 |
Claims  |
|
|
We claim:
1. A method for sequencing a strand of DNA, comprising the steps of:
providing said strand hybridized with a primer able to hybridize to said
strand, to give an hybridized mixture,
incubating said hydridized mixture with four deoxyribonucleoside
triphosphates, a DNA polymerase, and a first chain terminating agent,
wherein said DNA polymerase causes said primer to be elongated to form a
first series of first DNA products differing in the length of the
elongated primer, each said first DNA product having a said chain
terminating agent at its elongated end, the number of molecules of each
said first DNA products being approximately the same for substantially all
DNA products differing in length by no more than 20 bases, and providing a
second chain terminating agent in said hybridized mixture at a
concentration different from said first chain terminating agent, wherein
said DNA polymerase causes production of a second series of second DNA
products differing in the length of the elongated primer, each said second
DNA product having said second chain terminating agent at its elongated
end, the number of molecules of each said second DNA products being
approximately the same for substantially all second DNA products differing
in length from each other by from 1 to 20 bases, and being distinctly
different from the number of molecules of all said first DNA products
having a length differing by no more than 20 bases from that of said
second DNA products.
2. The method of claim 1 further including the steps of:
separating said first and second DNA products by gel permeation according
to molecular weight to form a first and second series of bands
respectively, each said first or second series band representing a said
first or second DNA product, respectively, of a given molecular weight,
wherein the intensity of each nearby first series band is approximately
the same for substantially all said first series bands, and wherein the
intensity of substantially all nearby second series bands is approximately
the same, and wherein the intensity of substantially all bands of the
first series is distinctly different from the intensity of each nearby
band of the second series, and
determining the position and intensity of each said first and second series
band.
3. The method of claim 1 further comprising the step of:
providing a third chain terminating agent in said hybridized mixture at a
concentration different from said first and second chain terminating
agents, wherein said polymerase causes production of a third series of
third DNA products differing in the length of the elongated primer, each
said third DNA product having said third chain terminating agent at its
elongated end, the number of molecules of each said third DNA product
being approximately the same for substantially all said third DNA products
which differ in length by no more than 20 bases, wherein the numbers of
molecules of substantially all said first, all said second and all said
third DNA products are distinctly different from the numbers of molecules
of all those DNA products of a different series which are of lengths
differing from them by no more than 20 bases.
4. The method of claim 3 further including the steps of:
separating said first, second and third DNA products by gel permeation
according to molecular weight to form a first, second and third series of
bands, respectively, each said first, second or third series band
representing a said first, second or third DNA product, respectively, of a
given molecular weight, wherein the intensity of each nearby first series
band is approximately the same for substantially all said first series
bands, wherein the intensity of substantially all nearby second series
bands is approximately the same, and the intensity of substantially all
nearby third series bands is approximately the same, and wherein the
intensity of substantially all nearby bands of different series is
distinctly different, and
determining the position and intensity of each said band.
5. The method of claim 3 further comprising the step of:
providing in said hybridized mixture a fourth chain terminating agent at a
concentration different from said first, second and third chain
terminating agents, wherein said DNA polymerase causes production of a
fourth series of fourth DNA products differing in the length of the
elongated primer, each said fourth DNA product having said fourth chain
terminating agent at its elongated end, the number of molecules of each
said fourth DNa product being approximately the same or substantially all
fourth DNa products differing in length by no more than 20 bases, wherein
the number of molecules of substantially all said first, all said second,
all said third, and all said fourth series of DNA products is distinctly
different from all the number of molecules of those DNa products of a
different series which differ in length from them by no more than 20
bases.
6. The method of claim 5, further including the steps of:
separating said first, second, third and fourth DNA products by gel
permeation according to molecular weight to form a first, second third and
fourth series of bands respectively, each said first, second, third and
fourth series band representing a said first, second, third or fourth DNA
product of a given molecular weight, wherein each said second, third and
fourth series produce second, third and fourth bands, wherein the
intensity of substantially all nearby first series bands, or substantially
all nearby second series bands, or substantially all nearby third series
bands, or substantially all nearby fourth series bands is approximately
the same, and wherein the intensity of substantially all nearby bands in a
different series is distinctly different, and
determining the position and intensity of each said band.
7. The method of claim 1, 2, 3, 4, 5 or 6 wherein said hybridized mixture
is provided with a manganese or iron ion, wherein said ion causes said
polymerase to be non-discriminatory for a said chain terminating agent.
8. The method of claim 5 wherein said DNA products are separated according
to molecular weight in less than four lanes of a gel.
9. The method of claim 8 wherein the intensity of each band is measured by
a gel reading apparatus.
10. The method of claim 1, 2, 3, 4, 5, or 6 wherein each said chain
terminating agent is a dideoxynucleoside triphosphate.
11. The method of claim 7, wherein said hybridized mixture is provided with
a chelator.
12. The method of claim 11, said chelator being citrate or isocitrate.
13. A method for sequencing a strand of DNA, comprising the steps of:
providing a DNA polymerase, and
incubating said polymerase and said strand of DNA in a solution comprising
a manganese ion and a chain terminating agent wherein said ion is provided
at a concentration effective for causing production of approximately equal
numbers of molecules of these DNA products which differ in size from each
other by no more than 20 bases.
14. A kit for sequencing DNA comprising:
a first container comprising a DNA polymerase,
a second container comprising a chain terminating agent, and
a compound comprising a manganese ion.
15. A kit for sequencing DNA comprising:
a first container comprising a DNA polymerase,
a second container comprising a chain terminating agent, and
a compound comprising an iron ion.
16. The kit of claim 14 or 15 wherein said polymerase is a T7-type DNA
polymerase, suitable for DNA sequencing, Klenow, or Taq polymerase.
17. The kit of claim 14, 16, or 15, wherein each said chain terminating
agent is a dideoxynucleoside triphosphate.
18. The kit of claim 16 further comprising a deoxyribonucleoside
triphosphate.
19. The kit of claim 14 further comprising a chelator.
20. The kit of claim 19, said chelator being citrate or isocitrate.
21. A method for sequencing DNA comprising:
providing a DNA polymerase in a reaction medium in which said DNA
polymerase is substantially non-discriminating for a chain terminating
agent, and
incubating said DNA in said reaction medium with said DNa polymerase, a
primer, four deoxyribonucleosie triphosphates and a chain terminating
agent to elongate said primer to form a series of DNA products differing
in the length of the elongated primer and having said chain terminating
agent at the elongated end, the number of molecules of each said DNA
product being approximately the same for substantially all those DNA
products differing in size by no more than 20 bases.
22. The method of claim 21 wherein said DNA polymerase is a T7-type DNA
polymerase provided in a solution comprising manganese ions.
23. The method of claims 7, 13, or 22 wherein said manganese ions are at a
concentration between 5 .mu.M and 20 .mu.M.
24. A method for sequencing a strand of DNA, comprising the steps of:
providing said strand hybridized with a primer able to hybridize to said
strand, to give a hybridized mixture, and
incubating said hybridized mixture with four deoxyribonucleoside
triphosphates, a DNA polymerase, and a plurality of chain terminating
agents in a reaction medium in which said polymerase causes said primer to
be elongated to form a series of DNA products differing in the length of
the elongated primer, each DNA product having a first said chain
terminating agent at its elongated end, wherein the number of molecules of
said DNA products is approximately the same for substantially all DNA
products differing in length by no more than 20 bases.
25. The method of claim 24, wherein said reaction medium comprises
manganese ions.
26. The method of claim 25, wherein said manganese ions are at a
concentration between 5 .mu.M and 20 mM.
27. The method of claim 25, wherein said reaction medium comprises a
chelator and manganese ions at a concentration between 0.005 and 100 mM.
28. The method of claim 13 wherein said solution comprises a chelator and
said manganese ion is at a concentration between 0.005 nd 100 mM.
29. The method of claim 13 wherein said manganese ion is at a concentration
between 5 .mu.M and 20 mM.
30. The method of claim 1, 2, 3, 4, 5 or 6, wherein said hybridized mixture
is provided with a manganese ion, wherein said ion causes said polymerase
to be non-discriminating for a said chain terminating agent.
31. The method of claim 1, 2, 3, 4, 5 or 6, wherein said DNA polymerase is
selected from the group consisting of the large fragment of E. coli DNA
polymerase I and Taq polymerase.
32. The method of claim 1, 2, 3, 4, 5 or 6, wherein said DNA polymerase is
a T7-type DNA polymerase.
33. A method for sequencing a strand of DNA, comprising the steps of:
providing a DNA polymerase, and
incubating said polymerase and said strand of DNA in a solution comprising
an iron ion and a chain terminating agent wherein said ion is provided at
a concentration effective for causing production of approximately equal
numbers of molecules of DNA products differing in size by no more than 20
bases.
34. A method for sequencing a strand of DNA, comprising the steps of:
providing said strand hybridized with a primer able to hybridize to said
strand to give an hybridized mixture,
incubating said hybridized mixture with four deoxyribonucleoside
triphosphates, a DNA polymerase, and a plurality of different chain
terminating agents each capable of terminating the DNa synthesis at a
different nucleotide base, to cause said primer to be elongated to form a
series of DNA products differing in length with each DNA product having a
chain terminating agent at its elongated end, the amount of each said
chain terminating agent being distinctly different from that of each other
chain terminating agent, and said incubation causing the total amount of
said series terminating with one chain terminating agent to be distinctly
different from the total amount of said series terminating with each
different chain terminating agent, and
separating said series according to length, whereby the DNA products of
approximately the same length containing one chain terminating agent are
distinctly different in amount from those containing any other chain
terminating agent. |
|
 |
|
 |
Claims |
|
|
|
Description |
|
|
This invention relates to DNA sequencing and in particular to automated
methods for DNA sequencing.
DNA sequencing is generally carried out by the method of Sanger et al.
(Proc. Nat. Acad. Sci. USA 74:5463, 1977) and involves enzymatic synthesis
of single strands of DNA from a single stranded DNA template and a primer.
Referring to FIG. 1, four separate syntheses are carried out. A single
stranded template is provided along with a primer which hybridizes to the
template. The primer is elongated using a DNA polymerase, and each
reaction terminated at a specific base (guanine, G, adenine, A, thymine,
T, or cytosine, C) via the incorporation of an appropriate chain
terminating agent, for example, a dideoxynucleotide. Enzymes currently
used for this method of sequencing include: the large fragment of
Escherichia coli DNA polymerase I ("Klenow" fragment), reverse
transcriptase, Taq polymerase, and a modified form of bacteriophage T7 DNA
polymerase.
Still referring to FIG. 1, the four DNA synthesis reactions result in
formation of four series of DNA products, each product having one defined
terminus and one variable terminus. The defined terminus starts with the
primer molecule. The variable terminus ends with a chain terminating agent
specific for the nucleotide base (either G, A, T, or C) at which the
synthesis reaction terminated. The four different series of products are
each separated on the basis of their molecular weight, in four separate
lanes in a high resolution polyacrylamide gel, to form four series of
bands, with each band on the gel corresponding sequentially to a specific
nucleotide in the DNA sequence. Thus, the relative positions of the bands
identify the positions in the DNA sequence of each given nucleotide base.
Generally, the DNA products are labelled so that the bands produced are
readily detected. As shown in FIG. 1, the intensity of the bands is
generally non uniform, within a single lane, because band intensity is
directly related to the total number or concentration of DNA products of
the same molecular weight in a specific lane, and this number varies from
one product to another even when they are of approximately the same
molecular weight and even when they contain the same chain terminating
agent.
Using the above methodology, automated systems for DNA sequence analysis
have been developed. One instrument, manufactured by EG&G, uses a .sup.32
P-label and a DNA polymerase, and the resulting DNA products separated by
gel electrophoresis. Toneguzzo et al , 6 Biotechniques 460, 1988. A
.sup.32 P-detector at the bottom of the gel scans for radioactivity as it
passes through the bottom of the gel Four synthesis reactions are required
for each template to be sequenced, as well as four lanes on each gel, a
separate lane being used for products terminated by each specific chain
terminating agent, as shown for example in FIG. 1.
Kanbara et al , 6 Biotechnology 816, 1988, have replaced the .sup.32
P-labelled primer, described above, with a fluorescent-labelled primer The
resulting fluorescently labelled products are excited with a laser at the
bottom of the gel and the fluorescence detected with a CRT monitor. This
procedure also requires four synthesis reactions and four lanes on the gel
for each template to be sequenced.
Applied Biosystems manufactures an instrument in which four different
primers are used, each labelled with a different fluorescent marker. Smith
et al., 13 Nuc. Acid. Res. 2399, 1985; and 321 Nature 674, 1986. Each
primer is used in a separate reaction containing one of four
dideoxynucleotides. After the four reactions have been carried out they
are combined together and run in a single lane on a gel. A laser at the
bottom of the gel is used to detect fluorescent products after they have
been permeated or electrophoresed through the gel. This system requires
four separate annealing reactions and four separate synthesis reactions
for each template, but only a single lane on the gel. Computer analysis of
the sequence is made easier by having all four bands in a single lane.
DuPont provides an instrument in which a different fluorescent marker is
attached to each of four dideoxynucleoside triphosphates. Prober et al.,
238 Science 336, 1987. A single annealing step, a single polymerase
reaction (containing each of the four labelled dideoxynucleosides
triphosphates) and a single lane in the sequencing gel are required. The
four different fluorescent markers in the DNA products are detected
separately as they are electrophoresed through the gel.
Englert et al., U S. Pat. No. 4,707,237 (1987), describes a multichannel
electrophoresis apparatus having a detection means, disposed substantially
across the whole width of the gel, which can sense labelled DNA products
as they migrate past the detector means in four separate lanes, and
identifies the channel or lane in which the sample is located. Preferably,
radioisotopic labels are used.
Inherent to procedures currently used for DNA sequence analysis is the
necessity to separate either radioactively or fluorescently-labelled DNA
products by a gel permeation procedure such as polyacrylamide or other gel
electrophoresis, and then detect their locations relative to one another
along the axis of permeation or movement through the gel. The accuracy of
this procedure is determined in part by the uniformity of the signal in
bands which have permeated approximately the same distance through the
gel. Differences or variations in signal intensities between nearby bands
create several problems. First, they decrease the sensitivity of the
method, which is limited by the ability to detect the bands containing the
weakest signals. Second, they create difficulties in determining whether a
band with a weak signal is a true signal due to the incorporation of a
chain terminating agent, or an artifact due to a pause site in the DNA,
where the polymerase has dissociated. Third, they decrease the accuracy in
determining the DNA sequence between closely spaced bands since the strong
signal of one band may mask the weak signal of its neighbor.
SUMMARY OF THE INVENTION
All of the foregoing problems are overcome in the present invention, where
approximately the same amounts of DNA products of similar molecular
weights are produced in a sequencing reaction, and thus nearby bands in
the sequencing gel, in the same lane, are of approximately the same
intensity.
The ability to produce nearby bands of approximately the same intensity is
useful since it permits the results of any sequencing reaction to be read
more easily and with greater certainty. Further, since the DNA products
from a sequencing reaction with a specific chain terminating agent form
bands which are of approximately the same intensity as that of nearby
bands, band intensity itself provides a specific label for the series of
bands so formed. The number of DNA products of approximately the same
molecular weight produced by a given chain terminating agent varies
depending upon the concentration of the chain terminating agent. Thus, by
using a different concentration of each of the four chain terminating
agents for the synthesis, the DNA products incorporating one chain
terminating agent are distinguished from DNA products of approximately the
same molecular weight incorporating other chain terminating agents in that
they differ in number or amount; consequently, the bands of DNA products
can be identified as to chain terminating agent simply by their intensity
as compared to the intensities of nearby bands. As a result, two or more
series of DNA products, each series having a different chain terminating
agent, can be subjected to gel permeation in a single lane and identified,
i.e., distinguished from each other, by the intensity of each band as
compared to the intensity of nearby bands. Moreover, the syntheses of DNA
products incorporating different chain terminating agents need not be
carried out separately, in separate containers, but may all be carried out
simultaneously in a single reaction vessel, and the same label, e.g.,
radioisotopic, fluorescent, etc. can, if desired, be used for all chain
terminating agents instead of a different label for each, thus simplifying
the procedure.
It should be noted, however, that there is a gradual decrease in intensity
of all bands of DNA products as they permeate through the gel, those that
have travelled the shortest distance displaying less intensity than those
which have travelled the farthest distance. Neverless, the relative
intensity of each band as compared to nearby bands at any location along
the axis of permeation remains approximately the same throughout. This
conservation of relative intensity throughout the extent of permeation
makes possible the present invention.
By "nearby bands" is meant those in the same lane within about 20-30 mm
either ahead of or behind the band in question, measured along the axis of
permeation. In general, the nearby bands include DNA products differing
from the one in question by no more than 20 bases (i.e., with a mass
differing by no more than about 6,000 daltons).
In general, the invention features a DNA polymerase for use in DNA
sequencing reactions, which, in a sequencing reaction, causes DNA products
of slightly different molecular weight to be produced in approximately
equal numbers. Thus, when such DNA products are separated in a gel matrix
they form bands, with nearby bands being of approximately the same
intensity. Without being bound to any particular theory, the inventors
regard this uniformity in intensity as being due to the polymerase not
discriminating between normal nucleoside triphosphates and chain
terminating agents, such as dideoxynucleoside triphosphates.
In a first aspect, the invention features a method for sequencing a strand
of DNA, including the steps of: providing the strand of DNA; annealing the
strand with a primer able to hybridize to the strand to give an annealed
mixture; incubating the annealed mixture with a deoxyribonucleoside
triphosphate, a DNA polymerase, and a first chain terminating agent under
conditions in which the polymerase causes the primer to be elongated to
form a first series of first DNA products differing in length of the
elongated primer, each first DNA product having a chain terminating agent
at its elongated end; the number of each first DNA product being
approximately the same for substantially all DNA products differing in
length from 1 to 20 bases. Preferably, the method further includes the
steps of: separating the first DNA products by gel permeation according to
molecular weight to form a first series of bands, each first series band
representing a first DNA product of a given molecular weight, wherein the
intensity of each nearby first series band is approximately the same for
substantially all first series bands; and determining the position of each
first band.
By "substantially all" is meant that at least 9 out of 10 (or 19 out of 20)
nearby bands have approximately the same intensity. That is, only
occasional bands will have a different intensity. This different intensity
results from artifacts. One example of such an artifact is the compression
of two or more DNA products of different molecular weight within one band.
The result of two such compressions are shown in FIG. 2 where the
artifactual bands are marked with an asterisk. By approximately the same
is meant that band intensity varies by at most 2 fold, most preferably at
most 1.2 fold. By gel permeation is meant to include existing
polyacrylamide gels used for DNA sequencing, and any other mechanism for
separating DNA products according to their molecular weight.
In one embodiment, production of nearby bands of approximately the same
intensity is achieved by incubating a DNA polymerase in a solution
containing manganese or iron ions.
In one preferred embodiment, the method further includes the steps of
providing a second chain terminating agent in the annealed mixture at a
concentration different from the first chain terminating agent, wherein
the DNA polymerase causes production of a second series of second DNA
products, each second DNA product having the second chain terminating
agent at its elongated end, the number of each second DNA product being
approximately the same for substantially all DNA products differing in
length from 1 to 20 bases, wherein the number of substantially all the
first and all the second DNA products differing in length from 1 to 20
bases is distinctly different. Most preferably, the second series of
second DNA products form a second series of bands when separated by gel
permeation according to molecular weight, wherein the intensity of
substantially all nearby second series bands is approximately the same,
and the intensity of substantially all bands of the first series is
distinctly and distinguishably different from the intensity of each nearby
band of the second series, and the method further includes the step of
determining the position- and intensity of each band, the intensity being
representative of a particular band series.
By distinctly different is meant that a band of one series can be
distinguished from a nearby band (i.e., a band with a length differing
from 1 to 20 bases) in the other series. That is, a machine which measures
the number of DNA products of a specific molecular weight can distinguish
the two series of DNA products from each other.
In another preferred embodiment, the method includes providing two other
chain terminating agents wherein the polymerase causes production of a
second and third series of second and third DNA products, the number of
each second and third DNA products being approximately the same for
substantially all DNA products differing in length from 1 to 20 bases,
wherein the number of substantially all the first, all the second and all
the third DNA products differing in length from 1 to 20 bases is
distinctly different. Most preferably, each second and third series of the
second and third DNA products form a different series of second and third
bands, when separated by gel permeation according to molecular weight,
wherein the intensity of substantially all nearby second series bands is
approximately the same, the intensity of substantially all nearby third
series bands is approximately the same, and wherein the intensity of
substantially all nearby bands of different series is distinctly
different; and the method further includes the steps of determining the
position and intensity of each band, the intensity being representative of
a particular band series.
In yet another preferred embodiment, the method includes providing in the
annealed mixture four different deoxyribonucleoside triphosphates and four
different chain terminating agents, wherein the DNA polymerase causes
production of second, third and fourth series of second, third and fourth
DNA products, the number of each second, third and fourth DNA products
being approximately the same for substantially all DNA products differing
in length from 1 to 20 bases, wherein the number of substantially all the
first, all the second, all the third and all the fourth DNA products
differing in length from 1 to 20 bases is distinctly different. Most
preferably, each second, third and fourth series produce series of second,
third and fourth bands, when separated by gel permation according to
molecular weight, wherein the intensity of substantially all nearby second
series bands, or substantially all nearby third series bands, or
substantially all nearby fourth series bands is approximately the same,
and wherein the intensity of substantially all nearby bands in a different
series is distinctly different; most preferably, the method further
includes the steps of determining the position and intensity of each band,
the intensity being representative of a particular band series.
In other preferred embodiments, the annealed mixture is provided with a
manganese or iron ion, wherein the ion causes the polymerase to be
non-discriminatory for a chain terminating agent; the DNA products are
separated according to molecular weight in less than four lanes of a gel;
the intensity of each band is measured by a gel reading apparatus; the DNA
polymerase is chosen from a T7-type DNA polymerase, the large fragment of
E. coli DNA polymerase I, and Taq polymerase; and the chain terminating
agent is a dideoxynucleoside triphosphate.
In related aspects, the invention features a method for sequencing a strand
of DNA, including the steps of either (a) providing a DNA polymerase, and
incubating the polymerase and the strand of DNA in a solution including an
ion of manganese or iron and a chain terminating agent; or (b) providing a
DNA polymerase which is substantially non-discriminating for a chain
terminating agent.
In another related aspect, the invention features a method for producing a
DNA polymerase for DNA sequencing, including the step of mixing the DNA
polymerase in a solution including a manganese or iron ion.
In another aspect, the invention features a solution including a T7-type
DNA polymerase, or a Taq polymerase, and a manganese or iron ion.
Preferably the ion is at a concentration from 0.005 to 100 millimolar.
In another aspect, the invention features a kit for sequencing DNA having a
DNA polymerase, a chain terminating agent, and a manganese or iron ion.
In preferred embodiments, the polymerase is a T7-type DNA polymerase, the
large fragment of E. coli DNA polymerase I, or Taq polymerase; the chain
terminating agent is a dideoxynucleotide; and the kit further includes a
deoxyribonucleoside triphosphate.
In another aspect, the invention features a method for automated sequencing
of DNA including providing a polymerase which is substantially
non-discriminating for a chain terminating agent and causes production of
a series of DNA products differing in molecular weight and terminating
with the same chain terminating agent, wherein the DNA products produce
substantially all nearby bands of approximately the same intensity.
By substantially non-discriminating is meant that chain terminating agents
are incorporated uniformly along the length of the DNA, regardless of the
DNA sequence. By approximately the same is meant that the intensity
differs by at most two- to three-fold.
In another aspect, the invention features an automated DNA sequencing
apparatus having a reactor for providing at least two series of DNA
products formed from a single primer and a DNA strand, each DNA product of
a series differing in molecular weight and having a chain terminating
agent at one end; separating means for separating the DNA products to form
a series of bands, the intensity of substantially all nearby bands in a
series being approximately the same, and the intensity of substantially
all nearby bands in a different series being different, band reading means
for determining the position and intensity of each band after separating;
and computing means for determining the DNA sequence of the DNA strand
directly from the position and intensity of the bands.
In preferred embodiments, the reactor includes a manganese or iron ion, and
a T7-type DNA polymerase.
In another aspect, the invention features a solution or kit including a
pyrophosphatase, a DNA polymerase, and a chain terminating agent or dITP;
and a method for DNA sequencing, including providing pyrophosphatase in
the sequencing reaction. Inclusion of pyrophosphatase in a sequencing
reaction reduces the level of pyrophosphate and improves the uniformity of
band intensity of nearby bands.
In any of the above aspects, the manganese or iron ion may be provided in
the presence of a chelate, such as citrate or isocitrate. Such chelates
are thought to provide a more controlled level of the desired ion in a DNA
sequencing reaction.
In a final aspect, the invention features a T7 DNA polymerase .DELTA. Lys
118--Arg 145, and DNA encoding this polymerase. This polymerase has no
detectable exonuclease activity.
We have found conditions under which DNA polymerases can be modified to
change their ability to incorporate a chain terminating agent at the
elongating terminus of a primer DNA in the presence of a DNA template.
This ability allows DNA sequencing to be performed with lower
concentrations of chain terminating agents, thus greatly lowering the
costs of a DNA sequencing reaction. Further, we have found that DNA
polymerases having this ability produce nearby bands in a sequencing gel
which are of approximately uniform intensity. That is, the polymerase is
no longer discriminating, to any great extent, between incorporating chain
terminating agents and normal deoxynucleoside triphosphates. We have shown
that at least three polymerases can be modified in this way, including a
modified T7 DNA polymerase, the large fragment of E. coli DNA polymerase
I, and Taq polymerase. Other polymerases having homology to these
polymerases will also work in the invention.
Another advantage of this invention is that the concentration of any given
chain terminating agent to be used in a sequencing reaction is readily
calculated, since band intensity is directly related to the concentration
of any chain terminating agent and is the same for each such agent.
The modified polymerases of this invention are particularly useful in DNA
sequencing reactions since only a single sequencing reaction containing
all four chain terminating agents at four different concentrations is
necessary. Thus, less than four different sequencing reactions can be used
for any particular DNA template.
Other features and advantages of the invention will be apparent from the
following description of the preferred embodiments thereof, and from the
claims.
Description of the Preferred Embodiments
The drawings will first briefly be described.
DRAWINGS
FIG. 1 is a schematic representation of DNA sequencing by the method of
Sanger et al., supra.
FIGS. 2-7 are graphical representations of relative band intensities of six
different sequencing gels scanned by an Applied Biosystems Model 370A DNA
Sequencing System, each from a single gel lane containing a sequencing
reaction mixture resulting from using a genetically modified T7 DNA
polymerase in the presence of various mixtures of manganese or magnesium
and various dideoxynucleosides.
In each of these Figures, the DNA sequenced was mGPl-2 (encoding T7 RNA
polymerase, Tabor et al., Proc. Nat. Acad. Sci. USA, 84:4767, 1987), and
the primer was the fam primer of Applied Biosystems. In each case the
unprocessed (raw) output for the fam primer is shown. The start and end of
each output are indicated. In addition, the positions of the sequences are
shown, with respect to their corresponding position in wild type T7 DNA.
(Dunn et al., J. Mol. Biol. 8:452, 1983) The points on each graph marked
by an asterisk represent regions of compression, where at least two DNA
products of different molecular weight migrate at the same position on the
gel. Compressions are generally described by Tabor et al., Proc. Nat.
Acad. Sci. USA, 84 4767, 1987.
FIG. 8 is a graph showing the optimum concentration of magnesium and
manganese for DNA polymerase activity for a genetically modified T7 DNA
polymerase in the presence and absence of 4.0 mM isocitrate.
FIG. 9 is a graph showing the effect of different concentrations of
isocitrate in the presence of 10 mM magnesium or manganese on DNA
polymerase activity for a genetically modified T7 DNA polymerase.
FIG. 10 is a schematic map of pGP5 8, a plasmid that encodes for a
genetically modified T7 DNA polymerase lacking amino acids Lys 118 through
Arg 145, that lacks exonuclease activity.
FIG. 11 is a diagrammatic representation of an automatic sequencing
apparatus of this invention.
DNA Polymerase
DNA polymerases useful in this invention include those belonging to a class
of homologous polymerases including T7 type DNA polymerases (such as T7,
T3, .PHI.I, .PHI.II, H, W31, gh-l, Y, All22, or SP6), the large fragment
of E. coli DNA polymerase I and Taq polymerase. By homologous polymerases
is meant an enzyme that discriminates against dideoxynucleoside
triphosphates compared to deoxynucleoside triphosphates in the presence of
magnesium; however, when magnesium is replaced by manganese the
discrimination against dideoxynucleoside triphosphates is reduced. These
polymerases are used in a DNA sequencing reaction under conditions in
which they produce nearby bands of approximately uniform intensity (with
about a 1.5- to 2.0-fold variation in intensity) when the DNA products of
the sequencing reaction are run in a gel. By nearby is meant to include
bands representing DNA products of molecular weight differing by as much
as 6000, i.e., 20 bases. The actual value of this intensity will decrease
along the length of the gel, as described below and shown in the Figures.
Band intensity reflects the number of DNA products within a certain band.
Labels, such as fluorophores or radioisotopes, are used to produce a
readily detectable band of intensity reflective of the number of such DNA
products. Thus, in this invention, nearby bands derived from one
sequencing reaction with one chain terminating agent have approximately
the same number of DNA products and thus a uniform band intensity. The
sequencing conditions include incubation of the polymerase in the presence
of specific divalent or trivalent cations such as manganese (II and III),
ferrous and ferric ions; monovalent and divalent cations which have no
detectable effect, or are detrimental to DNA synthesis, include: K, Na,
Ba, Be, Ca, Ce, Cr, Co, Cu, Ni, Si and Zn. The anion is unimportant, for
example, chloride, acetate, and sulfate are suitable. Under these
conditions the requirement for chain terminating agents, such as
dideoxynucleosides, is lessened by almost 1000-fold for enzymes such as
large fragment of E. coli DNA polymerase I and Taq polymerase, and by
about 10-fold for a modified T7 polymerase. A chelator may also be
provided in this solution in order to help regulate the concentration of
available divalent metal ions. For example, citrate or isocitrate may be
provided. These chelates are thought to maintain the level of, for
example, free manganese ions at a concentration of between 10 and 100
.mu.M over a wide range of manganese concentrations. That is, the chelator
acts as a buffer.
The DNA polymerases of this invention do not discriminate significantly
between dideoxynucleoside analogs and deoxynucleosides along the length of
the DNA template. That is, in the presence of manganese or iron these
polymerases are unable to discriminate between a nucleotide that has a 3'
hydroxyl group versus one that does not (i.e., has two hydrogens at the 3'
position of the ribose). However, these polymerases do discriminate
against modifications at other positions on the nucleosides, even in the
presence of manganese or iron. For example, the polymerases do
discriminate against some dideoxynucleoside analogs which have fluorescent
groups attached compared to deoxynucleosides. However, the polymerase do
not discriminate to a different extent at neighboring, or nearby
nucleotides, on the basis of the presence or absence of the modification
to the dideoxynucleoside. Thus, while they discriminate strongly against
these analogs, requiring higher concentrations for a DNA sequencing
reaction compared to unmodified dideoxynucleosides, the intensity of
nearby bands will still be uniform. For example, there is a 10 fold
discrimination against dideoxy ITP (ddITP), compared to dideoxy GTP
(ddGTP), in the presence of Mn However, all the bands produced in a
sequencing reaction are of equal intensity with ddITP since there is no
differential discrimination along the length of the DNA template.
Thus, the polymerases of this invention provide a uniform efficiency of
incorporation of chain terminating agents, even if they discriminate
against overall incorporation.
Chain terminating agents useful in this invention include
dideoxynucleosides having a 2', 3' dideoxy structure. Other agents useful
in the invention are those able to specifically terminate a DNA sequencing
reaction at a specific base, and are not discriminated against by the
polymerase under the above conditions.
In order to determine whether any particular DNA polymerase, in combination
with any particular chain terminating agent, or other component of a
sequencing reaction mixture, is useful in this invention, a standard
sequencing reaction is performed, as described below and shown in the
drawings, and the extent of band formation, and the uniformity of nearby
bands in a sequencing gel, reviewed. If the polymerase reaction does not
extend the primer by at least 20 bases, it is not suitable under the
conditions used. Adjacent band uniformity within a two-fold or less range
is useful in this invention, preferably the uniformity is within a 1.0-1.5
fold range. Similarly, determination of optimum cation concentration, or
of other potential cations useful in the invention, is determined by use
of this sequencing reaction under various conditions. For example, cations
are tested in ranges from 0.005-100 mM. An example of such an experiment
follows:
DNA synthesis is measured using a 17-mer primer of sequence
5'-GTAAAACGACGGCCAGT-3' (New England Biolabs catalog number 1211) that has
been labeled with .sup.32 P at its 5' end and annealed to single-stranded
mGPl-2 DNA. Tabor et al., proc. Nat. Acad. Sci. USA 84:4767 (1987) and
Tabor et al., J. Biol. Chem. 262:16212 (1987). Any other template is
equally useful in this reaction. This primer-template is used in a
| | |
