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Claims  |
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I claim:
1. A method for determining a nucleotide sequence of a polynucleotide, the
method comprising the steps of:
(a) ligating a probe to an end of a polynucleotide, the probe having a
nuclease recognition site of a nuclease whose cleavage site is separate
from its recognition site;
(b) identifying one or more nucleotides at the end of the polynucleotide by
the identity of the probe ligated thereto or by extending a strand of the
polynucleotide or probe;
(c) cleaving the polynucleotide with a nuclease recognizing the nuclease
recognition site of the probe such that the polynucleotide is shortened by
one or more nucleotides: and
(d) repeating said steps (a) through (c) until said nucleotide sequence of
the polynucleotide is determined.
2. The method of claim 1 wherein said nuclease is a type IIs restriction
endonuclease.
3. The method of claim 2 further including a step of blocking recognition
sites of said nuclease on said polynucleotide.
4. The method of claim 3 wherein said step of ligating includes treating
said polynucleotide and said probe with a ligase.
5. The method of claim 4 wherein said polynucleotide has a protruding
strand at at least one end and wherein said probe has a protruding strand
at one end, the protruding strand of said probe being complementary to the
protruding strand at one end of said polynucleotide.
6. The method of claim 5 wherein said protruding strand of said
polynucleotide has a 5'-phosphoryl group and wherein said complementary
protruding strand of said probe lacks a 5'-phosphoryl group.
7. The method of claim 6 wherein said step of ligating includes treating
said polynucleotide and said probe in succession with (i) a ligase to
ligate said protruding strand having said 5'-phosphoryl group to said
probe, (ii) a kinase to phosphorylate said complementary protruding strand
of said probe, and (iii) a ligase to ligate said complementary protruding
strand of said probe to said polynucleotide.
8. The method of claim 5 wherein said step of ligating includes providing
said probe as a mixture such that said complementary protruding strand of
said probe includes every possible sequence of nucleotides the length of
said protruding strand.
9. The method of claim 5 further including the step of removing unligated
probe from said polynucleotide after said step of ligating.
10. The method of claim 5 wherein said step of identifying includes
identifying a nucleotide in said protruding strand of said polynucleotide
by the identity of said probe ligated thereto.
11. The method of claim 10 further including the step of capping said
polynucleotide which fails to ligate to said probe.
12. The method of claim 11 wherein said step of capping includes extending
said polynucleotide with a DNA polymerase in the presence of
chain-terminating nucleoside triphosphates.
13. The method of claim 12 wherein said chain-terminating nucleoside
triphosphates are dideoxynucleoside triphosphates.
14. The method of claim 5 wherein said step of identifying includes
identifying a nucleotide in said protruding strand of said polynucleotide
by extending a strand of said polynucleotide or said probe with a nucleic
acid polymerase in the presence of chain-terminating nucleoside
triphosphates.
15. The method of claim 14 wherein said step of identifying further
includes extending a strand of said polynucleotide.
16. The method of claim 15 wherein said chain-terminating nucleoside
triphosphates are labeled.
17. The method of claim 14 wherein said step of identifying further
includes extending a strand of said probe and wherein said
chain-terminating nucleoside triphosphates are labeled.
18. The method of claim 1 wherein said polynucleotide has a protruding
strand at at least one end and wherein said probe has a protruding strand
at one end, the protruding strand of said probe being complementary to the
protruding strand at one end of said polynucleotide.
19. The method of claim 18 wherein said step of ligating includes treating
said polynucleotide and said probe with a ligase.
20. The method of claim 19 wherein said nuclease is a type IIs restriction
endonuclease.
21. The method of claim 20 wherein said step of ligating includes providing
said probe as a mixture such that said complementary protruding strand of
said probe includes every possible sequence of nucleotides the length of
said protruding strand.
22. The method of claim 21 further including the step of removing unligated
probe from said polynucleotide after said step of ligating.
23. The method of claim 1 wherein said step of identifying includes
identifying a nucleotide in said protruding strand of said polynucleotide
by extending a strand of said polynucleotide or said probe with a nucleic
acid polymerase.
24. The method of claim 23 wherein said step of identifying further
includes extending a strand of said polynucleotide in the presence of
chain-terminating nucleoside triphosphates.
25. The method of claim 24 wherein said chain-terminating nucleoside
triphosphates are labeled.
26. The method of claim 25 wherein said chain-terminating nucleoside
triphosphates are labeled with fluorescent dyes.
27. The method of claim 26 wherein said fluorescent dyes have spectrally
resolvable fluorescence emission bands.
28. The method of claim 1 wherein said polynucleotide has a protruding
strand at one end and is attached to a solid phase support by another end
and wherein said probe has a protruding strand at one end, the protruding
strand of said probe being complementary to the protruding strand at one
end of said polynucleotide.
29. The method of claim 28 wherein said step of ligating includes treating
said polynucleotide and said probe with a ligase.
30. The method of claim 29 wherein said nuclease is a type IIs restriction
endonuclease.
31. The method of claim 30 wherein said step of ligating includes providing
said probe as a mixture such that said complementary protruding strand of
said probe includes every possible sequence of nucleotides the length of
said protruding strand.
32. The method of claim 31 further including the step of removing unligated
probe from said polynucleotide after said step of ligating.
33. The method of claim 32 wherein said step of identifying includes
identifying a nucleotide in said protruding strand of said polynucleotide
by extending a strand of said polynucleotide with a nucleic acid
polymerase in the presence of chain-terminating nucleoside triphosphates.
34. A method for determining a nucleotide sequence of a polynucleotide, the
method comprising the steps of:
(a) ligating a probe to an end of a polynucleotide having a protruding
strand to form a ligated complex, the probe having an end with a
complementary protruding strand to that of the polynucleotide and the
probe having a nuclease recognition site of a nuclease whose cleavage site
is separate from its recognition site;
(b) cleaving the ligated complex with a nuclease, the nuclease recognizing
the recognition site and cleaving the ligated complex such that an
augmented probe is released leaving a protruding strand on the
polynucleotide;
(c) identifying one or more nucleotides in the protruding strand of the
polynucleotide by the identity of the probe ligated thereto or by
extending a strand of the polynucleotide or probe in the presence of
nucleoside triphosphates; and
(d) repeating steps (a) through (c) until the nucleotide sequence of the
polynucleotide is determined.
35. The method of claim 34 wherein said nuclease is a type IIs restriction
endonuclease and wherein said polynucleotide is provided with recognition
sites of said nuclease blocked.
36. The method of claim 35 wherein said recognition sites of said
polynucleotide are blocked with a methylase.
37. The method of claim 35 wherein said step of ligating includes treating
said polynucleotide with a ligase.
38. The method of claim 37 wherein said protruding strand of said
polynucleotide has a 5'-phosphoryl group and wherein said complementary
protruding strand of said probe lacks a 5'-phosphoryl group.
39. The method of claim 38 wherein said step of ligating includes treating
said polynucleotide and said probe in succession with (i) a ligase to
ligate said protruding strand having said 5'-phosphoryl group to said
probe, (ii) a kinase to phosphorylate said complementary protruding strand
of said probe, and (iii) a ligase to ligate said complementary protruding
strand of said probe to said polynucleotide.
40. The method of claim 39 wherein said step of identifying includes
identifying a nucleotide in said protruding strand of said polynucleotide
by the identity of said probe ligated thereto.
41. The method of claim 37 wherein said polynucleotide is attached to a
solid phase support.
42. The method of claim 41 wherein said step of ligating includes providing
said probe as a mixture such that said complementary protruding strand of
said probe includes every possible sequence of nucleotides the length of
said protruding strand.
43. The method of claim 42 further including the step of removing unligated
probe from said polynucleotide after said step of ligating.
44. The method of claim 43 wherein said step of identifying includes
identifying a nucleotide in said protruding strand of said polynucleotide
by the identity of said probe ligated thereto.
45. The method of claim 43 wherein said step of identifying includes
identifying a nucleotide in said protruding strand of said polynucleotide
by extending a strand of said polynucleotide or said probe with a nucleic
acid polymerase in the presence of chain-terminating nucleoside
triphosphates.
46. The method of claim 43 wherein said solid phase support is a
microparticle.
47. The method of claim 46 wherein said type IIs restriction endonuclease
is Fok I.
48. A method for determining a nucleotide sequence of a polynucleotide, the
method comprising the steps of:
(a) providing a polynucleotide in double stranded form such that the
polynucleotide is attached to a solid phase support and has a protruding
strand at one end;
(b) ligating a probe to the protruding strand of the polynucleotide to form
a ligated complex, the probe having an end with a complementary protruding
strand to that of the polynucleotide and the probe having a type IIs
endonuclease recognition site;
(c) identifying a nucleotide in the protruding strand of the polynucleotide
by the identity of the ligated probe;
(d) cleaving the ligated complex with a type IIs endonuclease that
recognizes the type IIs endonuclease recognition site and cleaves the
ligated complex to give an augmented probe and a new protruding strand on
the polynucleotide; and
(e) repeating steps (a) through (d) until the nucleotide sequence of the
polynucleotide is determined.
49. The method of claim 48 wherein said probe comprises a first single
stranded oligonucleotide and a second single stranded oligonucleotide, the
first single stranded oligonucleotide having an end with complementary
nucleotides to those in said protruding strand of said polynucleotide and
the second single stranded oligonucleotide being complementary to a
portion of the first single stranded oligonucleotide such that the first
and second single stranded oligonucleotides are capable of forming a
duplex containing a type IIs endonuclease recognition site, and wherein
said step of ligating includes (i) annealing the first single stranded
oligonucleotide to said protruding strand of said polynucleotide under
conditions that promote the formation of a perfectly matched duplex
therebetween, (ii) ligating the first single stranded oligonucleotide to
said polynucleotide, (iii) annealing the second single stranded
oligonucleotide to the first single stranded oligonucleotide, and (iv)
ligating the second single stranded oligonucleotide to said
polynucleotide.
50. The method of claim 49 wherein said step of ligating includes providing
said first single stranded oligonucleotide as a mixture such that said
complementary nucleotides in said end of said first single stranded
oligonucleotide includes every possible sequence of nucleotides the length
of said end.
51. The method of claim 50 further including the step of removing unligated
said first and second single stranded oligonucleotides from said
polynucleotide after said step of ligating.
52. The method of claim 51 wherein said probe comprises four components,
each component being capable of indicating the presence of a different
nucleotide in said protruding strand of said polynucleotide upon ligation.
53. The method of claim 52 wherein each of said components of said probe is
labeled with a different fluorescent dye and the different fluorescent
dyes are spectrally resolvable.
54. A method for determining a nucleotide sequence of a polynucleotide, the
method comprising the steps of:
(a) providing a polynucleotide in double stranded form such that the
polynucleotide is attached to a solid phase support and has a protruding
strand and a recessed strand at one end;
(b) identifying a nucleotide in the protruding strand of the polynucleotide
by extending the recessed strand with a nucleic acid polymerase;
(c) ligating a probe to the one end of the polynucleotide, the probe having
a type IIs restriction endonuclease recognition site;
(d) cleaving the polynucleotide with a type IIs restriction endonuclease
that recognizes the type IIs endonuclease recognition site leaving a new
protruding strand on the polynucleotide; and
(e) repeating steps (a) through (d) until the nucleotide sequence of the
polynucleotide is determined.
55. The method of claim 54 wherein said nucleic acid polymerase extends
said recessed strand in the presence of chain-terminating nucleoside
triphosphates.
56. The method of claim 55 further including the step of removing unligated
probe from said polynucleotide after said step of ligating.
57. The method of claim 56 further including a step of blocking recognition
sites of said type IIs restriction endonuclease on said polynucleotide.
58. The method of claim 57 wherein said recognition sites of said
polynucleotide are blocked with a methylase.
59. The method of claim 57 wherein said probe has a 5' protruding strand
one nucleotide less in length than said protruding strand of said
polynucleotide and wherein said step of ligating includes providing said
probe as a mixture such that the protruding strand of the probe includes
every possible sequence of nucleotides the length of the protruding
strand.
60. The method of claim 59 wherein said chain-terminating nucleoside
triphosphate is a labeled dideoxynucleoside triphosphate and wherein said
step of identifying includes identifying said one or more nucleotides by
the label on the labeled dideoxynucleoside triphosphate incorporated into
said recessed strand of said polynucleotide.
61. The method of claim 60 further including the steps of excising said
labeled dideoxynucleotide and extending said recessed strand with a
nucleic acid polymerase.
62. The method of claim 61 wherein said step of excising is carded out with
T4 DNA polymerase in the presence of deoxyribonucleoside triphosphates.
63. A method for determining a nucleotide sequence of a population of
identical polynucleotdes, the method comprising the steps of:
(a) providing a first probe having a first nuclease recognition site of a
first nuclease whose cleavage site is separate from its recognition site,
the first nuclease having a reach;
(b) providing a second probe having a second nuclease recognition site of a
second nuclease whose cleavage site is separate from its recognition site,
the second nuclease having a reach;
(c) providing a conversion probe for converting a polynucleotide of the
population from one cleavable by the first nuclease to one cleavable by
the second nuclease;
(d) ligating a mixture of the first probe and the conversion probe to the
polynucleotides of the population to form a subpopulation of ligated
complexes comprising the conversion probe;
(e) cleaving the subpopulation of ligated complexes comprising the
conversion probe with the second nuclease and determining a portion of the
nucleotide sequence of the resulting polynucleotides by:
i) ligating the second probe to an end of the polynucleotides of the
subpopulation;
ii) identifying one or more nucleotides at the end of the polynucleotides
of the subpopulation by the identity of the probe ligated thereto or by
extending a strand of the polynucleotide or the second probe;
iii) cleaving the polynucleotides of the subpopulation with the second
nuclease such that the polynucleotides of the subpopulation are shortened
by one or more nucleotides;
iv) repeating steps i)-iii) until the number of nucleotides cleaved from
the polynucleotides of the subpopulation is equal to or greater than the
reach of the first nuclease;
v) capping the ends of the polynucleotides of the subpopulation;
(f) cleaving the polynucleotides with the first nuclease; and
(g) repeating steps (d)-(f) until the nucleotide sequence of the population
of polynucleotides is determined.
64. The method of claim 63 wherein said polynucleotides of said population
have protruding strands at one end and are attached to a solid phase
support by another end and wherein said first probe, said second probe,
and said conversion probe have protruding strands at one of their
respective ends, such that the protruding strands of said first probe,
said second probe, and said conversion probe are complementary to the
protruding strands of the polynucleotides to which they are ligated.
65. The method of claim 64 wherein said first and second nucleases are type
IIs restriction endonucleases, and wherein said method further includes a
step of blocking recognition sites of said first and second nucleases on
said polynucleotides by treating said polynucleotides with one or more
methylases.
66. The method of claim 65 wherein said steps of ligating include providing
said first probe, said second probe, and said conversion probe as mixtures
such that their respective complementary protruding strands include every
possible sequence of nucleotides the length of their respective protruding
strands.
67. The method of claim 66 wherein said steps of ligating include treating
with a ligase.
68. A method for determining a nucleotide sequence of a population of
identical polynucleotdes, the method comprising the steps of:
(a) ligating a mixture comprising a first probe and a conversion probe to
the ends of the population of polynucleotides so that a subpopulation of
ligated complexes comprising conversion probes is formed, the first probe
containing a nuclease recognition site of a first nuclease whose cleavage
site is separate from its recognition site, the first nuclease having a
reach, and the conversion probe containing a nuclease recognition site of
a second nuclease whose cleavage site is separate from its recognition
site, the second nuclease having a reach;
(b) cleaving the subpopulation of ligated complexes comprising the
conversion probe with the second nuclease and determining a portion of the
nucleotide sequence of the polynucleotides by applying the method of any
of claims 1 through 57 until the polynucleotides of the subpopulation are
shortened by a number of nucleotides equal to of greater than the reach of
the first nuclease;
(c) capping the polynucleotides of the subpopulation;
(d) cleaving the polynucleotides with the first nuclease;
(e) repeating steps (a)-(d) until the nucleotide sequence of the population
of polynucleotides is determined. |
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Claims |
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Description |
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FIELD OF THE INVENTION
The invention relates generally to methods for determining the nucleotide
sequence of a polynucleotide, and more particularly, to a method of
step-wise removal and identification of terminal nucleotides of a
polynucleotide.
BACKGROUND
Analysis of polynucleotides with currently available techniques provides a
spectrum of information ranging from the confirmation that a test
polynucleotide is the same or different than a standard sequence or an
isolated fragment to the express identification and ordering of each
nucleoside of the test polynucleotide. Not only are such techniques
crucial for understanding the function and control of genes and for
applying many of the basic techniques of molecular biology, but they have
also become increasingly important as tools in genomic analysis and a
great many non-research applications, such as genetic identification,
forensic analysis, genetic counseling, medical diagnostics, and the like.
In these latter applications both techniques providing partial sequence
information, such as fingerprinting and sequence comparisons, and
techniques providing full sequence determination have been employed, e.g.
Gibbs et al, Proc. Natl. Acad. Sci., 86: 1919-1923 (1989); Gyllensten et
al, Proc. Natl. Acad. Sci, 85: 7652-7656 (1988); Carrano et al, Genomics,
4: 129-136 (1989); Caetano-Anolles et al, Mol. Gen. Genet., 235: 157-165
(1992); Brenner and Livak, Proc. Natl. Acad. Sci., 86: 8902-8966 (1989);
Green et al, PCR Methods and Applications, 1: 77-90 (1991); and Versalovic
et al, Nucleic Acids Research, 19: 6823-6831 (1991).
Native DNA consists of two linear polymers, or strands of nucleotides. Each
strand is a chain of nucleosides linked by phosphodiester bonds. The two
strands are held together in an antiparallel orientation by hydrogen bonds
between complementary bases of the nucleotides of the two strands:
deoxyadenosine (A) pairs with thymidine (T) and deoxyguanosine (G) pairs
with deoxycytidine (C).
Presently there are two basic approaches to DNA sequence determination: the
dideoxy chain termination method, e.g. Sanger et al, Proc. Natl. Acad.
Sci., 74: 5463-5467 (1977); and the chemical degradation method, e.g.
Maxam et al, Proc. Natl. Acad. Sci., 74: 560-564 (1977). The chain
termination method has been improved in several ways, and serves as the
basis for all currently available automated DNA sequencing machines, e.g.
Sanger et al, J. Mol. Biol., 143: 161-178 (1980); Schreier et al, J. Mol.
Biol., 129: 169-172 (1979); Smith et al, Nucleic Acids Research, 13:
2399-2412 (1985); Smith et al, Nature, 321: 674-679 (1987); Prober et al,
Science, 238: 336-341 (1987); Section II, Meth. Enzymol., 155: 51-334
(1987); Church et al, Science, 240: 185-188 (1988); Hunkapiller et al,
Science, 254: 59-67 (1991); Bevan et al, PCR Methods and Applications, 1:
222-228 (1992).
Both the chain termination and chemical degradation methods require the
generation of one or more sets of labeled DNA fragments, each having a
common origin and each terminating with a known base. The set or sets of
fragments must then be separated by size to obtain sequence information.
In both methods, the DNA fragments are separated by high resolution gel
electrophoresis, which must have the capacity of distinguishing very large
fragments differing in size by no more than a single nucleotide.
Unfortunately, this step severely limits the size of the DNA chain that
can be sequenced at one time. Sequencing using these techniques can
reliably accommodate a DNA chain of up to about 400-450 nucleotides,
Bankier et al, Meth. Enzymol., 155: 51-93 (1987); and Hawkins et al,
Electrophoresis, 13: 552-559 (1992).
Several significant technical problems have seriously impeded the
application of such techniques to the sequencing of long target
polynucleotides, e.g. in excess of 500-600 nucleotides, or to the
sequencing of high volumes of many target polynucleotides. Such problems
include i) the gel electrophoretic separation step which is labor
intensive, is difficult to automate, and introduces an extra degree of
variability in the analysis of data, e.g. band broadening due to
temperature effects, compressions due to secondary structure in the DNA
sequencing fragments, inhomogeneities in the separation gel, and the like;
ii) nucleic acid polymerases whose properties, such as processivity,
fidelity, rate of polymerization, rate of incorporation of chain
terminators, and the like, are often sequence dependent; iii) detection
and analysis of DNA sequencing fragments which are typically present in
fmol quantities in spatially overlapping bands in a gel; iv) lower signals
because the labeling moiety is distributed over the many hundred spatially
separated bands rather than being concentrated in a single homogeneous
phase, and v) in the case of single-lane fluorescence detection, the
availability of dyes with suitable emission and absorption properties,
quantum yield, and spectral resolvability, e.g. Trainor, Anal. Biochem.,
62: 418-426 (1990); Connell et al, Biotechniques, 5: 342-348 (1987);
Karger et al, Nucleic Acids Research, 19: 4955-4962 (1991); Fung et al,
U.S. Pat. No. 4,855,225; and Nishikawa et al, Electrophoresis, 12: 623-631
(1991).
Another problem exists with current technology in the area of diagnostic
sequencing. An ever widening array of disorders, susceptibilities to
disorders, prognoses of disease conditions, and the like, have been
correlated with the presence of particular DNA sequences, or the degree of
variation (or mutation) in DNA sequences, at one or more genetic loci.
Examples of such phenomena include human leukocyte antigen (HLA) typing,
cystic fibrosis, tumor progression and heterogeneity, p53 proto-oncogene
mutations, ras proto-oncogene mutations, and the like, e.g. Gyllensten et
al, PCR Methods and Applications, 1: 91-98 (1991); Santamaria et al,
International application PCT/US92/01675; Tsui et al, International
application PCT/CA90/00267; and the like. A difficulty in determining DNA
sequences associated with such conditions to obtain diagnostic or
prognostic information is the frequent presence of multiple subpopulations
of DNA, e.g. allelic variants, multiple mutant forms, and the like.
Distinguishing the presence and identity of multiple sequences with
current sequencing technology is virtually impossible, without additional
work to isolate and perhaps clone the separate species of DNA.
A major advance in sequencing technology could be made if an alternative
approach was available for sequencing DNA that did not required high
resolution separations, provided signals more amenable to analysis, and
provided a means for readily analyzing DNA from heterozygous genetic loci.
SUMMARY OF THE INVENTION
The invention provides a method of nucleic acid sequence analysis based on
ligation and cleavage of probes at the terminus of a target
polynucleotide. Preferably, repeated cycles of such ligation and cleavage
are implemented in the method, and in each such cycle a nucleotide is
identified at the end of the target polynucleotide and the target
polynucleotide is shortened, such that further cycles of ligation,
cleavage, and identification can take place. That is, preferably, in each
cycle the target sequence is shortened by a single nucleotide and the
cycles are repeated until the nucleotide sequence of the target
polynucleotide is determined.
An important feature of the invention is the probe employed in the ligation
and cleavage events. A probe of the invention is a double stranded
polynucleotide which (i) contains a recognition site for a nuclease, and
(ii) preferably has a protruding strand capable of forming a duplex with a
complementary protruding strand of the target polynucleotide. At each
cycle in the latter embodiment, only those probes whose protruding strands
form perfectly matched duplexes with the protruding strand of the target
polynucleotide are ligated to the end of the target polynucleotide to form
a ligated complex. After removal of the unligated probe, a nuclease
recognizing the probe cuts the ligated complex at a site one or more
nucleotides from the ligation site along the target polynucleotide leaving
an end, usually a protruding strand, capable of participating in the next
cycle of ligation and cleavage. An important feature of the nuclease is
that its recognition site be separate from its cleavage site. As is
described more fully below, in the course of such cycles of ligation and
cleavage, the terminal nucleotides of the target polynucleotide are
identified.
In one aspect of the invention, more than one nucleotide at the terminus of
a target polynucleotide can be identified and/or cleaved during each cycle
of the method.
Generally, the method of the invention comprises the following steps: (a)
ligating a probe to an end of the polynucleotide, the probe having a
nuclease recognition site; (b) identifying one or more nucleotides at the
end of the polynucleotide; (c) cleaving the polynucleotide with a nuclease
recognizing the nuclease recognition site of the probe such that the
polynucleotide is shortened by one or more nucleotides; and (d) repeating
steps (a) through (c) until the nucleotide sequence of the polynucleotide
is determined. As is described more fully below, the order of steps (a)
through (c) may vary with different embodiments of the invention. For
example, identifying the one or more nucleotides can be carried out either
before or after cleavage of the ligated complex from the target
polynucleotide. Likewise, ligating a probe to the end of the
polynucleotide may follow the step of identifying in some preferred
embodiments of the invention. Preferably, the method further includes a
step of removing the unligated probe after the step of ligating.
Preferably, whenever natural protein endonucleases are employed as the
nuclease, the method further includes a step of methylating the target
polynucleotide at the start of a sequencing operation to prevent spurious
cleavages at internal recognition sites fortuitously located in the target
polynucleotide.
The present invention overcomes many of the deficiencies inherent to
current methods of DNA sequencing: there is no requirement for the
electrophoretic separation of closely-sized DNA fragments; no
difficult-to-automate gel-based separations are required; no polymerases
are required for generating nested sets of DNA sequencing fragments;
detection and analysis are greatly simplified because signal-to-noise
ratios are much more favorable on a nucleotide-by-nucleotide basis,
permitting smaller sample sizes to be employed; and for fluorescent-based
detection schemes, analysis is further simplified because fluorophores
labeling different nucleotides may be separately detected in homogeneous
solutions rather than in spatially overlapping bands.
The present invention is readily automated, both for small-scale serial
operation and for large-scale parallel operation, wherein many target
polynucleotides or many segments of a single target polynucleotide are
sequenced simultaneously. Unlike present sequencing approaches, the
progressive nature of the method--that is, determination of a sequence
nucleotide-by-nucleotide--permits one to monitor the progress of the
sequencing operation in real time which, in turn, permits the operation to
be curtailed, or re-started, if difficulties arise, thereby leading to
significant savings in time and reagent usage. Also unlike current
approaches, the method permits the simultaneous determination of allelic
forms of a target polynucleotide: As described more fully below, if a
population of target polynucleotides consists of several subpopulations of
distinct sequences, e.g. polynucleotides from a heterozygous genetic
locus, then the method can identify the proportion of each nucleotide at
each position in the sequence.
Generally, the method of the invention is applicable to all tasks where DNA
sequencing is employed, including medical diagnostics, genetic mapping,
genetic identification, forensic analysis, molecular biology research, and
the like.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1a illustrates a preferred structure of a labeled probe of the
invention.
FIG. 1b illustrates a probe and terminus of a target polynucleotide wherein
a separate labeling step is employed to identify one or more nucleotides
in the protruding strand of a target polynucleotide.
FIG. 1c illustrates steps of an embodiment wherein a nucleotide of the
target polynucleotide is identified by extension with a polymerase in the
presence of labeled dideoxynucleoside triphosphates followed by their
excision, strand extension, and strand displacement.
FIG. 1d diagrammatically illustrates an embodiment in which nucleotide
identification is carried out by polymerase extension of a probe strand in
the presence of labeled chain-terminating nucleoside triphosphates.
FIG. 1e diagrammatically illustrates an embodiment in which nucleotide
identification is carr | | |
