Note: Descriptions are shown in the official language in which they were submitted.
CA 02266755 1999-03-25
METHOD FOR SEQLJEI~'CING
OF NUCLEIC ACID POLYMERS
This application is a continuation-in-part of International Patent Application
No.
PCT/LJS97/07135 filed April 29. 1997, designating the Lnited States) which is
a continuation-in-
part of US Patent Applications I~os. 081640,672 Tled May 1. 1996. 08/684,498
filed July 19. 1996
and 08/807.138 filed February 27. 1997. All of these applications are
incorporated herein by
reference.
>JACKGROUND OF THE INVENTION
This application relates to DNA sequencing reactions, and in particular to
improved sequen-
cing reaction protocols making use of thermally stable polymerase enzymes
having reduced error
rates.
DNA sequencing can be performed in two distinct environments: a research
environment in
which each procedure is fairly unique and in which the sequence being
determined is generally not
known prior to completion of the sequence determination; and a diagnostic
environment in which
the same procedure is repeated on many samples and the sequences being
determined are generally
known. While the basic procedures used in these two environments can be the
same) requirements
Cor speed) cost-effectiveness and low risk of error in the diagnostic
environment make many of the
techniques actually employed too cumbersome to permit their effective
utilization. This hat limited
the availability of sequencing-based diagnostics, and has indeed led some to
question w hether
sequencing can ever be cost effective for routine diagnostic use.
The ideal DNA sequencing procedure for use in a diagnostic environment would
have the
following characteristics: ( I ) it would be able to utilize a DNA-containing
sample which had been
subjected to only minimal pretreatment to make the DNA accessible for
sequencing; (2) it would
require combining this sample with only a single reaction mixture, thus
reducing risk of etTOr and
contamination, and increasing the ease with which the procedure can be
automated; and (3) it would
CA 02266755 1999-03-25
require a short amount of time to perform the sequence determination. thus
decreasing the marginal
costs in tenors of equipment and labor for performing the test.
DNA sequencing, whether for research or diagnostics) is generally performed
using tech-
niques based on the "chain termination" method described by Singer et al..
Proc. Nat'1 Acad Sci.
(USA) 74(12): 5463-5467 (1977). Basically) in this process) DMA to be tested
is isolated. rendered
single stranded) and placed into four vessels. In each vessel are the
necessary components to
replicate the DNA strand) i.e.. a template-dependant DNA polymerise) a short
primer molecule
complementary to a known region of the DNA to be sequenced. and the standard
deoxynucleotide
triphosphates (dI~TTP's) commonly represented by A, C. G and T) in a buffer
conducive to
hybridization between the primer and the DNA to be sequenced and chain
extension of the
hybridized primer. In addition) cach vessel contains a small quantity of one
type (i.e.) one species)
of dideoxynucleotidc triphosphate (ddNTP). e.g. dideoxyadenosine triphosphate
(ddA).
In each vessel, the primer hybridizes to a specific site on the isolated DNA.
The primers are
then extended, one base at a time to form a new nucleic acid polymer
complementary to the isolated
LS pieces of DNA. When a dideoxynucleotide triphosphate is incorporated into
the extending polymer.
this terminates the polymer strand and prevents it from being further
extended. Accordingly) in
each vessel. a set of extended polymers of specific lengths arc formed which
are indicative of the
positions of the nucleotide corresponding to the dideoxynucleotide in that
vessel. These sets of
polymers are then evaluated using gel electrophoresis to determine the
sequence.
2U As Church and Gilbert observed. ''in a mammalian cell, the Dh'A
corresponding to any gene
sequence is surrounded by DNA corresponding to some million other sequences."
''The Genomic
Sequencing Technique'' in Medical Ge~tetics: Past , Present and Future) Alan
R. Liss) Inc.. pp. 17-
21. ( 1991 ). The same is true) to a greater or lesser extent) of any complex
DNA sample, c.g:
containing microbial genetic materials. plant genetic materials, complete cDNA
libraries ete. In the
25 past. DNA sequencing procedures have dealt with this complexity by adding
steps which
substantially purify the DNA of interest relative to other DNA species present
in the sample. This
purification has been accomplished by cloning of the DNA to be sequenced prior
to sequencing. or
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CA 02266755 1999-03-25
by amplification of a selected portion of the genetic material in a sample to
enrich the concentration
of a region of interest relative to other DNA. For example, it is possible to
amplify a selected
portion of a gene using a polymerise chain reaction (PCR) as described in U.S.
Patents Nos.
4,683,19, 4.683,195 and 4,683,202, which are incorporated herein by reference.
This process
involves the use of pairs of primers, one for each strand of the duplex DNA.
that will hybridize at a
site located near a region of interest in a gene. Chain extension
polymerization (without a chain
terminating nucleotide) is then carried out in repetitive cycles to increase
the number of copies of
the region of interest many times. The amplified polynucleotides are then
separated from the
reaction mixture and used as the startin5 sample for the sequencing reaction.
Gelfand et al. have
1 (~ described a thermostable enzyme. "Taq polymerise," derived from the
organism Tirernu~s aguaticus,
which is useful in this amplification process. (See US Patent Nos. 4,889.818;
5,352.600 and
5.079,352 which arc incorporated herein by reference) Taq polymerise has also
been disclosed as
useful in sequencing DNA when certain special conditions are met. US Patent
No. 5,075,216)
incorporated herein by reference.
I S Improvements to the original technique described by Singer ct al. have i
ncluded improv e-
menu to the enzyme used to extend the primer chain. For example) Tabor et al.
have described
enrymes such as T7 DVA polymerise which have increased processivity, and
increased levels of
incorporation of dideoxynucleotides. (See US Patent No. 4.795.699 and EP-A-0
386 8~7, which are
incorporated herein by reference). More recently) Reeve et al. have described
a thermostable
20 enzyme preparation, called Thermo SequenaseT'~, with improved qualities for
DNA sequencing.
Nature 376: 796-797 ( I 995); EP-A-U 655 506. which is incorporated hereiwby
reference. For
sequencing, the Thermo SequenaseT'~ product is used with an amplified DNA
sample containing
0.5-2 ~tg of single stranded DNA (or 0.5 to 5 ~tg of double stranded DNA) into
four aliquots: and
combining each aliquot with the Thermo Sequenase~"' enzyme preparation) one
dideoxynucleotide
25 termination mixture containing one ddNTP and all four dNTP't; and one dye-
labeled primer which
will hybridize to the DNA to be sequenced. The mixture is placed in a
thermocycler and run for 20-
30 cycles of annealing) extension and denaturation to produce measurable
amounts of dye-labeled
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CA 02266755 1999-03-25
extension products of varying lengths which are then evaluated by gel
electrophoresis. EP-A-U 655
506 further asserts that Thermo SequcnascT" and similar enzymes can be used
for amplification
reactions.
Notwithstanding the observations in the art that enzymes useful for
amplification can also be
used for sequencing. and vice versa) efforts to combine the amplification
reaction and the
sequencing reaction into a single step have been limited. Ruano and Kidd)
Proc. Nat'l. Acad Sri.
(USA) 88: 2815-2819 (1991 ) and U.S. Patent No. 5.427,911, which are
incorporated herein by
reference) describe a process which they call "coupled amplification and
sequencing" (CAS) for
sequencing of DNA. In this process. a sample is treated in a first reaction
stage with two primers
la and amplified for a number of cycles to achieve 10.000 to 100,000-fold
amplification. A ddNTP is
then added during the exponential phase of the amplification reaction, and the
reaction is processed
for additional thermal cycles to produce chain-terminated sequencing
fragments. The CAS process
does not achieve the criteria set forth above for an ideal diagnostic assay
because it requires an
intermediate addition of reagents (the ddI~'TP reagents). This introduces and
opportunity for error or
contamination and increases the complexity of any apparatus which would be
used for automation.
The problem of errors occumng during amplification has been addressed in one
approach
through the incorporation into the extending polymers of unusual nucleotides
(for example dUTP)
which are subject to enzymatic attack (for example with uracil-N-glycosylase)
and degradation. See
US Patent No. 5.418,149, which is incorporated herein by reference. Such
molecules can be in
utilized in most of the same ways that conventional amplification are used,
but can be eliminated as
contaminants from other reactions by incorporation of a pre-treatment step
utilizing an appropriate
enzyme to degrade the modified nucleic acid polymers.
It is an object of the present invention to provide a method for sequencing of
high-
complexity D~IA samples which is well-suited for use in the diagnostic
environment and for
automation and which provides a means for minimising errors caused by
contamination and nucleic
acid polymer carryover.
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CA 02266755 1999-03-25
It is a further object of the invention to provide a method for sequencing of
DNA which
utilizes a DNA-containing sample which had been subjected to only minimal
pretreatment to make
the DNA accessible for sequencing and which provides a means for minimizing
errors caused by
contamination and nucleic acid polymer carryover.
It is still a further object of the invention to provide a method for
sequencing of DNA which
requires combining a complex DNA-containing sample with only a single reaction
mixture, thus
reducing risk of error and contamination. and increasing the ease with which
the procedure can be
automated.
SUMMARY OF THE 1NVENT10N
The present invention provides a method for sequencing a region of interest in
a DMA
sample in w hick a single set of reagents is added to a minimally-treated
sample to produce useful
sequencing results. The invention is bated on the surprising observation and
discovery that the
addition of a reaction mixture containing the thermostable polymerise Thermo
SequenaseT~~) two
primers which bind to complementary strands of a target DNA molecule at sites
flanking the region
of interest) a mixture of nucleotide triphosphates (A) C. G and T) and one
dideoxynucleotide
triphosphate to a DNA sample which contains target and non-target D~VA in
substantially natural
abundance. including highly coritplex DNA samples such as genomic human DNA,
and the
processing of the combination through multiple cycles of annealing) extension
and denaturation
results in the production of a mixture which can be loaded directly onto a gel
for sequence analysis
of the region of interest. The reaction mixture also includes an
unconventional nucleotide and an
appropriate enzyme for degradation of nucleic acid polymers containing the
unconventional
nucleotide.
One aspect of the present invention is a method for sequencing a selected
region of a
target nucleic acid polymer comprising the steps of
(a) combining a natural abundance sample containing the target nucleic acid
polymer
with a reaction mixture comprising three types of deoxynucleotide
triphosphates. an unconventional
CA 02266755 1999-03-25
nucleotide triphosphate corresponding to the fourth type of base. a
didcoxynucleotide triphosphate.
first and second primers. an enzyme which degrades nucleic acid polymers
incorporating the
unconventional nucleotide. and a thermally stable polymerise enzyme which
incorporate
dideoxynucleotides into an extending nucleic acid polymer at a rate which is
no less than about 0.4
times the rate of incorporation of deoxynucleotides to form a reaction
mixture, said first and second
primers binding to the sense and antisense strands, respectively, of the
target nucleic acid polymer at
locations flanking the selected region;
(b) exposing the reaction mixture to an initial stage in which the enzyme that
degrades
nucleic acid polymers incorporating the unconventional nucleotide is active
for a period of time
sufficient to degrade nucleic acid polymers containing the unconventional
nucleic acid which may
be present in the sample;
(c) exposing the reaction mixture to a plurality of temperature cycles each of
which
includes at least a high temperature denaturation phase and a lower
temperature extension phase to
produce a product mixture comprising sequencing fragments which arc terminated
by incorporation
I S of the dideoxynucleotide; and
(d) evaluating the product 'mixture to determine the lengths of the sequencing
fragments
produced.
BBIEF DESCRIPTION OF TH nR a vyl~~ ,~
Fig. 1 illustrates the method of the invention Schematically;
2~ Figs. 2A and 2B show a comparison of sequencing tuns performed using Thetmo
Sequenaser'1 as the polymerise in the method of the invention with results
obtained using other
thermostable polymerises in a comparative experiment:
Fig. 3 shows the data trace of Fig. 2A in greater detail;
Fig. 4 illustrates a mufti-dye embodiment of the invention:
25 Fig. 5 illusiratcs a second mufti-dye embodiment of the invemion; and
Figs. 6A and 6B illustrate a third mufti-dye embodiment of the invention.
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CA 02266755 1999-03-25
DETAILED DES('RIPTIfIV OF THE INVENTION
The present invention answers the need for a simple and readily-automated
sequencing
procedure which can be used directly on samples which contain complex mixtures
of DNA. To
distinguish such mixtures from DNA preparations which have been sequenced in
the past. the
specification and claims of this application use the term ''natural abundance
sample" to describe
such a mixture. As used herein a "natural abundance sample" is a sample which
has been treated to
make DNA in the sample accessible for hybridization with oligonucleotide
primers, Cor example by
lysis, centrifugation to remove cellular debris and proteolytic digestion to
expose the DNA. but
which has not been subjected to a preferential purification or amplification
step to increase the
0 amount of target DNA relative to non-target DNA present in the initial
sample. The term "natural
abundance'' does not) however, require the presence of all the DNA from the
on«inal sample. Thus)
a complex sample containing just nuclear DIVA. or just mitochondria) DMA or
some subfraction of
nuclear or mitochondria) DNA obtained by isolation from a tissue sample but
not subjected to
preferential amplification would be a "natural abundance" sample within the
meaning of that term in
the specification and claims of this application. The term "natural abundance"
would also include a
DNA sample prepared by conversion, for example by reverse transcription, of a
total mRNA
preparation or the genome of an RNA virus to cDNA; DNA isolated from an
individual bacterial
colony jrowing on a plate or from an enriched bacterial culture; and a viral
DNA preparation where
substantially the entire viral genome is isolated. The term ''natural
abundance" does not encompass
a sample in which the isolated DNA is not a complex combination of DNA
molecules, and thus
would not encompass, for example, a purified plasmid preparation containing
only a single species
of plasmid.
Natural abundance samples of mammalian DNA can be prepared from fluid samples,
e.g.,
blood or urine or tissue samples by any of a number of techniques. including
lysis) centrifugation to
2J remove cellular debris and proteolytic digestion to expose the DNA; salt
precipitation or standard
SDS-proteinase K-phenol extraction. Natural abundance samples can also be
prepared using kits,
for example the Gentra Pure Gene DNA Isolation Kit.
_7_
CA 02266755 1999-03-25
The method of the invention utilizes the properties of eniymes like Thermo
Sequenaser".
namely the ability to incorporate dideoxynucleotides into an extending
polynucieotide at a rate
w hich is no less than about 0.4 times the rate of incorporation of
deoxynucleotides) to provide a
method for the sequencing of a nucleic acid polymer from a natural abundance
sample in a single set
of thermocycling reactions which can be carried out in a single vessel. Thus,
the method of the
invention is ideally suited for automation.
Fig. J illustrates the fundamental simplicity and elegance of the method of
the invention in
flow chart form. As shown in Fig. 1. a sample containing a target nucleic acid
polymer which
includes a region to be sequenced is combined with a reaction mixture
containing two primers) a
l0 mixture of dV1'P's. a chain terminating nucleotide triphosphate. i.e.. a
dideoxynucleotide
triphosphate, and a thermostablc polymerise with a high affinity for ddhTP
incorporation in a
buffer suitable for hybridization and template-dependant polymerization. The
mixture is processed
for a number of thermal cycles sufficient to produce detectable amounts of
sequencing fragments.
generally from 2U to 50 cycles. During each cycle) the primers each anneal to
the respective strand
1S of target DNA present in the sample, and primer chain extension using the
polymerise enzymes and
the nucleotide triphosphate feedstocks proceeds until terminated by
incorporation of a chain-
terminating nucleotide triphosphate. This results in the production of
sequencing fragments
comparable to those generated in a conventional sequencing reaction. Analysis
of these fragments
prow ides information concerning the sequence of the selected region of the
target DNA. Those
20 extension products which are not terminated prior to reaching the region
complementary to the other
primer can serve as template for generation of sequencing fragments in later
cycles. although this
generally occurs to a very small extent. Finally, the product mixture
containing dideoxy-terminated
fragments is loaded onto an electrophoresis gel for analysis of the positions
of the base
corresponding to the chain-terminating nucleotide triphosphate with in the
target nucleic acid
25 polymer.
The operation of the invention can be understood in the context of a
hypothetical 200 nt
DNA fragment having equal amounts of each base. This means chat there will be
50 potential
_g_
CA 02266755 1999-03-25
truncation events during the cycle. For each cycle, some of the products would
be full length (and
thus able to hybridize with one of the two primers to produce more sequencing
fragments) and some
would be truncated at the points where the ddNTP was added. !f each of these
truncation events has
a statistical likelihood of occurring 1 time in 500 as a result of the
relative concentration of dd:VTP
compared to d:~ITP and the relative incorporation by the enzyme, then overall
a truncation product
will occur in slightly less than ten percent of the reactions. Table 1 shows
the relative amounts of
full-length and chain-termination products theoretically formed after 10, 20
and 30 cycles of a
reaction according to the invention using this 200 nl polynuclcotide assuming
various ratios of
truncated to full-length product.
I TABLE
p 1
truncation truncation truncation
ratio ratio ration
=0. I =0.3 = 0.5
- ~
Cycles truncatedfull-len truncatedfull-Icn~thtruncated full-len
th th
32 613 86 202 57 57
2U 41.000 376.000 17,400 40.462 3.300 3.300
tS 30 25.6 X 230 X 3.5 X 8.2 X 190,000 190.000
l0 106 ~ 10~ 10
The absolute and relative amounts of nucleotide triphosphates and chain-
terminating
nucleotide triphosphates may be optimized for the particular enzyme employed.
In actual practice,
it has been found that useful results are obtained with Thermo SequenascT"~
when the reaction is run
for 3~ to 45 cycles. using a dideoxy:deoxy mole ratio of 1:100 to 1:300. In
general) each nucleotide
triphosphate will be included in the reaction mixture at concentrations of
from ?SO 1rM to 1.5 mM.
and the chain-terminating nucleotide triphosphate will be included at a lev el
of from 0.5 pM to 30
1rM to produce compositions in which the mole ratio of the chain terminating
nucleotide
triphosphate to the corresponding nucleotide triphosphate is from t :~0 to
1:1000, preferably from
1:100 to 1:500. This will result in incorporation of a chain-terminating
nucleotide triphosphate into
from 30 to almost 100 percent of the extending polymer chains formed durin'
the thermal cycling of
the reaction mixture.
_c~_
CA 02266755 1999-03-25
A key factor in successfully performing the method of the invention is the
utilization of
Thermo Scquenase~~~'~ or a comparable enzyme as the thermostablc polymerise in
the reaction
mixture. Such enzymes are characterized by a high affinity for incorporating
dideoxynucleotides
into the extending nucleotide chain. In general, for purposes of the present
invention, the poly-
merase used should be one which incorporates dideoxynucleotides into an
extending nucleic acid
polymer at a rate which is no less than about 0.4 times the rate of
incorporation of deoxynucleotides.
Thermo SequenaseT'~t is known to favor the incorporation of
dideoxynucleotides. and is suitable for
use in the invention.. Tabor et al. have also described enzymes having
increased processiv ity and
high and increased levels of incorporation of dideoxynucleotides. (See EP 0
655 506) . Roche sells
a polymerise under the trademark TAQ-FS which meets these criteria as well.
Figs. 2A and 3B and Fig. 3 illustrate the importance of this characteristic of
the polymerise
enzyme employed. Figs. 2A and :i shows a sequencing data trace for an actual
heterozygous patient
sample of natural abundance DNA which was obtained using Thermo Sequenase~'~
and primers
flanking exon 2 of the Von Hippcl-Lindau ;ene in a process according to the
invention. Large) well-
1$ defined peaks corresponding to the termination fragments were obtained
which made sequence
evaluation of the sample very straightforward. In addition) the peaks for
homozygous peaks are all
approximately the same size, and are readily distinguishable from peaks for
bases at heterozygous
locations. This result was obtained performing the test in a single reaction
vessel, with a single
unaugmented reaction mixture. in a total of 45 thermal cycles. Comparable
results could be
obtained using fewer reaction cycles, for example 35 cycles as shown in
Example 1 herein.
In contrast. Fig. 2B shows the trace obtained when a combination of Vent and
Sequitherml"~
were used instead of Thermo SequenaseT~~ for a total of 45 thermal cycles. In
this trace, the peaks
for the termination fragments are much smaller and less well defined.
Furthermore) the peaks are
quite variable in height and did not permit identification of heterozygous
peaks based on peak
height. Perfornung the same experiment using Taq polymerise alone resulted in
a data trace that
contained no usable peaks.
- 10-
CA 02266755 1999-03-25
in the method of the invention. a natural abundance sample containing) or
suspected to
contain. a target DNA sequence is combined in a reaction mixture with an
appropriate polymerise)
all four types of dcoxynucieotide triphosphates, a dideoxynucleotide
triphosphate, and tint and
second primers. The primers used in the method of the present invention can be
any pair of primers
which hybridize with the sense and antisense strands of the target DNA
flanking a selected region
that is to be sequenced. and which do not both hybridize to neighboring
iocations in human DNA or
other DNA potentially found in the sample. As used herein, the term
''flanking" will be understood
to mean the positioning of primers at the 5'-ends of the selected region on
each DNA strand, such
that extension of the primers leads to replication of the region between the
primers. The primers are
preferably selected such that the primer pair flanks a region that is about
500 by or less. although
primers spanning largo regions of DNA can be utilized with adjustments to the
sequencing mixture
(generally an increase in the relative amount of deoxynucleotide
triphosphates) to increase the
amount of longer sequencing fragments produced.
Primers can be selected to hybridize with highly conser~~ed regions which arc
the same in all
variants of the target DNA or can be prepared as degenerate primers to take
known sequence
variations at the primer site into account. Thus, the first and second primers
of the invention may
each be a discrete oligonucleotide species, or may be a set of oligonucleotide
primers with similar
but not identical sequences.
One or both of the primers may be labeled with a detectable label at the 5'-
end thereof)
particularly a fluorescent label such as fluorescein or a cyanine dye such as
Cy 5.5. If labels are
used on both primers, the labels selected should be spectroscopically-
distinct. i.e.. they should have
either a different excitation spectrum or a different emission spectrum such
that one primer can be
distinguished from the other. When both primers are labeled with different
detectable labels) for
example with two different fluorophores as in the process described by Wiemann
et al.)
"Simultaneous On-Line DNA Sequencing on Both Stands with Two Fluorescent
Dyes," Anal.
Binchem 224: 117-121 ( 1995)) the sequence of both strands of the sample can
be determined in a
single reaction.
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CA 02266755 1999-03-25
The nucicotide triphosphate feedstock mixture is a standard mixture of three
of the four con-
ventional deoxynucleotide bases (A, C. G and T), plus an unconventional
nucleotide corresponding
to the fourth base in a buffer suitable for template-dependent primer
extension with the enzyme
employed. As will be appreciated by persons skilled in the art, the specific
concentrations of the
nucleotide triphosphates and the nature of the buffer will vary depending on
the enzyme employed.
Standard buffers and reagent concentrations for v arious known polymerase
enzymes may be
employed in the invention.
'The term "unconventional nucleotides" refers to unnatural or analog type
nucleotide
triphosphates that can be polymerized in a template dependent manner into the
sequencing
fragments. Unnatural forms of modified nucleotides include alkylated
nucleotides and nucleotides
modified by aikylhydroxylation. Specific examples of modified nucleotides
includes but are not
limited to V-7 methylguanine, deoxyuridine. dcoxyinosine, deoxy-5.6-
dihydroxythymine (from
OsO,.treated DhiA). S'.6'-dihydroxydihydrothymine. and deoxy-3'-
methyladenosine.
Unconventional nucleotides may also include natural fot'rrts of nucleotides
which arc not
i$ conventionally incorporated in DNA. Thus, for purposes of the present
invention) uracii and
hypoxanthine are unconventional nucleotides.
An important characteristic of the unconventional nucleotides use din the
invention is
existence of an enzyme which will degrade polynucleotides containing the
unconventional
nucleotides to make them unsuitable as substrates for further gcneratio of
sequencing fragments at
temperatures below those normally associated with generation of sequencing
fragments using a
thermostabie polymerace. The reaction mixture in accordance with the invention
includes an
appropriate enzyme. frequently a glycosylase. Examples of specif c cn~ymes
include: uracil N-
glycosylase, hypoxanthine-DNA ~lycosylase. 3-methyladenine-DNA glycosylase I
and II)
hydroxymethyl uracil-DNA glycosylase and foramido-pyrimidine DNA glycosylases.
The enzyme
is preferably a thermolabile enzyme so that it is active for destruction of
carry-over polynucleotides
prior to the first sequencing reaction, but inactive thereafter. Care should
be taken in the selection
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CA 02266755 1999-03-25
of reaction conditions so that the temperature is not lowered after synthesis
of sequencing fragments
to a temperature which will permit significant degradation of the desired
products prior to analysis.
The reaction mixture used in the present invention also includes at least one
type (or one
species) of chain-terminating nucleotide triphosphatc. Separate reactions for
the four different types
of bases may be run either concurrently or successively. Running all four
bases concurrently
comports with conventional sequencing practice. However. a preferred
embodiment of the present
invention combines the single vessel methodology of this application with
"single track sequencing''
which is described in commonly assigned US Patent Application No. 08/577.858.
In single track
sequencing, the determination of the positions of only one (or in any event
less than 4)
nucleotides) of a target sequence is frequently sufficient to establish the
presence of and determine
the qualitative nature of a target microorganism by providing a finger-print
or bar-code of the target
sequence that may be sufficient to distinguish it from all other known
varieties of the sequence.
Throughput is increased by rcducinb the number of reactions and
electrophoresis runs required to
identify a sequence. By selection of the order of bases teued, and
intermediate analysis) it may be
l5 unnecessary to run all four bases to determine the presence and specific
qualitative nature of any
target microorganism present in the sample.
The present method can be used in combination with any type of detection
system that is
compatible with the label employed on the primers. For example. a Pharmacia
A.L.F. sequences
may be employed when fluorescein-labeled primers are used, while a Visible
Genetics MicroGene
Blaster is appropriate when the label used is Cy~.S. When multiple labels are
used) the sample can
be processed on multiple instruments, or it can be evaluated on an instrument
which is capable of
detecting signals from multiple labels. An example of such an instrument is
the Prism 377
Sequences (Applied Biosystems lnc.) which detects and distinguishes between 4
dyes in a single
lane. Spectroscopically distinouishablc dyes which are recognized by the Prism
377 are the FAM)
ROX. TAMRA and JOE dyes known in the art.
The possibility of multi-dye detection leads to a wide range of applications
for the invention
which lead to improved accuracy in sequencing and to improved instrumental
throughput. For
- l3-
CA 02266755 1999-03-25
example. Fig. 4 illustrates a method for obtaining both the forward and
reverse sequences of a target
nucleic acid sequence using two primers, each with a spectroscopically-
distinguishable label. The
natural abundance sample is mixed with forward and reverse primers) each with
a distinguishable
label (1 and 2). The reaction is performed with four termination reactions,
one each for A. C. G and
T. Each reaction is loaded into a single well of an automated sequencing
instrument that detects and
distinguishes at least the two labels employed. The results detected from
label 1 are combined to
give the forward sequence. The results detected from label 2 are combined to
give the reverse
sequence. The two sequences can be used to check each other and correct any
ambiguities in base
calling. In addition, the opposite sequence can be used to confirm sequence
proximal to a primer
which is found empirically to be difficult to determine on commercially
available automated DNA
sequencers.
Fig. S demonstrates the use of multiple labels in a different format. In this
cast, assuming
the instrument can distinguish between 4 labels. 4 genes or gene fragments in
a single sample can be
sequenced contemporaneously. 4 pairs of primers are added to patient sample
genomic DNA. Each
I S pair is specific for a different target, possibly 4 exons of the same gene
of interest (P, Q. R and S).
One member of each pair is conjugated to a detectable label (l. 2. 3) or 4)
and each label is
distinguishable from the others. This mixture is divided into tour termination
reaction tubes. one
each for ddA, ddC, ddG and ddT) and thermally processed. Termination reaction
products are
loaded in one lane) thus employing the method as disclosed in US Patent
Application Setial No.
08/634.284, assigned to the assignee of the instant invention and incorporated
herein by reference.
The results from each label arc combined to give the sequence of the exon for
which that primer is
specific.
Another embodiment takes advantage of the Cact that a single ddA termination
reaction
identifies the A nucleotides from each strand. Fig. 6A, thus identifying the
complementary base (in
this case T) in the opposite strand. (i.e. the A termination sites on the
opposite strand correspond to
T nucleotide sites in the first strand). The complementary base can be located
in the "missing" sites
of the opposite strand. Note that sequence from the opposite strand must be
inverted before it can
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CA 02266755 1999-03-25
be added in to the missing sites because it starts at the opposite end of the
target gene and chain
extension is in the opposite direction.
In Fig. 6B. a second termination reaction for ddC is added. This allows
identification of C
and its complement G in each strand. When these results are added to the first
reaction. ~ full DNA
sequence is obtained. Thus on the basis of 2 termination reactions employing
one ddNTP chain
terminator each, the full 4 lane sequence of a gene can be obtained.
The base-calling and compiling of sequences illustrated in Fig. 6A and 6B can
be facilitated
using GeneObjects software (Visible Genetics Inc.) Toronto) and employing
techniques disclosed in
US Patent Application Nos. 08/497,202 and 08/670.534) incorporated herein by
reference.
The method of the present invention is advantageously applied in many contexts
including:
(1) detection of mutations) particularly mutations of medical significance. in
samples derived from a
human patient, animal. plant or microorganism: (2) determination of HLA type
ancillary to
transplant procedures: (3) detection and identification of microorganisms,
particularly pathogenic
microorganisms. in a sample: and (4) in-situ sequencing reactions to produce
sequencing fragments
within a histological specimen which are then removed from a selected location
on the tissue
preparation and loaded onto a gel for sequence analysis. 1"hi~ latter approach
is particularly useful
for evaluation of archived samples in retrospective studies where the outcome
of a disease condition
is known) but the potentially causative mutation is not. This method can be
used with labeled
primers for single base sequencing (or multiple-base sequencing using multiple
tissue samples).
The basic method of the invention can also be enhanced by various
modifications without
departing from the scope of the present invention. For example. improvements
in reproducibility
and sensitivity can be obtained by using a combination of an enzyme having a
high affinity for
incorporation of dideoxynucleotide triphosphates into the extending polymer,
c.g.) Thermo
Sequenase~"'. and one having a low affinity for incorporation of
dideoxynucleotide triphosphates
into the extending polymer) e.g.. Taq polymerase. under conditions where both
enzymes are actively
catalyzing template-dependent primer extension polymerization. As noted above)
the high affinity
enzyme produces almost entirely termination products, with very few of the
polymers actually being
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CA 02266755 1999-03-25
extended to full length. On the other hand, the low affinity enzyme produces
almost exclusively full
Icngth product) with relatively few termination products. Addition of the low
aftinity enzyme to the
reaction mixture increases the sensitivity of the method by producing more
full length material to be
sequenced without increasing the processing time or adding processing steps.
The increase in
sensitivity can be controlled by varying the ratio of high affinity to low
affinity encyme present in
the mixture.
It will be noted. however. that including oC low affinity enzyme to produce
full length
product will also result in the formation of a very intense labeled full-
length product peak. This
peak may make analysis of the bases near the end of the sequence difficult. To
obtain the benefits
of increased sensitivity while making less full length product) it may be
desirable to utilize a low
affinity enzyme which is more thermolabile than Taq polymerise. such that the
low affinity enzyme
is essentially inactivated by the end of the first 15 to 25 cycles. This would
allow the production of
longer fragments early in the assay and the generation of more terminated
fragments late in the
assay.
1S The reaction mixture of the invention may also incorporate other additives
which enhance
the formation of sequencing fragments'. For example. a product called
TaqStartT"' Antibody is a
monoclonal antibody which hinds to and blocks the activities of Taq
polymerise. This antibody is
added to PCR reactions using Taq polymerise to block encyme activity during
set-up at ambient
temperature to prevent or reduce the Cormation of non-specific amplification
products. TaqStartr"
Antibody can be used in the present invention with Thermo SequenaseT" to
reduce nonspecific
primer extension reactions.
The method of the invention may also be used in conjunction with Johnson &
Johnson
techniques known as "PCR In' A POUCH" which is described in US patent No.
5.460,780
incorporated herein by reference.
While the preferred method for sequencing nucleic acid polymers in accordance
with the
invention involves the use of a single step procedure as described above, the
invention also
encompasses other uses of unconventional nucleotides during the preparation of
sequencing
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CA 02266755 1999-03-25
fragments to provide a mechanism for reducing the risk of carry-over
contamination in laboratories
performing sequencing reactions through the addition of a preliminary
degradation step prior to each
sequencing procedure. Thus, in the broadest sense. the invention is a method
for sequencing a
nucleic acid polymer in a sample. in which the sample is combined with a
reaction mixture
containing an unconventional nucleotide, three conventional nucleotide bases.
at least one type of
one chain-terminating nucleotide triphosphate, a polymerise enzyme. and an
enzyme for degrading
polynucleotides containing the unconventional base. The sequencing reaction
can be done as a
single step from natural abundance DNA, as described above. or can be
performed on a previously
amplified (preferably using an unconventional nucleotide) sample. The
sequencing fragments can be
obtained using any known method, including cycle sequencing. CAS,
bidirectional sequencing or
conventional one extension sequencing.
The invention will now be further described with reference to the following
non-limiting
examples.
EXAMPLE 1
DNA Amplification by PCR with dUTP and uracyl-n-glycosylase
PCR Vtastcr Mix
HK-UNG Dilution Buffer 10 lrl
HK-UNG 1 pl
lUX PCR Buffer II 55 ~.1
rrtM MgCl2 6b lr 1
dNTP Mix 66 lrl
PCR Primers 13 pl
Sterile water 284 pl
ZS AmpliTaq DNA Polymerise C~ 5 U/pl 2.8 pl
Where
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CA 02266755 1999-03-25
HK-UVG Dilution Buffer is 50 mM Tris-HCI (pH 7.5)) 100 mM NaCI. O.l m-M EDTA.
1 mM DTT, and 0.1 % Triton X-100
HK-C; NG is Thermolabile Uracyl-N-Glycosylase, Epicentre Technologies
1 UX PCR Buffer II: 100 mM Tris-HCl pH 8.3 C 25°C; 500 mM KCl
dl~'TP Mix: ?.5 mM dATP) 2.5 mM dCTP. 2.5 mM dGTP. and 2.5 mM dUTP
PCR Primers: 2.0 IrM primer CT1590, 2.0 ~tM primer KLl-Cy5.5
Primer KL1-Cy5.5~'-TCC GGA GCG AGT TAC GAA GA-3', labeled with Cy5.5
Primer CT15905'-ATG CCC GGG ATT GGT TGA TC-3', unlabeled
45 lrl of the PCR Master Mix was put in a 200.1r1 thin-walled PCR tube.
5 ul of a recombinant plasmid (pL'C8 containing the cryptic plasmid of
Chlamydia trachomatis, serovar LI) at 400 copies/pl.
The tube was closed and put in the thermal eyelet. The PCR was started with
the following
conditions
37°C 1 ~ min
75°C 15 min
94°C 5 min
37 cycles of the following
94°C 30 sec
61 °C 30 sec
z0 72°C 45 sec
Then
72°C ~ min
hold at 4°C
Sequencing of the dUTP-containing amplicon by CLIP with dC;TP
CLIP'.Vlaster Mix
Sequencing Buffer 22 pl
CLIP Primers 12 Irl
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CA 02266755 1999-03-25
ThermoSequenase @ 32 U/pl 2.2 pl
Sterile water 84.8 pl
Where
Sequencing BufferTris-HCI 260 mM pH 9.1 @ 25'C; MgCl2 39 mM in ddH20
CLIP Primers: 2.U ~tM primer CT1431 F-Cy5.5, 2.0 ~tM primer CT1538R
Primer CT1431F-Cy~.55'-GTG CAT AAA CTT CTG AGG AT-3'. labeled with Cy5.5
Primer CT1538R5'-GTA AAC GCT CCT CTG AAG TC-3') unlabeled
11 ftl of the CLIP Master Mix was added to a tube 2 pl of the dUTP-containing
amplicon, created
by the PCR reaction above, was added to the 11 pi of CLIP Master Mix) this is
the Sample Master
Mix 20 ~tl of sterile mineral oil was added to four 200 lrl thin-walled PCR
tubes
3 ~t1 of the Sample Master Mix was added to each of the four tubes containing
the mineral oil
3 ltl of A-Sequencing Mix (75U pM dATP. 750 p.Vt dCTP, 750 pM dGTP. 750 pM
dUTP, 2.5 uM
ddATP) was added to first of the four tubes
3 ~tl of C-Sequencing Mix (75U NM dATP. 75U 1rM dCTP. 750 lrM dGTP. 750 pM
dUTP, 2.5 NM
ddCTP) was added to second of the four tubes
3 Itl of G-Sequencing Mix (750 pM dATP. 750 NM dCTP. 750 ~M dGTP. 750 uM
dUTP, 2.5 pM ddGTP) was added to third of the four tubes
3 NI of T-Sequencing Mix (75U ~M dATP. 75U lrM dCTP. 750 pM dGTP. 750 lr VI
dUTP. 2.5 pM ddTTP) was added to fourth of the four tubes
The tubes were closed and put in the thermal eyelet. The sequencing reactions
were started with the
following conditions
94°C 5 min
37 cycles of the following
94°C 1 U sec
60°C 15 sec
70°C 45 sec
Then
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CA 02266755 1999-03-25
70°C J min
hold at 4°C
The tubes were taken out of the thermal cycler, and 6 pl of loading dye was
added to each tube (A) C, G) and T). 2 pl of each tubes containing the loading
dye were migrated on
a MICROGENE BLASTER sequencer at 50°C for 2U minutes at a voltage of
1,300 volts.
After the run) the bases where determined.
Sequencing an H1V dTTP-containing amplicon by CLIP with dUTP
CLIP Master Mix
Sequencing Buffer 18.5 irl
HK-UNG 1.0 pi
dTTP-Containing Amplicon 5.0 pl
AmpIiTAQ FS @ 15 U/pl =1.0 irl
Sterile water 69.5 pl
Where
Sequencing Buffer: Tris-HCI 260 mM pH 8.3 @ 25°C; MgCl2 32.5 mM in
ddH20
dTTP-Containing Amplicon: a 1.3-kb amplicon at a concentration of 100 ng/pl,
containing the
sequence of the protease of H1V-1.
HK-UNG: Thermolabile Uracyl-N-Glycosylase, Epicentre Technologies
7 pl of A-Termination Mix (64U mM dATP, 640 mM dCTP. 640 mM dGTP, 640 mM
dUTP, 1 pM ddATP. 163 mM primer PR 170F-Cy5.5. I 63 nM primer PR 170F-A-Cy5.5.
131 nM primer PR543R-Cy5.0, 200 nM primer PR543R) was added to a 200 pl
thin-walled PCR tube.
7 pl of C-Termination Mix (64U mM dATP. 640 mM dCTP. 640 mM dGTP, 640 m.VI
dUTP. 21tM ddCTP. 163 mM primer PR170F-Cy5.5. 163 nM primer PR170F-A-Cy5.5,
131 nM primer PR543R-Cy5Ø 200 nM primer PR543R) was added to a 2001rl
-..
'0 -
CA 02266755 1999-03-25
thin-walled PCR tube.
7 ~tl of G-Termination Mix (640 mM dATP) 640 m:'Vl dCTP) 640 mM dGTP. 640 mM
dUTP, 21rM ddGTP) 163 mM primer PR 17UF-Cy5.5. I 63 nV1 primer PR 170F-A-
Cy5.5,
131 nM primer PR543R-CyS.U. 2U0 nVI primer PR543R) was added to a 200 ftl
thin-walled PCR tube.
7 ~tl of T-Termination Mix (640 mM dATP) 64U mM dCTP. 640 mM dGTP. 64U mM
dUTP. 2 ~tM ddTTP. 163 m.M primer PR170F-Cy5.5. 163 nM primer PR170F-A-Cy5.5.
131 nM primer PR543R-Cy5.0) 200 nM primer PR543R) was added to a 2UU ftl
thin-walled PCR tube.
Where
Primer PR170F-Cy5.5: 5'-GAG CCR ATA GAC AAG GAA YTR TAT-3') labeled with Cy5.5
Primer PR170F-A-Cy5.5: 5'-GAG MCG ATA GAC AAG GRV CTG TAT-3', labeled with
Cy5.5
Primer PR543R-Cy5.5: 5'-ACT TTT GGG CCA TCC ATT CCT-3'. labeled with Cy5.0
Primer PR543R-Cy5.5: 5'-ACT TTT GGG CCA TCC ATT CCT-3', unlabeled
5 lrl of the CL,1P Master Mix was added to each of the four tubes containing
the TetirtinatioWlixes.
The tubes were closed and put in the thermal cycler. The sequencing reactions
were started with the
following conditions
37°C 15 min
2U 75°C 15 min
94°C 5 min
30 cycles of the following
94°C 2U sec
56°C 20 sec
70°C 90 sec
Then
70°C 5 min
-21 -
CA 02266755 1999-03-25
hold at 4°C
The tubes were taken out of the thermal cycler, and l2 pl of loading dye was
added to each tube (A.
C) G, and T). 2 ~tl of each cubes containing the loading dye were migrated on
a Clipper
(two dye automated scquencer) at 54°C for 35 minutes at a voltage of
1.400 volts. After the run) the
bases where determined on both orientation (forward and reverse).
EXAMPLE 3
Cycle Sequencing
Sequencing M13 with dUTP
Sequencing Master Mix
HK-UNG diluted 1/10 1.0 pl
Sequencing Buffer 2.U lrl
Primer at 3 uM 1.0 lrl
M13 ssDI~A I.0 pl
ThermoSequenase @ 3.2 U/ltl 2.5 ~tl
Sterile w ~ater 5.5 lrl
Where
Sequencing BuCCer: Tris-HCl 260 mM pH 8.3 C~ 25°C: YIgCI? 39 mM in
ddH20
PrimerM 13 Universal-CyS.~: 5'-GTA AAA CGA CGG CCA GT-3', labeled with Cy5.5
M13 ssDNA: single stranded DNA (M13) at 200 ng/pl
HK-UNG: Thermolabile LTracyl-N-Glycosylase, Epicentre Technologies
Aliquot 3 pl of A-Termination Mix (750 pM dATP) 750 pM dCTP. 750 NM Dgtp. 750
pM dUTP)
2.5 ~tM ddATP) in a 2(l0 ul thin-walled PCR tube.
Aliquot 3 pl of C-Termination Mix (750 pM dATP. 75U lrM dCTP, 75U NM dGTP.
750 pM dUTP) 2.5 ttM ddCTP) in a 200 lrl thin-walled PCR tube.
Aliquot 3 a l of G-Termination M ix (750 N M dATP. 7501r M dCTP. 75011 M dGTP.
7501rM dUTP. 2.S 1tM ddGTP) in a 200 Nl thin-walled PCR tube.
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CA 02266755 1999-03-25
Aliquot 3 ~1 of T-Termination Mix (750 ~'.vI dATP. 750 ~M dCTP, 750 IrM dGTP.
75U ~M dUTP. 2.5 ~rM ddTTP) in a 20U ~1 thin-walled PCR tubc.
Aliquot 3 ul of the Sequencing Master Mix in each of the termination tubes.
Close the tubes and put
them in the thermal cylcer. Stan the sequencing reaction with the following
conditions
37°C 1 S min
75°C 15 min
94°C 2 min
35 cycles of the following
94°C d0 sec
50°C 20 sec
72°C 60 sec
Then
72°C 2 min
hold at 4°C
Take the tubes out of the thermal cycler. and add 6 ul of loading dye to each
tube (A) C. G) and T).
Migrate 2 ~1 of each tubes containing the loading dye a MICROGENE BLASTER
sequencer at
54°C for 35 minutes at a voltage of 1,4(>n volts. After the run)
analyse the data to determine the
sequence.
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