is stimulated three-fold by use of 100% o~yye~ The
reaction will oceur slowly in the absence of added
transition mQtals due to the presQnCe of traeQ
guantities of transition metals ~1-2~M) in most buffer
preparations.
As e~eeted, inhibitors of the modification
reaction inelude anaerobie eonditions (e.g., N2) and
metal ehelators ~e.g. EDTA, citrate,
nitrilotriacetate). In addition, the enzymes catalase
and superoxide dismutase may inhibit the reaction,
con~istent with the essQntial role of reactive oxygen
species in the generation of modified T7 DNA polymerase.
As an alternative procedure, it is possible to
- genetieally mutatQ the T7 gene S to speeifically
inactivato the e~on~elease domain of the protein. The
T7 gene S protein purified from such mutants is ideal
for us~ in DNA s~guencing without the need to chemically
inactivate the exonuelease by oxidation and without the
seeondary damage that inevitably occurs to the protein
during chemical modification.
Genetically modified T7 DNA polymerase can be
isolated by rAn~omly mutagenizing the gene 5 and then
1 335962
screening for those mutants that have lost exonuclease
activity, withou~ loss of polymerase activity.
Mutagenesis is performed as follows. Single-stranded
DNA containing genQ S (e.g., cloned in pEMBL-8, a
plasmid containing an origin for single stranded DNA
replication) under the control of a T7 RNA polymerase
promoter is prepared by standard procedure, and treated
with two different chemical mutagens: hydrazine, which
will mutate C's and T's, and formic acid, which will
mutate G's and A's. MyQrs et al. 229 Science 2~2,
198S. The DNA is mutagenised at a dose which results in
an averaqe of one base being altered per plasmid
molecule. The single-strandQd mutagenized plasmids are
then primed with a universal 17-mer primer (see above),
and used as templates to synthesize the op~osite
strands. The synthesized strands contain r~omly
incorporated bases at positions corresponding to the
mutated bases in the templates. The double-stranded
mutagenized DNA is then used to transform the strain
K38/pGPl-2, which is strain K38 containing the plasmid
pGPl-2 (Tabor et al., suPra)~ Upon heat induction this
strain expresse~ T7 RNA polymerase. The transformed
cells are plated at 30C, with approximately 200
colonies per plate.
Screening for cells having T7 DNA polymerase
lac~ing eYonuolease activity is based upon the following
f ~n~n~ . The 3' to S' eYo~uelQase of DNA polymerases
servea a proofrc~ng function. When bases are
misincorporated, the eYon~lclea~e will remove the newly
incorporated base which is recognized as "abnormal".
This is the case for the analog of dATP, etheno-dATP,
which is readily incorporated by T7 DNA polymerase in
place of dATP. Rowever, in the presence of the 3' to 5'
exonuclease of T7 DNA polymerase, it i~ excised as
1 335~62
- 29 -
rapidly as it is incorporated, resulting in no net DNA
synthesi~. As ~hown in figure 6, using the altQrnating~
copolymQr poly d(AT) as a template, native T7 DNA
polymerase catalyzes extensive DNA synthesis only in the
s presence of dATP, and not etheno-dATP. In contrast,
modified T7 DNA polymerasQ, beeause of its lack of an
assoeiated eYon-lelQa~e, stably ineorporates etheno-dATP
into DNA at a rate comparablo to dATP. Thu~, using poly
d(AT) a~ a template, and dTTP and etheno-dATP as
preeursors, native T7 DNA polymerase i~ unable to
synthe~ize DNA from this template, while T7 DNA
polymerase whieh has lo~t it~ exonuelQase aetivity will
be able to use this t~mplate to synthesize DNA.
Tho proeedure for ly~ing and sereening large
number of eolonies i~ deseribed in Raetz ~72 Proe. Nat.
Aead. Sei. 227~, 197~). Briefly, the K38/pGPl-2 cells
transformed with the mutagenized gene S-eontaining
plasmid~ are transferred from the petri dish, where they
are present at approximately 200 eolonies per plate, to
a pieee of filter paper ("repliea plating"). The ilter
paper dise~ are then plaeed at ~2C for 60 min to induce
thQ T7 RNA polymera~Q, which in turn expre~se~ the gene
5 protein. Thioredoxin i~ eonstitutively produced from
the ehromosomal gane. ~y~ozyme i8 added to the filter
paper to lyae th~ eell~. After a freez- thaw stap to
ensuro e-ll ly-i~, the filter paper dise3 are ineubated
with poly d(AT), ta32P]dTTP and etheno-dATP at 37~C
for 60 min. Th- filter paper dises are then wa~hed with
aeid to remov~ the ~)nin~orporated [32P]dAT~. DNA will
preeipitate on the filter paper in aeid, while
nueleotid~-will be soluble. The washQd filter paper is
then u~ed to QXpO~e X-ray film. Colonie~ which have
indueed an aetive T7 DNA polymeras- whieh i~ defieient
1 3359~
- 30 -
in its eYonuclQase will have incorporated acid-insoluble
32p, and will be visible by autoradiography. Colonies
expressing native T7 DNA polymerase, or expressing a T~
DNA polymerase defective in polymerase activity, will
not appear on the autoradiograph.
Colonies which appear positive are recovered
from the master petri dish containing the original
colonie~. CQ11S containing each potential positive
clone will be induced on a larger scale (one liter) and
T7 DNA polymera~e purified from each preparation to
ascertain the levels of exonuclQase associated with each
mutant. ThosQ low in exonuclease are appropriate for
DNA seguencing .
Directed mutag~ne~is may also be used to
isolate genetic mutants in th~ eYon~clea~e domain of the
T7 gene S protein. The following is an example of this
procedure.
T7 DNA polymerase with reduced eYonuclease
activity (modified T7 DNA polymerase) can al80 be
distinguished from native T7 DNA polymerase by its
ability to synthasize through region~ of secon~ry
StrUctUrQ. Thu~, with mo~ifiod DNA PO1Ym~ra8Q~ DNA
synthesis from a labeled primer on a template having
secondary structure will result in significantly longer
extcnsions, compared to unmodified or native DNA
polymerase. Th~ as~ay provides a basis for screening
for th- conver~ion of small percentage~ of DNA
polymera~- moleculea to a modified form.
~he above a~say was u~ed to screen for altered
T~ DNA polymera~o a~ter treatment with a numbor of
chemical r~agQnt~. Three reactions resulted in
conversion of th~ enzyme to a modified form. The first
is treatment with iron and a reducing agent, a~
1 335962
described above. The other two involve treatment of the
enzyme with pho~ooxidizing dyes, Rose Bengal and
methylene blue, in ~he presence of light. The dyes must
be titrated carefully, and even under optimum conditions
the speeifieity of inaetivation of exonuclease activity
over polymerase aetivity i8 low, compared to the high
speeificity of thQ iron-induced oxidation. Since these
dyes are guite speeifie for modification of histidine
residues, thi~ result strongly implicates histidine
ra~idues as an e~sential sp~cies in the exonuelease
active site.
There ar~ 23 histidine residues in T7 gene 5
protein. Eight of these r2sidues lie in the amino half -
of the protein, in the rQgion where, based on the
homology with the large fragment of E. coli DNA
polymerase r, the eYon~lclease domain may be located
(Ollis et al. Nature 313, 818. 198~). As described
below, seven of the eight histidine residues were
mutated individually by synthesis of appropriate
oligonucleotides, which were then incorporatet into gene
s. These eorrespond to mutant~ 1, and 6-10 in table 1.
The mutations were construeted by first cloning
the T7 gene S from pGPS-3 (Tabor et al., J. Biol. Chem.
282, 1987) into th~ Smar and HindIII sites of the vector
M13 mpl8, to giv~ mGPS-2. (The vector used and the
source of gene S are not critical in this procedure,)
Single-strand~d mGPS-2 DNA was prepared from a strain
that ineorporatas deoxyuracil in place of deoxythymidine
(KUn~Q1, Proe. Natl. Acad. Sci. USA 82, ~88, 198S).
This procedure providQs a strong seleetion for survival
of only the synthesized strand (that containing the
mutation) when transfeeted into wild-type E.coli, since
the strand containing uracil will be preferentially
degraded.
~ 1 335962
- 32 -
Mutant oligonucleotides, 15-20 bases in length,
were synthesized by standard procedures. Each
oligonucleotide was annealed to the template, extended
using native T7 DNA polymerase, and ligated using T4 DNA
S ligase. Covalen~ly closed circular molecules were
isolated by agarose gel electrophoresis, run in the
presence of O.S~g/ml ethidium bromide. The resulting
purified molecul~s were then used to transform E. coli
71.18. DNA from the resulting plaques was isolated and
the relevant region seguenced to confirm each mutation.
The following summarizes the oligonucleotides
used to genQrate genQtic mutants in the gene 5
exonuclease. Th~ mutations created are underlined.
Amino acid and b~e pair numbers are takQn from Dunn et
lS al., 166 J. Molec. Biol. 4?' ~ 1983. The relevant wild
type sequences of the region of gene S mutated are also
shown.
Wild type seguQnce:
l~i (aa~ 122 123
~eu ~au Arg Ssr Gly Lys Leu Pro Gly ~ys Arg Pke Gly S~r Nis Ala ~eu Glu
CTT CTG CGT TCC GGC AAG TTG CCC GGA AAA CGC TTT GGG TCT CAC GCT TTG GAG
14677 (S7 bp)
X~ ~a~ H~a 123 ~ S-r 123
- Pr~er us-d: S' CGC TTT GGa, TC~ 5~ C GCT TTG 3'
Mutant ~ uenro:
123
Leu Leu Arg Ssr Gly Ty:r ~eu Pro Gly Ly~ Arg Phe Gly Ser ~ Al.~ reu Glu
CTT CTG CGT TCC GGC AAG TSG CCC GGA AA.A CGC TTT GG~ TC~ ;C GCT TTG GAG
M~t~t~o~ 2: Deletion o~ Ser 122 and Hla 123
Pr~mer uaed: 5' GGA AAA CGC TTT GG GC~ TTG GAG GCG 3'
6 baae deletion
Mutant ~e~uence:
122 123
rOU Leu Arg Ser Gly ry:~ r.eu Pro Gly Ly:r Ar~ Phe Gly ~- Ala reu Glu
CTT CTG CGT TCC GGC AAG TTG CCC GGA AA~ CGC TTT GG~ --- GCf' TTG GAG
_ 33 _ 1 33596~
M~tation 3: Ser 122, Hi~ 123 _ Ala 122, Glu 123
Primer u ed: 5' CGC T$T GGG ~CT ÇA~ GCT TTG G 3'
Mutant qequence:
122 123
Leu Leu Arg Ser Gly Lys Leu Pro Gly Lya Arg Phe Gly ~1~ 9L~ Ala Leu Glu
CTT CTG CGT TCC GGC AAG TTG CCC GGA AAA CGC TTT GGG ~CT 9AG GCT TTG GAG
Mutatlon 4: Lys 118, Arg 119 -~ Glu 118, Glu 119
Primer u~ed: 5' 5' G CCC GG~ ~AA gaG TTT GGG TCT CAC GC 3'
Mutant sequence:
118 119
Leu Leu Arg Ser Gly Lya Leu Pro Gly 9LY ~L~ Phe Gly Ser His Ala Leu Glu
CTT CTG CGT TCC GGC AAG TTG CCC GG~ ~AA 9a~ TTT GGG TCT CAC GCT TTG GAG
t~o~ 5:. Arg 111, Ser 112, ~y~ 114 -~ Glu 111, Ala 112, Glu 114
Primer u ed : 5' G GGT CTT CTG gaa CC GGC ~AG TTG CCC GG 3'
Mutant aequence:
111 112 114
Leu Leu ~ Al~ Gly ~ Leu Pro Gly Lya Arq Phn Gly Ser ~i3 Ala Leu
Glu
CTT CTG 9aa QCC GGC ÇAG TTG CCC GGA AAA CGC TTT GGG TCT CAC GCT TTG GAG
-- at~o~ 6: His 59, His 62 -~ Ser 59, Ser 62
Prl~er u~ed: S' ATT GTG TTC 5CC AAC GG~ 5CC AAG TAT GAC G 3'
Wild-type ~equence:
aa: 55 59 62
- Leu Ile Val Phe Nia Aan Gly N~s Lya Tyr Aap Val
CTT ATT GTG TTC CAC AAC GGT CAC AAG TAT GAC GTT
T7 bp: 14515
Mutant ~c~.~r-~:
59 62
Leu Ile Val Phe ~e~ Aan Gly S - r ~ya Tyr A~p Val
CTT ATT GTG TTC ~CC AAC GG~ ~rC AAG TAT GAC GTT
--
~ 34 ~ 1 3 3 5 9 6 2
~ut~t~on 7~ 82 ~ Ser 82
Prlmer uaed: S' GAG TTC 5CC CTT CCT CG 3'
Wlld-type ~equenc~:
aa: 77 82
L~u A~n Arg Glu Phe Nis Le~u Pro Arg Glu A~n
TTG AAC CGA GAG TTC CAC CTT CCT CGT GAG AAC
T7 bp: 14581
Mutant equence: 82
Leu Asn Arg Glu Pho ~ r eu Pro Arg Gl u A~n
TTG AAC CGA GAG TTC 5CC :SS CCT CGT GAG AAC
~t~t~s~ 8: Arg 96, His 99 _ Leu 96, S~r 99
Pri~ r u~ed: S' C5~ TTG ATS 5CS TCC AAC CTC 3'
W~ld-type ~qu~nce:
aa: 93 96 99
V~l ~cu Ser Arg Lou ~ S~r Asn L~u Ly~ A~p Thr Asp
GTG TTG TCA CG$ TTG ATT CAT TCC AAC CTC AAG GAC ACC GAT
T7 bp: 14629
Mutant ~equ¢nce:
. .... .. - 96 99
V~l L~u S~r ~,~ Lcu ~1~ ~ Sor A~n ~eu Lya A~p Thr A~p
GTG TTG TCA C5~ TTG ATT 5CT TCC AAC CTC AAG GAC ACC GAT
. . .
~h'.~t10~ 9: Hi~ 190 -~ S-r l90
Pris~r u~ed: S' CT GAC AAA 5CT TAC $TC CCS 3'
W~ld-typ
- ~: 185 190
~u L u Sor ASp Ly~ Tyr Pho Pro Pro Glu
CTA CTC TCT GAC AAA CAT TAC TTC CCT CCT GAG
T7 bp: 14905
Mutant C~c~ce:
190
~u L~u Scr A~p Ly~l ~ Tyr Phe Pro Pro Glu
CTA CTC TCT GAC AAA 5CT TAC TTC CCT CCT GAG
~ 35 l 33 5 9 62
-
?'ut~tio~ 13: HiS 218 ~ Ser 218
Pri3~r u od: 5' GAC AST GAA 5CT CGT GCT GC 3'
~ild-typ~ aequenc~:
aa: 21~ 218
V~l Asp Il~ Glu ~is Arg Ala Ala rrp L~u Lau
GTT GAC ATS GAA CAT CGT GCT GCA TGG CTG CTC
T7 bp: 14992
Mutant ~e~uenc~: 218
V~l Asp Ila Glu ~e~ Arg Ala Al~ Trp r-u r~u
GTT GAC ATT GAA ~CT CGT GCT GCA TGG CTG CTC
~tat~ D~ ion of amLno aci~s 118 to 123
Pr~ r u d: 5' C GGC AAG TTG CCC GG~ GCT TTG GAG GCG TGG G 3'
18 ~as~ tion
~ pe 5~1" ~ ~
CTT CTG CGT TCC GGC AAG TTG CCC GGAY AAA CAGsg TPThT Gly S~s is Ala L 1l 51u
Mutant a~ - a-:
L-u ~u Arg S r Gly Ly~ L u Pro Gly~ 6 ~mino acids)~ Ala L~u Glu
CTT CTG CGT TCC GGC AA~ TTG CCC GG~ ~ ~(13 b~StS) ~ ~ GCT TTG GAG
t t t C~ 12: l~iS 123 --~ Glu 123
Pri~ r usod: 5' GGG TCT 9A~ GCT TTG G 3'
Mutant at~
r~u L~u Arg Ser Gly Lys L-u Pro Gly Lys Arg P~o Gly S s ~L~ Al- Lcu Glu
CTT CTG CGT TCC GGC AAG TTG CCC GGA AAA CGC TTT GGG TCT CAG GCT TTG GAG
~ 1 335962
- 36 -
Mut-t~on 13: (A~g 131, Lys 136, Lys 140, Lys 144, A~g 14S
Glu 131, Glu 136, Glu 140, Glu 144, Glu 145)
PrLm~r u ed: S' GGT TAT 9a~ ~3C GGC GAG ATG 9AG GGT GAA TAC ~AA GAC GAC TTT ~AG ~ A~G
CST GAA G 3'
Wild~ re:
129 (aa) l31 136 140 144 145
Gly ~y-: A~g L U Gly Glu ~t Ly~ Gly GlU ~y~ t~y~ A~p A~p Ph~ ry~ A~g M~t ~u Glu Glu
GGT TAT CGC TTA GGC GAG ATG AhG GGT G~A TAC AAA GAC GAC TTT AAG CGT ATG CTT GAA
14737 (T7 bp)
Mutant s~
129 (aa) 131 136 140 144 145
Gly ~y~ ~ ~u Gly G1Y ~t ~ aly G1U ~Y~ ~1~ A~p A~p Ph~ ~ . S~. M~t r~u Glu Glu
GGT TAT 9a~ C~C GGC GAG ATG ÇAG GGT GAA TA- ~AA GAC GAC TTT ~AG 9a~ ATG CTT GAA G
14737 (T7 bp~
1 335962
- 36a -
Each mutant genQ S protein was produced by
infection of the mutant phage into K38/pGPl-2, as
follows. The cells WQrQ grown at 30C to an
A~g0~r.o. The temperature was shifted to ~C for 30
min., to inducQ T7 RNA polymQrase. IPTG was added to
O.S mM, and a lysatQ of each phage was added at a
moi-l0. Inected cQlls w~re grown at 37C for 90 min.
The cells were th~n harvQs1Qd and extracts prepared by
standard procQdurQ~ for T7 gQn~ S protQin.
Extract3 wer~ partially purified by passage
over a phosphocQllulose and DEAE A-50 column, and
assayed by measuring the polymerase and eYont~lQasQ
activities di~Qctly, as dQscribed abov~. ThQ results
arQ shown in Tabl~ l.
.
Table 1
SUMMARY OF ~X~NuCLEASE AND POLYMERASE
ACTIVIT~ES OF T7 GENE S MUTAN~S
FYonllcleasQ Polymerase
Mutant activi tY, % activi tY, %
ype~ tlOOla ' tlOO]b
. _ _ _ . . . _ .. _: _ ~ . _ ... . . . .
Mu~t I
~I~123-~Ser123) 1~2S ~90
Mu~nt2
(~S~ 12~ H~123) 0.2-0.4 ~90
Mu~nt3
(Ser122,H~123-~ ~ 12~ G1u123) <2 ~90
~ 335962
- 37 -
Table 1
SUMMARY OF EXONUCLEASE AND POLYMERASE
ACTIVITIES OF T7 GENE 5 MUTANTS
Exonuclease Polymerase
Mutant activity, ~ activity,
Mutant 4
(Lys 118, Arg 119 ) Glu 118, Glu 119) <30 >90
Mutant S
(Arg 111, Ser 112, Lys 114
Glu 111, Ala 112, Glu 114) >75 ~go
Mutant 6
(~s 59, ~s 62 ~ Scr 59, Ser 62) >1S >90
Mutant 7
(F~s 82 ~ Ser 82) >7S >90
Mutant 8
(Arg 96, His 99 ~ Leu 96, S~ 99) >7S >90
Mutant 9
(~s l90 ~ Ser 190) >7S >90
Mu~ant 10
(His 218 ~ Ser 218) >7S ~90
~utant 1 1
(~ Lys 118, Arg 119, Phe 120,
Gly 121, Ser 122, H~s 123) <0.02 ~90
`,II;tant 12
(His 123 ~ Glu 123) <30 ~90
.
Mutant 1 3
(Arg 131, Lys 136, Lys 140, Lys 144, Arg 14S
Glu 131, Glu 13~, Glu 140, Glu 144, Glu 14S) <30 ~90
a. ~xonuclease ac~ivi~y was measured on single stranded ~3HlT7
~NA. 100% e~onuclease activity corresponds tO 5, 000 units/mg.
b. PolymerasQ activity was measured usin~ sinqle-stranded calf thymus
DN~. 100~ polymerase activity corresponds to 8,000 units/mg.
1 335962
- 38 -
Of the seven histidines tested, only one (His
123: mutant 1) ha~ the enzymatic activities
characteristic of modified T7 DNA polymerasQ. T7 gene s
protein was purified from this mutant using
DEAE-cellulose, phosphocellulose, DEAE-Sep~adeY and
hydroxylapatite chromatography. While the polymerase
activity was nearly normal (~90% the level of the native
enzyme), the ~Yonuclease activity was reduced ~ to
10-fold.
A variant of this mutant was constructed in
which both His 123 and Ser 122 were deleted. The gene 5
protein purified from this mutant ha~ a 200-500 fold
lower exon~l~leafi~ activity, again with retention of >90%
of the polymerasQ activity.
The~e data strongly suggest~that His 123 lies in
the active ~ite of the exonuclease domain of T~ gene 5
protein. Furthermore, it is likely that the His 123 is
in fact the residue baing modified by the oxidation
involving iron, oxygen and a reducing agent, since such
oxidation has been shown to modify histidine residuQs
in other protein~ (Levine, J. Biol. Chem. 2S8: 11823,
1983: and Hodgson et al. Biochemistry 14: 529~, 1975).
The level of residual exonuclease in mutant 11 is
comparable to the levels obtainable by chemical
modification.
Althouqh mutations at His residues are
dQscrib~d, mutations at nearby si~tes or even at distant
sitQs may also produce mutant enzymQs suitable in this
invention, e.g., ly8 and arg (mutants ~ and lS).
Similarly, although mutations in some His residues have
little effect on exonuclease activity that does not
necessarily indicate that mutations near these residues
will not affect eYonuclease activity.
1 33~962
- 39 -
Mutations whieh are especially effective include those
having deletion~ of 2 or more amino acids, preferably
6-8, for example, near the His-123 region. Other
mutations should reduce exonuclease aetivity further, or
eompletely.
As an example of the use of thesQ mutant
strains the following i8 illustrative. A pGP5-6
(mutation ll)-eontaining strain has been deposited with
the ATCC (see below). The strain is grown as de w ribed
above and inducsd as de~eribed in Taber et al. J. Biol.
Chem. 262:16212 (1987). K38/pTrx-2 cells may be added
to inerease the yield of genetically modified T7 DNA
polymerase.
The above noted deposited strain also eontains
plasmid pGPl-2 which expressQs T7 RNA polymerase. This
plasmid is deseribed in Tabor et al., Proe. Nat. Aead.
Sci. USA 82:1074, 198S and was deposited with the ATCC
on Mareh 22, 1985 and assigned the number 40,17S.
Referring to Fig. 10, pGPS-6 ineludes the
following segments:
1. EeoRI-Saer-SmaI-BamHI polylinker sequencQ from M13
mplO (21bp).
2. T7 bp 1~309 to 167~7, that contain~ the T7 gene 5,
with the following modifieations:
T7 bp 14703 is chan~s~ from an A to a G,
CrQating a SmaI 3it~.
T7 bp 1430~ to 14321 inclusive are deleted (18
bp)-
3. SalI-PstI-HindIII polylin~er sequence from M13 mp 10
(15 bp)
4. pBR322 bp 29 (HindIII site) to pBR322 bp 37S (3amHI
site).
1 335962
- 40 -
5. T7 bp 228SS to T7 bp 22927, that contains the T7 RNA
Polymerase promoter ~10, with BamHI linkers inserted
at each end (82 bp).
6. pBR322 bp 37S (BamHI site) to p3R322 bp 4361 (EcoRI
site).
DNA Sequencinq Usina Modified T7-type DNA Polymarase
DNA synthesis rQactions using modified T7-type
DNA polymerase result in chain-terminated fragments of
uniform radioactive intensity, throughout the range of
several bases to thousands of bases in length. There i~
virtually no background due to terminations at sites
inde~endent of chain terminating agent incorporation
(i.e. at pause ~ites or secondary structure impediments).
Sequencing reactions using modified T7-type DNA
polymerase consist of a pulse and chase. By pulse is
meant that a short labelled DNA fragment is synthesized;
by chase is meant that the short fragment is lengthened
until a chain terminating agent is incorporated. The
rationale for each step differs from conventional DNA
seguencing reactions. In the pulse, the reaction is
incubated at O-C-3~oC for O.S-4 min in the presence of
high level~ of three nucleotide triphosphates (e.g.,
dGTP, dCTP and dTTP) and limiting levels of one other
labelled, carri~r-free, nucleotide triphosphate, e.g.,
- ~35S] dATP. Under these conditions the modified
polymerase i~ unable to exhibit its processive character,
and a population of radioactive fragments will be
synthesized ranging in size from a few bases to several
hundred bases. The purpose of the pulse is to
radioactively label each primer, incorporating m~im~l
radioactivity while using minimal levels of radioactive
~3~
nucleotides. In this example, two conditions in the
pulse reaction (low temperature, e.g., from 0-20C, and
limiting levels of dATP, e.g., from O.l~M to l~M)
prevent the modified T1-type DNA polymerase from
exhibiting its procQssive character. OthQr essential
environmental compo~ents of the mixture will have similar
effects, e.g., limiting more than one nucleotide
triphosphate or increa~ing the ionic strength of the
reaction. If the primer is already labQlled (e.g., by
kinasing) no pu18~ step i8 required.
In the chase, thQ rQaction is incubated at ~SC
for 1-30 min in th~ presence of high levels (S0-SOO~M)
of all four deoxynuclQoside triphosphate~ and limiting
lQvels (l-SO~M) of any one of the four chain
terminating agents, e.g~., dideoaynucleosidQ
triphosphates, such that DNA synthesis is terminated
after an averagQ of S0-600 bases. The purpose of the
chase is to Qxtend each radioactivQly labeled primer
under conditions of processive DNA synthQsis, terminating
each extension exclusively at correct sites in four
separate reactions using each of the four
dideoxynucl~oside triphosphates. Two conditions of the
chase (high temperature, e.g., from 30-S0C) and high
levels (abov~ SO~M) of all four dQoxynucleosidQ
triphosphate~) allow the modified T7-type DNA polymerase
to exhibit it~ procQ~sive character for ten~ of thousands
of ba~es: thu~ the same polymerase molecule will
synthe~ize from the primer-template until a
dideOa~ Cl~Otid- i8 incorporated. At a chase
temperature of ~5C synthesis occur~ at >700
nucleotides/sec. Thu~, for sequencing reactions the
chase is complete in less than a second. ssb increases
processivity, for example, when using dITP, or whQn using
low temperatures or high ionic strength, or low levels of
triphosphates throughout the sequencing reaction.
1 335962
-- 42 --
E i ther t a3 5S ] dATP ~ ~c3 2p ] dATP
fluorescently labelled nucleotides can be used in the DNA
sequeneing reaetions with modified T7-type DNA
polymerase. lf the fluoreseent analog is at the 5' end
of the primer, then no pulse step is reguired.
Two components determine the avQrage extensions
of the synthesis reactions. First i8 the length of time
of the pulse reaetion. Sinee the pulse is done in the
absenee of ehain terminating agents, the longer the pulse
lo reaction time, the longer the primer extensions. At 0C
the polymerase extensions average 10 nucleotides/see.
Seeond is the ratio of deoxyribonueleoside triphosphates
to ehain terminating agQnts in the ehase reaction. A
modified T7-type DNA polymerase does not discriminate
against the incorp~ration of these analog~, thu~ the
average length of extension in the chase is four times
tha ratio of the deoxynucleoside triphosphate
concentration to the chain terminating agent
concentration in the chase reaction. Thus, in order to
shorten the average size of the extensions, the pulse
- time is shortened, e.g., to 30 see. and/or the ratio of
chain terminating agent to deoxynucleoside triphosphate
concentration is raised in the chase reaction. This can
be done either by raising the concentration of the chain
2s terminating agen~ or lowering the concentration of
deoxynueleoside ~riphosphate. To increase the average
length of the ex~ensions, the pulse time is increased,
e.g., to 3-~ min; and/or the concentration of chain
terminating agen~ i8 lowered (e.g., from 20~M to 2~M)
in the chase reaetion.
Example 2: DNA ~equencin~ usin~ modified T7 DNA
polymerase
The following i~ an example of a seguencing
protocol using dideoxy nucleotides as terminating agents.
~ 33~9~2
- 43 -
9~1 of single-stranded M13 DNA (mGP1-2,
propared by standard procedures) at 0.7 mM concentration
is mixed with 1 ~1 of complementary sequencing primer
(standard universal 17-mer, 0.5 pmole primer / ~1) and
2.5 ~1 5X annealing buffer (200 mM Tris-HCl, pH 7.5,
50 mM MgC12) heated to 6SC for 3 min, and slow cooled
to room temperaturQ over 30 min. In thQ pulse reaction,
12.S ~1 of the above annealed mix was mixed with 1
~1 dithiothreitol 0.1 M, 2 ~1 of 3 dNTP~ (dGTP,
dCTP, dTTP) 3 mM eaeh ~P.~ Bioch- icals, in TE), 2.S
~1 t~3SS~dATP, ~lSOO Ci/mmol, New England Nuelear)
and 1 ~1 o~ mod~fied T7 DNA polymerase de~eribed in
Example 1 (0.~ mg/ml, 2SOO units/ml, i.e. 0.~ ~g, 2.S
unit~) and ineubated at O-C, for 2 min, after vortexing
and centrifuginq in a mierofuge for 1 8QC. The time of
incubation can vary from 30 SQC to 20 min and
temperature ean vary from 0C to 37C. Longer times are
used for determining seguenees distant from the primer.
~.S ~1 aliquots of the above pulse reaction
are added to each of four tubes containing the chase
mixe~, preheated to 4SC. The four tubes, labeled G, A,
T, C, each eontain trace amounts of either dideoxy (dd)
G, A, T, or C ~Y-L 3iochemicals). ThQ speeifie chase
solutions are givQn below. Eaeh tube eontains l.S ~1
dATP lmM, O.S ~1 SX annealing buffer (200 mM Tris-HCl,
pH 7.S, SOmM.MgC12~, and 1.O ~1 ddNTP 100 ~M
(where ddNTP~eorLe~onds to ddG,A,T or C in the
rc~e~ive t~b~). Eaeh ehasQ reaetion i8 ineubated at
~SC (or 30-C-SO-C) for 10 min, and then 6 ~1 of stop
solution (90% formamide, lOmM EDTA, 0.1% xyleneeyanol)
i~ added to eaeh tube, and the tube placed on iCQ. The
chase timQs can vary from 1-30 min.
~` ~
1 33~2
Th~ sequencing re~ctions are run on standard,
6% polyacrylamide sequencing gel in 7M urea, at 30 Watts
for 6 hours. Prior to running on a gel the reactions
are heated to 75C for 2 min. The gel is fixQd in 10
acQtic acid, 10% methanol, dried on a gel dryer, and
exposQd to Kodak OMl high-contrast autoradiography film
overnight.
Example 3 DNA se~uencinq usin~ limitinq concentrations
_ dNTPs
In this example DNA s~quence analysis of mGPl-2
DNA is performQd using limiting 1QVQ18 of all four
deoxyri~on~cleoside triphosphatQ~ in the pulse
reaction. This method has a numb~r of advantagQs over
the protocol in QxamplQ 2. First, the pulse reaction
runs to coDpletion, wherQas in the previou3 protocol it
was necessary to interrupt a time course. As a
conæequence the reactions are easi~r to run. Second,
with this method it is easier to control the extent of
the elongations in the pulsQ, and so the efficiency of
labeling of sequencQs near thQ primer (the first 50
baSQ8) i8 increased approximatQly 10-fold.
7 ~1 of O.75 mM single-~tranded M13 DNA
(mGPl-2) was mixed with 1~1 of complementary
soquencing primer (17-mer, 0.5 pmole primer/~l) and
2 ~1 5X annQaling buffer (200 mM Tris-HCl pH 7.5, 50
mM MgC12~ 2SO mM NaCl) heated at 65C for 2 min, and
~lowly e~ooled to room temperaturQ over 30 min. In the
pulse reaetion 10 ~1 of the above annealQd mix was
mixed with 1 ~1 dithiothreitol 0.1 M, 2 ~1 of 3
dNTPs (dGTP, dCTP, dTTP) 1.5 ~M each, 0.5 ~1
ta35S]dA~P, (alO~M) tabout lO~M, 1500 Ci/mmol,
New England Nuelear) and 2 ~1 modified T7 DNA
polymQrasQ (0.1 mg/ml, 1000 units/ml, i. Q ., 0.2 ~g, 2
units) and incubated at 37C for S min. (The
1 335962
- 45 -
temperature and time of incubation can be varied from
20C-~SC and 1 60 min., respectively.)
3.S ~1 aliquots of the above pulse reaetion
were added to each of four tubes containing the chase
mixes, whieh were preheated to 37C. The four tubes,
labeled G, A, T, C, eaeh contain traee amounts of either
dideoxy G, A, T, C. The speeifie chase solutions are
given below. Eaeh tube eontains 0.5 ~1 SX annealing
buffer (200 mM Tris-HCl pH 7.5, 50 mM MgC12, 2S0 mM
NaCl), 1 ~1 ~dNTPs ~dGTP, dATP, dTTP, dCTP) 200 ~M
each, and 1.O ~1 ddNTP 20 ~M. Eaeh ehase reaetion
is ineubated at 37C for S min (or 20-C-~SC and 1-60
min reg~ee~ively), and then ~ ~1 of a stop solution
(9S% formamide, 20 mM EDTA, O.OS% xylene-eyanol) added
to eaeh tube, and the tube plaeed on iee prior to
running on a standard polyacrylamidQ sequeneing gel as
deseribed abovQ.
Example ~: Replaeement of dGTP with dITP for DNA
sequencin~
In order to seguenee through regions of
eompression in DNA, i.e., regions having compact
seeondary strueture, it is eommon to USQ dITP (Mills et
al., 76 Proe. Natl. Aead. Sei. 2232, 1979) or
deazaguanosine triphosphatQ (deaza GTP, Mizusawa et al.,
1~ Nue. Aeid Re~. 1319, 1986). We have found that both
ana~og~ function well with T7-type polymerases,
espe;eially with dITP in the prQsQnce of ssb. Preferably
theie reaetion~ are performed with the above described
genetie~lly modi~ied T7 polymerase, or the ehase
reaetion is for 1-2 min., and/or at 20C to reduce
exon~elease degradation.
Modified T7 DNA polymerase efficiently utilizes
dITP or deaza-GTP in plaee of dGTP. dITP is sub~tituted
for dGTP in both the pulsQ and chase mixes at a
concentration two to five times that at whieh dGTP i~
1 33596~
.
- 46 -
used. In the ddG chase mix ddGTP is still used tnot
ddITP).
The ehase reaetions using dITP are sensitive tO
the residual low levels (about 0.01 units) of
exonuelease aetivity. To avoid this problem, the ehase
reaction times should not exceed 5 min when dITP is
used. It is reeommended that the four dITP rQaetions be
run in eonjunetion with, rather than to the exelusion
of, the four reaetion~ u~ing dGTP. If both dGTP and
dITP are rout~nely us~d, the number of reguired mixe~
can be minimtzQd by: (1) Leaving dGTP and dITP out of
the ehase mixes, which means that thQ four cha~e mixes
ean be used for both dGTP and dITP chasQ re w tion~. (2)
Adding a high coneentration of dGTP or dITP (2~1 at
0.5 mM and 1-2.5 mM re~pQctively) to thQ appropriate
pulse mix. The two pulse mixes then eaeh eontain a low
coneentration of dCTP,dTTP and t~3SS]dATP, and a
high eoneentration of either dGTP or dITP. This
modifieation doe~ not u~ually adversely effeet the
quality of the s~guaneing reaetions, and reduee~ the
re~uired number of pulse and ehase mixes to run
rQaetions using both dGTP and dITP to ~ix.
The sequeneing reaetion is a- for example 3,
exeept that two of th- pul~e mixe- eontain a) 3 dNTP mix
2s for daTP: 1.5 ~ dCTP,dTTP, and 1 mM dGTP and b) 3
.dNTP mix for dITP: l.S ~M dCTP,dTTP, and 2 mM dITP.
~In th- eh~- r-~etion dGTP i~ removed from the ehase
mixe~ (i.e. th- eha~- mixe~ eontain 30 ~M dATP,dTTP
and dCTP, and on~ of th- four dideoxynueleotidQs at 8
~M), and the ehas- time using dITP doe~ not exeeed S
min.
_ 47 _ 1 3359~2
Deposits
Strains K38/pGPS-5/pTrx-2, K38/pTrx-2 and M13
mGPl-2 have been deposited with the ATCC and assigned
numbers 67,287, 67,286, and ~0,303 respectively. Thes~
deposits were made on January 13, 1987. Strain
s K38/pGPl-2/pGP5-6 was deposited with the ATCC. On
December ~, 1987, and assign~d the number 67571.
Applicants' and their a~signees acknowledge
their responsibility to replace the~ cultures should
they die before the end of the term of a patent is~ued
hereon, 5 years after th~ last request for a culture, or
30 year~, whichever i~ the longer, and its
ra~ponsi~il$ty to notify th~ depo~itory of the issuance
of such a patent~ at which time the deposit~ will be
~ made irrQvocably available to the publlc. Until that
time the deposit~ will be made irr~vocably available to
the Commissioner of Patents under the terms of 37 CFR
Section 1-14 and 35 USC Section 112.
Other ~m~o~i~entS
Other embodiments are within the following
claims.
Oth~r uses of the modifiQt DNA polymerasQs of
this invention, which take advantage of their
processivity, and lac~ of eYo~lcleas- activity, include
the direct ensymatic amplification of genomic DNA
SQ~l~ . Thi- ha~ baen describ~d, for other
polymera---, by 8ai~i et al., 230 Science 1350, 1985;
and Scharf, 233 Science 1076, 1986.
R~f-rrin~ to Fig. 6, enzymatic amplification of
a ~pecific DNA region entail~ the use of two primer~
which ann~a1 to oppo~ite strands of a double ~tranded
DNA sequence in the r~gion of interest, with their 3'
ands directed toward one another (see dar~ arrowr). The
actual proce~lre involves multiple (10-~0, preferably
16-20) cycles of d~naturation, annealing, and D~A
1 335962
- 48 -
synthe~is. Using this procedure it is possible to
amplify a specific region of human genomic DNA over
200,000 times. As a result the specific gene fragment
represents about one part in f iVQ, rather than the
initial one part in a million. This greatly facilitates
both the cloning and the direct analysis of genomic
DNA. For diagnostic USQ8, it can speed up the analysi~
from several wee~ to 1-2 days.
Unli~Q ~lenow fragment, where the amplification
procQs~ i8 limit~d to fragment~ under two hundred basQs
in length, modified T7-type DNA polymerases should
(preferably in con~uction with ~. coli DNA binding
protQin, or ~b, to prevent "En~pb~c~ formation of
single strandQd DNA~ permit the amplification of DNA
fragment~ thousands of base~ in length.
The modifi~d T7-type DNA polymerases are also
suitable in stand~rd reaction mixtures: for a) filling
in S' protruding termini of DNA fragments generated by
re~triction enzyMe cleavage; in order to, for example,
produce blunt-ended double stranded DNA from a linear
DNA molecule having a single stranded region with no 3'
protruding termini: b) for labeling the 3~ termini of
restriction fragmentr, for mapping mRNA start sitQs by
Sl nuclea~e analyJi~, or s~quencing DNA u~ing the Maxam
and Gilbert chemical modification procedura; and c) for
in vitro mutag n6~i~ of cloned DNA fragmQnts. For
exampl-, a chemically synthe~ized primer which contain~
specific mi~match~d base~ i~ hybridized to a DNA
templat-, and then ext~s~ by the modified T7-type DNA
polym~ra~-. In thi~ way the mutation become~
permanently~incorporated into the synthe~ized strand.
It i8 advantageou~ for the polymerase to synthesize rom
the primer through the entire length of the DNA. This
~ 3~
- 49 -
is most efficiently done using a processivQ DNA
polymera~Q. Alternatively mutagene~i~ is performed by
mi~incorporation during DNA synthesis (see above). This
application is usad to mutageniz~ specific regions of
S cloned DNA fragments. It is important that the enzyme
used lacX exonuclease activity. By standard reaction
mixture i~ meant a buffered solution containing the
polymerase and any necQssary deoxynucleosides, or other
compound~.
..
