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Claims  |
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What is claimed is:
1. A method for imaging a single labeled nucleotide on an individual double
stranded nucleic acid molecule, comprising:
(a) nicking a double stranded nucleic acid molecule elongated and fixed
onto a surface so that the double stranded nucleic acid molecule remains
accessible for enzymatic reactions with enzymes for the addition of a
labeled nucleotide creating a nicked strand;
(b) enzymatically adding a single nucleotide comprising a label; and
(c) imaging the added label.
2. The method of claim 1, in which the step of nicking the double stranded
nucleic acid molecule is performed by the enzyme DNase.
3. The method of claim 1, in which the surface is a planar surface.
4. The method of claim 1, in which the step of adding nucleotides
comprising a label is performed by a polymerase.
5. The method of claim 4, in which the polymerase is DNA Polymerase I, the
Klenow fragment of DNA Polymerase I lacking the 5'-3' exonuclease
activity, T7 Sequenase v. 2.0 or Taq polymerase.
6. The method of claim 1, in which the step of imaging the label is
performed using a camera and a microscope.
7. The method of claim 6, in which the step of imaging the label further
comprises using laser illumination.
8. The method of claim 1, in which the step of imaging further comprises
using a computer.
9. The method of claim 1, further comprising analyzing the image using a
mathematical algorithm.
10. The method of claim 9, in which the mathematical algorithm is a
Bayesian estimation method.
11. The method of claim 1, further comprising the steps:
(a) modifying the label after imaging the label in order to visualize the
subsequently added labeled nucleotides; and
(b) repeating the steps of enzymatically adding a nucleotide comprising a
label, imaging the label, and modifying the label to image multiple,
consecutively added nucleotides.
12. The method of claim 11, further comprising enzymatically displacing the
nicked strand of the nucleic acid molecule.
13. The method of claim 12, in which enzymatically displacing the nicked
strand is performed using the Klenow fragment of DNA Polymerase I.
14. The method of claim 11, further comprising enzymatically opening the
nicked sites on the double stranded nucleic acid molecule.
15. The method of claim 14, in which the step of opening the nicked sites
is performed by an enzyme having 5'-3' exonuclease activity.
16. The method of claim 15, in which the enzyme having 5'-31 exonuclease
activity is DNA Polymerase I or T7 exonuclease.
17. The method of claim 11, in which the label is photolabile.
18. The method of claim 11, in which the step of modifying the label after
imaging is performed by photobleaching or photolysis.
19. A method for determining the nucleotide sequence of an individual
double stranded nucleic acid molecule, comprising:
(a) nicking a double stranded nucleic acid molecule elongated and fixed
onto a surface so that the double stranded nucleic acid molecule remains
accessible for enzymatic reactions with enzymes for the addition of
labeled nucleotides forming a 3' terminus to create an extended strand;
(b) extending the strand by adding at least one nucleotide comprising a
label to the 3' terminus of the nicked site; and
(c) imaging the added label.
20. The method of claim 19, in which the step of nicking the double
stranded nucleic acid molecule is performed by the enzyme DNase.
21. The method of claim 19, further comprising enzymatically displacing the
nicked strand of the nucleic acid molecule.
22. The method of claim 21, in which enzymatically displacing the nicked
strand is performed using the Klenow fragment of DNA Polymerase I.
23. The method of claim 19, further comprising opening the nicked sites on
the double stranded nucleic acid molecule.
24. The method of claim 23, in which the step of opening the nicked sites
on the double stranded nucleic acid molecule is performed by an enzyme
having 5'-3' exonuclease activity.
25. The method of claim 24, in which the enzyme having 5'-3' exonuclease
activity is DNA Polymerase I or T7 exonuclease.
26. The method of claim 19, in which the step of extending the strand by
adding a nucleotide comprising a label is performed by a polymerase.
27. The method of claim 26, in which the polymerase is DNA Polymerase I,
the Klenow fragment of DNA Polymerase I lacking the 5'-3' exonuclease
activity, T7 Sequenase v. 2.0, or a Taq polymerase.
28. The method of claim 23, in which the step of opening the nicked sites
on the double stranded nucleic acid molecule and extending the strand by
adding a nucleotide comprising a label is performed by T7 exonuclease gene
6 and T7 Sequenase v. 2.0, respectively.
29. The method of claim 19, in which the step of imaging the label is
performed using a camera and a microscope.
30. The method of claim 29, in which the step of imaging the label further
comprises using laser illumination.
31. The method of claim 19, in which the label is photolabile.
32. The method of claim 19, further comprising modifying the label after
imaging in order to visualize subsequently added labels.
33. The method of claim 3, in which the step of modifying the label after
imaging is performed by photobleaching or photolysis.
34. The method of claim 19, in which the nucleotides comprise a mix of
labeled and unlabeled nucleotides.
35. A system for determining the nucleotide sequence of an individual
double stranded nucleic acid molecule, comprising:
(a) the double stranded nucleic acid molecule elongated and fixed onto a
surface so that the nucleic acid molecule remains accessible for enzymatic
reactions and/or hybridization reactions;
(b) a polymerase fixed on the surface;
(c) nucleotides comprising a label fixed on the surface; and
(d) a device for imaging the label to produce an image.
36. The system of claim 35, in which the polymerase is DNA Polymerase I,
the Klenow fragment of DNA Polymerase I without the 5'-3' exonuclease
activity T7 Sequenase v. 2.0, or Taq polymerase.
37. The system of claim 35, further comprising a nucleic acid nicking
enzyme.
38. The system of claim 36, in which the nicking enzyme is a DNase.
39. The system of claim 35, in which the label is a fluorescent label.
40. The system of claim 35, further comprising a nick opening enzyme fixed
on the surface.
41. The system of claim 40, in which the nick opening enzyme is T7
exonuclease gene 6, DNA Polymerase I, the Klenow fragment of DNA
Polymerase I or a 5'-3' exonuclease.
42. The system of claim 35, in which the device for imaging comprises a
fluorescence microscope, a camera and a source of illumination.
43. The system of claim 35, in which the source of illumination is a laser.
44. The system of claim 35, in which the device for imaging the label
processes the image using Bayesian estimation, comprising:
(a) accumulating signals of an addition site of the image;
(b) filtering the signals according to fluorescence intensity;
(c) correlating the signals with the backbone of the nucleic acid molecule;
(d) tabulating addition sites of the image using Bayesian inference
estimation of the signals; and
(e) aligning and assembling the addition sites to determine a nucleotide
addition.
45. A method of determining the nucleotide sequence of an individual
nucleic acid molecule, comprising:
(a) exposing a nucleic acid molecule annealed with at least one primer
elongated and fixed onto a surface so that the nucleic acid molecule
remains accessible for enzymatic reactions with enzymes for the addition
of labeled nucleotides to a polymerase and dideoxy nucleotides comprising
a base and a label; and
(b) imaging the labeled nucleotides added onto the primer;
to determine the nucleotide sequence of the nucleic acid molecule by the
addition of the labeled dideoxy nucleotide.
46. A method of determining the nucleotide sequence of an individual
nucleic acid molecule, comprising:
(a) exposing a nucleic acid molecule annealed with at least one primer
elongated and fixed onto a surface so that the nucleic acid molecule
remains accessible for enzymatic reactions with enzymes for the addition
of a labeled nucleotide to a polymerase and nucleotides comprising a base
and a label; and
(b) imaging the labeled nucleotide added onto the primers;
to determine the nucleotide sequence of the nucleic acid molecule by the
addition of the labeled dideoxy nucleotide.
47. A method of determining the nucleotide sequence of an
individual-nucleic acid molecule, comprising:
(a) exposing a nucleic acid molecule annealed with at least one primer
elongated and fixed onto a surface so that the nucleic acid molecule
remains accessible for enzymatic reactions with enzymes for the addition
of labeled nucleotides to a polymerase and dideoxy nucleotides;
(b) exposing the nucleic acid molecule annealed with at least one primer to
a polymerase and nucleotides including nucleotides comprising a label to
produce a labeled primer extension nucleic acid molecule; and
(c) imaging the labeled primer extension nucleic acid molecule to produce
an image;
to determine the nucleotide sequence of the nucleic acid molecule by the
absence of a primer extension product corresponding to the dideoxy
nucleotides used in step (a).
48. A method of determining the nucleotide sequence of an individual
nucleic acid molecule, comprising:
(a) elongating and fixing the nucleic acid molecule onto a surface so that
the nucleic acid molecule remains accessible for enzymatic reactions with
enzymes for the addition of labeled nucleotides;
(b) annealing at least one primer to the elongated and fixed nucleic acid
molecule;
(c) exposing the nucleic acid molecule annealed with a primer to a
polymerase and dideoxy nucleotides;
(d) exposing the nucleic acid molecule to a polymerase and nucleotides
including nucleotides comprising a label to produce at least one labeled
primer extension nucleic acid molecule; and
(e) imaging the labeled primer extension nucleic acid molecule to produce
an image;
to determine the nucleotide sequence of the nucleic acid molecule by the
absence of a primer extension product corresponding to the dideoxy
nucleotides used in step (c).
49. A system for determining the nucleotide sequence of an individual
nucleic acid molecule, comprising:
(a) an elongated and fixed nucleic acid molecule on a surface so that the
nucleic acid molecules remain accessible for enzymatic reactions with
enzymes for the addition of labeled nucleotides;
(b) at least one primer annealed to the nucleic acid molecule;
(c) a polymerase enzyme fixed on the surface to produce a primer extension
product;
(d) dideoxy nucleotides fixed on the surface;
(e) nucleotides comprising a label fixed on the surface; and
(f) a device for imaging the elongated and fixed nucleic acid molecule to
detect the presence of labeled nucleotides in the primer extension product
to produce an image;
whereby the absence of the image of the primer extension product for a
particular dideoxy nucleotide corresponds to the nucleotide sequence at
one position of the nucleic acid molecule.
50. A system for determining a single nucleotide polymorphism in a
population of nucleic acid molecules, comprising the system of claim 47,
in which the nucleic acid molecules are elongated and fixed onto at least
four surfaces and the surfaces are individually exposed to
dideoxynucleotides comprising different bases.
51. A method for imaging multiple labeled nucleotides on an individual
double stranded nucleic acid molecule, comprising:
(a) nicking a nucleic acid molecule elongated and fixed onto a surface so
that the double stranded nucleic acid molecule remains accessible for
enzymatic reactions with enzymes for the addition of labeled nucleotides;
(b) enzymatically adding multiple nucleotides comprising at least four
bases and at least four labels; and (c) simultaneously imaging the added
labels. |
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Claims |
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Description |
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INTRODUCTION
The present invention relates to methods and systems for determining the
nucleotide sequence of individual nucleic acid molecules using optical
techniques, referred to herein as "single molecule optical sequencing."
The present invention also relates to methods for imaging single or
multiple labeled nucleotides added onto an individual double stranded
nucleic acid molecule mounted on a solid surface. Bayesian inference
estimation methods are utilized to analyze a population of images and to
produce statistically accurate nucleotide sequences.
The present invention also relates to methods and systems for determining
single nucleotide polymorphisms in a population of individual double
stranded nucleic acid molecules.
BACKGROUND
The analysis of nucleic acid molecules at the genome level is an extremely
complex endeavor which requires accurate, rapid characterization of large
numbers of often very large nucleic acid molecules via high throughput DNA
mapping and sequencing. The construction of physical maps, and ultimately
of nucleotide sequences, for eukaryotic chromosomes currently remains
laborious and difficult. This is due, in part, to the fact that current
procedures for mapping and sequencing DNA were originally designed to
analyze nucleic acids at the gene, rather than at the genome, level
(Chumakov, et al., 1992, Nature 359:380; Maier, et al., 1992, Nat. Genet.
1:273).
DNA Sequencing
Approaches to DNA sequencing have varied widely, and have made it possible
to sequence entire genomes, including portions of the human genome. The
most commonly used method has been the dideoxy chain termination method of
Sanger (1977, Proc. Natl. Acad. Sci. USA 74:5463). However, this method is
time-consuming, labor-intensive and expensive, requiring the analysis of
four sets of radioactively labeled DNA fragments resolved by gel
electrophoresis to determine the DNA sequence.
To overcome some of these deficiencies, automated DNA sequencing systems
were developed which used four fluorescently labeled dideoxy nucleotides
to label DNA (Smith et al., 1985, Nucleic Acids Res. 13:2399-2412; Smith
et al., 1986, Nature 321:674; Prober et al., 1987, Science 238:336-341,
which are incorporated herein by reference). Automated slab gel
electrophoresis systems enable large-scale sequence acquisition (Roach et
al., 1995, Genomics 26:345-353; Venter et al., 1996, Nature 381:364-366;
Profer et al., 1987, Science 238:336-341; Lake et al., 1996, Science
273:1058; Strathmann et al., 1991, Proc. Natl. Acad. Sci. USA
88:1247-1250; and the complete genomic sequence of Saccharomyces
cerevisiae in the Stanford database). Current large-scale sequencing is
largely the domain of centers where costly and complex support systems are
essential for the production efforts. Efforts to deal with sequence
acquisition from a large population (usually less than 1,000) is limited
to relatively small numbers of loci (Davies et al., 1995, Nature
371:130-136). However, these methods are still dependent on Sanger
sequencing reactions and gel electrophoresis to generate ladders and
robotic sample handling procedures to deal with the attending numbers of
clones and polymerase chain reacting products.
Some recently developed methods and devices for automated sequencing of
bulk DNA samples that utilize fluorescently labeled nucleotides are
described in U.S. Pat. No. 5,674,743; International Application Nos.
PCT/GB93/00848 published Apr. 22, 1993 as WO 93/21340; PCT/US96/08633
published Jun. 4, 1996 as WO 96/39417; and PCT/US94/01156 published Jan.
31, 1994 as WO 94/18218. None of the recently developed methods is capable
of sequencing individual nucleic acid molecules.
Techniques for sequencing large genomes of DNA have relied upon the
construction of Yeast Artificial Chromosomes ("YAC") contiguous sequences.
Preliminary physical maps of a large fraction of the human genome have
been generated via YACs (Cohen et al., 1993, Nature 366:698-701). However,
extensive high resolution maps of YACs have not been widely generated, due
to the high frequency of rearrangement/chimerism among YACs, the low
complexity of fingerprints generated by hybridization approaches, and the
extensive labor required to overcome these problems. Ordered maps of YACs
have been optically made by using a spermine condensation method (to avoid
shearing the DNA) and fixing the clones in molten agarose onto derivatized
glass surfaces (Cai et al., 1995, Natl. Acad. Sci. USA 92:5164-5168).
There have been several proposals for the rapid attainment of sequence data
from clones that minimize or obviate the need for shotgun sequencing
approaches or subcloning of large insert clones (Smith et al., 1994,
Nature Genet. 7:40-47; Kupfer et al., 1995, Genomics 27:90-100; Chen et
al., 1993, Genomics 17:651-656 and Roach et al., 1995, Genomics
26:345-353). Several of these approaches advocate the generation of
"sequence sampled maps" (Smith et al., 1994, Nature Genet. 7:40-47 and
Venter et al., 1996, Nature 381:364-366) which require fingerprinting of
clones, or large numbers of subclones, to achieve good target coverage
while simultaneously generating a fine-scale map.
A recent development has been the proposal of DNA sequencing of aligned and
oriented Bacterial Artificial Chromosomes ("BAC") contiguous sequences
(Venter et al., 1996, Nature 381:364-366); (see also Smith et al., 1994,
Nature Genetics 7:40-47; Kupfer et al., 1995, Genomics 27:90-100; and Chen
et al., 1993, Genomics 17:651-656). BACs offer the advantage of
considerably greater stability than YACs, are more easily physically
managed due to their smaller size (.about.500 kb to 2 Mb versus .about.100
to 200 kb, respectively), and are more compatible with automated DNA
purification procedures (Kim et al., 1996, Proc. Natl. Acad. Sci. USA
93:6297-6301; Kim et al., 1994, Genomics 24:527-534; and Schmitt et al.,
1996, Genomics 33:9-20). Further approaches for the optical analysis of
BAC clones were also developed (Cai et al., 1998, Proc. Natl. Acad. Sci.
USA 95:3390-3395).
Limitations of these approaches described above include low throughput, DNA
fragmentation (preventing subsequent or simultaneous multimethod
analyses), and difficulties in automation. Despite the potential utilities
of these and other approaches, it is increasingly clear that current
molecular approaches were developed primarily for characterization of
single genes, not entire genomes, and are, therefore, not optimally suited
to the analysis of polygenic diseases and complex traits, especially on a
population-wide basis (Risch et al., 1996, Science 273:1516-1517).
Visualization and Surface Mounting of Single DNA Molecules
Single molecule approaches represent a subset of current physical and
genetic mapping approaches constitute the two major approaches to genomic
analysis, and are critical to mapping and cloning of disease genes and to
direct sequencing efforts. Such methods of visualization of single DNA
molecules include fluorescence microscopy in solution (Yanagida et al.,
1986, in Applications of fluorescence in the biomedical sciences Taylor et
al. (eds), Alan Liss, New York, pp 321-345; Yanagida et al., 1983, Cold
Spring Harbor Symp. Quantit. Biol. 47:177; Matsumoto et al., 1981, J. Mol.
Biol. 132:501-516; Schwartz et al., 1989, Nature 338:520-522; and Houseal
et al., 1989, Biophys. J. 56:507-516); FISH (Manuelidis et al., 1982, J.
Cell. Biol. 95:619; Lawrence et al., 1988, Cell 52:51; Lichter et al.,
1990, Science 247:64; Heng et al., 1992, Proc. Natl. Acad. Sci. USA
89:9509; van den Engh et al., 1992, Science 257:1410); visualization by
scanning tunneling microscopy or atomic force microscopy techniques
(Keller et al., 1989, Proc. Natl. Acad. Sci. USA 86:5356-5360; see, e.g.,
Karrasch et al., 1993, Biophysical J. 65:2437-2446; Hansma et al., 1993,
Nucleic Acids Research 21:505-512; Bustamante et al., 1992, Biochemistry
31:22-26; Lyubchenko et al., 1992, J. Biomol. Struct. and Dyn. 10:589-606;
Allison et al., 1992, Proc. Natl. Acad. Sci. USA 89:10129-10133;
Zenhausern et al., 1992, J. Struct. Biol. 108:69-73); visualization of
circular DNA molecules (Bustamante et al., 1992, Biochemistry 31:22-26);
DNA bending in transcription complexes by scanning force microscopy (Rees
et al., 1993, Science 260:1646-1649); direct mechanical measurement of the
elasticity of single DNA molecules using magnetic beads (Smith et al.,
1992, Science 258:1122-1126); alignment and detection of DNA molecules
involving either elongation of end-tethered surface bound molecules by a
receding air-water interface (U.S. Pat. Nos. 5,079,169; 5,380,833; Perkins
et al., 1994, Science 264:819; and Bensimon et al., 1994, Science
265:2096-2098), and elongation of non-tethered molecules by `fluid
fixation` (Samad et al., 1995, Nature 378:516-517; Cai et al., 1995, Proc.
Natl. Acad. Sci. USA 92:5164-5168; Meng et al., 1995, Nature Genet.
9:432-438; Wang et al., 1995, Proc. Natl. Acad. Sci. USA 92:165-169; and
Schwartz et al., 1993, and Science 262:110-114); (See also Reed et al., "A
Quantitative Study Of Optical Mapping Surfaces By Atomic Force Microscopy
And Restriction Endonuclease Digestion" in press, Analytical Biochemistry;
Cai et al., "High Resolution Restriction Maps Of Bacterial Artificial
Chromosomes Constructed By Optical Mapping", 1998, Proc. Natl. Acad. Sci.
USA 95:3390-3395; Samad and Schwartz, "Genomic Analysis by Optical
Mapping" in Analytical Biotechnology--Genomic Analysis in press; Schwartz
et al., 1997, Current Opinion in Biotechnology, 8:70-74; Samad, 1995,
Genomics Research 59:1-4; and Primrose, 1995, Principles of Genome
Analysis: A guide to mapping and sequencing DNA from different organisms,
Blackwell Science Ltd., Oxford England, pp. 76-77; and Bautsch et al.,
1997 "Long-Range Restriction Mapping of Genomic DNA" in Genomic Mapping: A
Practical Approach, Chapter 12, Paul H. Dear ed., Oxford University Press,
New York, pp. 281-313).
New modes of molecular investigation have emerged from advances in
molecular fixation techniques, labeling, and the development of scanning
probe microscopies (Keller et al., 1989, Proc. Natl. Acad. Sci. USA
86:5356-5360; Bensimon et al., 1994, Science 265:2096-2098; Guthold et
al., 1994, Proc. Natl. Acad. Sci. USA, 91:12927-12931; Hansma et al.,
1996, Nucleic Acids Res. 24:713-720; Cai et al., 1995, Proc. Natl. Acad.
Sci. USA 92:5164-5168; Meng et al., 1995, Nature Genet. 9:432-438; Weier
et al., 1995, Hum. Mol. Genet. 4:1903-1910; Wang et al., 1995, Proc. Natl.
Acad. Sci. USA 92:165-169; Schwartz et al., 1993, Science 262:110-114;
Schena et al., 1995, Science 270:467-470; Heller et al., 1997, Proc. Natl.
Acad. Sci. USA 94:2150-2155; Erie et al., 1994, Science 266:1562-1566; and
Leuba et al., 1994, Proc. Natl. Acad. Sci. USA 91:11621-11625). In
particular, molecular fixation techniques have relied on the application
of outside forces such as electrical fields, a travelling meniscus
(Michalet et al., 1997, Science 277:1518) or end-tethering of molecules
with beads (Strick et al., 1996, Science 271:1835-1837) to fix DNA to
solid surfaces. Biochemistries have been performed on surface-mounted DNA
molecules, but the procedures used bulk deposition and analysis (Schena et
al., 1995, Science 270:467-470; Heller et al., 1997, Proc. Natl. Acad.
Sci. USA 94:2150-2155; Craig et al., 1990, Nucleic Acids Res.
18:2653-2660; and Nizetic et al., 1991, Proc. Natl. Acad. Sci. USA
88:3233-3237).
Once the nucleic acid molecules are fixed, they must be imaged and
analyzed. Although the spatial resolution of conventional light microscopy
is limited, cooled, charged-coupled (CCD) imaging devices have stimulated
the development of new optical approaches to the quantitation of nucleic
acids, that may supplant electrophoresis-based techniques in many
applications (Schena et al., 1995, Science 270:467-470; Lipshutz et al.,
1995, Biotechniques 19:442-447; and Chee et al., 1996, Science
274:610-614). Yanagida and coworkers (Yanagida et al., 1996, in
Applications of fluorescence in the biomedical sciences, Taylor et al.
(eds), Alan Liss, New York, pp. 321-345) first investigated the molecular
motions of fluorescently stained individual DNA molecules in solution by
image-enhanced fluorescence microscopy. Optical mapping was subsequently
developed for the rapid production of ordered restriction maps from
individual, fluorescently stained DNA molecules (Cai et al., 1995, Proc.
Natl. Acad. Sci. USA 92:5164-5168; Meng et al., 1995, Nature Genet.
9:432-438; Wang et al., 1995, Proc. Natl. Acad. Sci. USA 92:165-169;
Schwartz et al., 1993, Science 262:110-114; Schwartz et al., 1997, Curr.
Opinions in Biotechnology 8:70-74; Samad et al., Nature 378:516-517; and
Samad et al., 1995, Genomic Research 59:1-4).
In the original method, individual fluorescently labeled yeast chromosomes
were elongated and fixed in a flow of molten agarose generated between a
coverslip and a glass slide (Schwartz et al., 1993, Science 262:110-114).
Restriction endonuclease cleavage events were recorded as time-lapse
images, following addition of magnesium ions to activate the added
endonuclease. Cleavage sites appeared as growing gaps due to relaxation of
DNA coils at nascent ends, and maps were constructed by measuring fragment
sizes using relative fluorescent intensity or apparent length
measurements.
In another closed system, the DNA molecules (2-1,500 kb) were elongated and
fixed using the flow and adhesion forces generated when a fluid sample is
compressed between two glass surfaces, one derivatized with polylysine or
APTES (Meng et al., 1995, Nature Genet. 9:432-438 and Cai et al., 1995,
Proc. Natl. Acad. Sci. USA 92:5164-5168). Fixed molecules were digested
with restriction endonucleases, fluorescently stained (Rye et al., 1992,
Nucleic Acids Res. 20:2803-2812) and optically mapped (Meng et al., 1995,
Nature Genet. 9:432-438 and Cai et al., 1995, Proc. Natl. Acad. Sci. USA
92:5164-5168). However, closed systems have limited access to the samples
and cannot readily accommodate arrayed samples (Bensimon et al., 1994,
Science 265:2096-2098 and Meng et al., 1995, Nature Genet. 9:432-438).
To increase the throughput and versatility of optical mapping and
sequencing, multiple samples need to be arrayed on a single mapping
surface. Although robotic gridding techniques for DNA samples exist
(Heller et al., 1997, Proc. Natl. Acad. Sci. USA 94:2150-2155; Craig et
al., 1990, Nucl. Acids Res. 18:2653-2660; and Nizetic et al., 1991, Proc.
Natl. Acad. Sci. USA 88:3233-3237), such approaches were not designed to
work with single molecule substrates and could not be relied upon to
deposit molecules retaining significant accessibility to enzymatic action.
While single molecule techniques offer the potential advantage of an
ordering capability which gel electrophoresis lacks, none of the current
single molecule techniques can be used, on a practical level, as high
resolution genomic sequencing tools. The molecules described by Yanagida
(Yanagida, M. et al., 1983, Cold Spring Harbor Symp. Quantit.
Biol. 47:177; Matsumoto, S. et al., 1981, J. Mol. Biol. 132:501-516) were
visualized, primarily free in solution making any practical sequencing
impossible. Further, while the FISH technique offers the advantage of
using only a limited number of immobilized fragments, usually chromosomes,
it is not possible to achieve the siz | | |
