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1. FIELD OF THE INVENTION
This invention relates to methods and compositions for manipulating and
characterizing individual polymer molecules, especially nucleic acid
molecules, according to, for example, size and/or nucleotide sequence.
2. BACKGROUND OF THE INVENTION
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 acid at the gene, rather than at the genome, level
(Chumakov, I. et al., 1992, Nature 359:380; Maier, E. et al., 1992, Nat.
Genet. 1:273).
Traditionally, the separation and molecular weight distribution of nucleic
acid molecules has been accomplished, most commonly, via gel
electrophoresis (see, for example, Freifelder, 1976, Physical
Biochemistry, W. H. Freeman), which involves moving a population of
molecules through an appropriate medium, such that the molecules are
separated according to size. Such electrophoretic methods offer an
acceptable level of size resolution, but, especially for purposes of high
throughput mapping, suffer from a number of setbacks.
For example, such techniques require the preparation of DNA in bulk
amounts. First, with respect to genome mapping, such preparative
procedures may require sources such as genomic DNA or DNA from yeast
artificial chromosomes (YACs; Burke, D. T. et al., 1987, Science 236:806;
Barlow, et al., 1987, Trends in Genetics 3:167-177; Campbell et al., 1991,
Proc. Natl. Acad. Sci. U.S.A. 88:5744). Obtaining quantities of DNA from
these sources which is sufficient for detailed analyses, such as
restriction mapping, is time consuming and often impractical. Second,
because populations of molecules of like size migrate through the medium
at the same rate, it is impossible to separate individual molecules from
within a sample of particles by utilizing such a technique. Additionally,
while it is possible to resolve a wide size range of DNA molecule
populations gel electrophoresis techniques, optimal techniques can often
require the use of several different gel matrix compositions and/or
alternative electrophoresis procedures, depending upon the sizes of the
molecules of interest. For example, the separation of large molecules of
DNA may require such techniques as pulse field electrophoresis (see, e.g.,
U.S. Pat. No. 4,473,452). Further, standard gel electrophoresis techniques
involve the separation of populations of molecules according to size,
making it impossible to separate individual molecules within a
polydisperse mixture. In summary, therefore, the accurate, rapid,
practical, high throughput separation of individual DNA molecules,
especially those of highly disparate sizes, which would often be required
for genomic mapping purposes, is impossible via gel electrophoresis.
Techniques have been reported for the visualization of be single nucleic
acid molecules and complexes. Such techniques include such fluorescence
microscopy-based techniques as fluorescence in situ hybridization (FISH;
Manuelidis, L. et al., 1982, J. Cell. Biol. 95:619; Lawrence, C. A. et
al., 1988, Cell 52:51; Lichter, P. et al., 1990, Science 247:64; Heng, H.
H. Q. et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:9509; van den Engh,
G. et al., 1992, Science 257:1410) and those reported by, for example,
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);
tethering techniques, whereby one or both ends of a nucleic acid molecule
are anchored to a surface (U.S. Pat. Nos. 5,079,169; 5,380,833; Perkins,
T. T. et al., 1994, Science 264:819; Bensimon, A. et al., 1994, Science
265:2096); and scanning probe microscopy-based visualization techniques,
including scanning tunneling microscopy and atomic force microscopy
techniques (see, e.g., Karrasch, S. et al., 1993, Biophysical J.
65:2437-2446; Hansma, H. G. et al., 1993, Nucleic Acids Research
21:505-512; Bustamante, C. et al., 1992, Biochemistry 31:22-26;
Lyubchenko, Y. L. et al., 1992, J. Biomol. Struct. and Dyn. 10:589-606;
Allison, D. P. et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10129-10133;
Zenhausern, F. et al., 1992, J. Struct. Biol. 108:69-73).
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, for
example, high resolution genomic mapping 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), for
example, were visualized, primarily free in solution, in a manner which
would make any practical mapping 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 sizing resolution available with gel electrophoresis.
Single molecule tethering techniques, as listed above, generally involve
individual nucleic acid molecules which have, first, been immobilized onto
a surface via one or both of their ends, and, second, have been
manipulated such that the molecules are stretched out. These techniques,
however, are not suited to genome analysis. First, the steps involved are
time consuming and can only be accomplished with a small number of
molecules per procedure. Further, in general, the tethered molecules
cannot be stored and used again.
A combination of the sizing capability of gel electrophoresis and the
ordering capability of certain single molecule techniques such as, for
example, FISH, would, therefore, be extremely useful for genomic analyses
such as genomic mapping. Such analyses would be further aided by the
ability to manipulate the single molecules being analyzed. Additionally,
an ability to reuse the nucleic acid samples of interest would increase
the efficiency and throughput capability of the analysis. Currently,
however, there exists no single technology which embodies, in a practical
manner, each of these elements.
Citation of documents herein is not intended as an admission that any of
the documents cited herein is pertinent prior art, or an admission that
the cited documents are considered material to the patentability of the
claims of the present application. All statements as to the date or
representations as to the contents of these documents are based on the
information available to the applicant and does not constitute any
admission as to the correctness of the dates or contents of these
documents.
3. SUMMARY OF THE INVENTION
The present invention relates to methods and compositions for
characterizing and manipulating individual nucleic acid molecules,
including mammalian chromosome-sized individual nucleic acid molecules.
The methods and compositions described herein can be utilized for the
accurate, rapid, high throughput analysis of nucleic acid molecules at the
genome level, and may, for example, include the construction of high
resolution physical maps, referred to herein as "optical mapping", and the
detection of specific nucleotide sequences within a genome, referred to
herein as "optical sequencing."
Specifically, methods are described whereby single nucleic acid molecules,
including mammalian chromosome-sized DNA molecules, are elongated and
fixed in a rapid, controlled and reproducible manner which allows for the
nucleic acid molecules to retain their biological function and, further,
makes rapid analysis of the molecules possible. In one embodiment of such
a procedure, the molecules are elongated in a flow of a molten or
unpolymerized gel composition. The elongated molecules become fixed as the
gel composition becomes hardened or polymerized. In such an embodiment,
the gel composition is preferably an agarose gel composition. The
elongated molecules became fixed as the agarose.
In a second embodiment, the single nucleic acid molecules are elongated and
fixed in a controllable manner directly onto a solid, planar surface. This
solid, planar surface contains a positive charge density which has been
controllably modified such that the single nucleic acid molecules will
exhibit an optimal balance between the critical parameters of nucleic acid
elongation state, degree of relaxation stability and biological activity.
Further, methods, compositions and assays are described by which such an
optimal balance can precisely and reproducibly be achieved.
In a third embodiment, the single nucleic acid molecules are elongated via
flow-based techniques. In such an embodiment, a single nucleic acid
molecule is elongated, manipulated (via, for example, a regio-specific
restriction digestion), and/or analyzed in a laminar flow elongation
device. The present invention further relates to and describes such a
laminar flow elongation device.
The elongated, individual nucleic acid molecules can then be utilized in a
variety of ways which have applications for the analysis of nucleic acid
at the genome level. For example, such nucleic acid molecules may be used
to generate ordered, high resolution single nucleic acid molecule
restriction maps. This method is referred to herein as "optical mapping"
or "optical restriction mapping". Additionally, methods are presented
whereby specific nucleotide sequences present within the elongated nucleic
acid molecules can be identified. Such methods are referred to herein as
"optical sequencing". The optical mapping and optical sequencing
techniques can be used independently or in combination on the same
individual nucleic acid molecules.
Still further, the elongated nucleic acid molecules of the invention can be
manipulated using any standard procedure. For example, the single nucleic
acid molecules may be manipulated by any enzymes which act upon nucleic
acid molecules, and which may include, but are not limited to, restriction
endonucleases, exonucleases, polymerases, ligases or helicases.
Additionally, methods are also presented for the imaging and sizing of the
elongated single nucleic acid molecules. These imaging techniques may, for
example, include the use of fluorochromes, microscopy and/or image
processing computer software and hardware. Such sizing methods include
both static and dynamic measuring techniques.
Still further, high throughput methods for utilizing such single nucleic
acid molecules in genome analysis are presented. In one embodiment of such
high throughput methods, rapid optical mapping approaches are described
for the creation of high-resolution restriction maps. In such an
embodiment, single nucleic acid molecules are elongated, fixed and gridded
to high density onto a solid surface. These molecules can then be digested
with appropriate restriction enzymes for the map construction. In an
alternative embodiment, the single nucleic acid molecules can be
elongated, fixed and gridded at high density onto a solid surface and
utilized in a variety of optical sequencing-based diagnostic methods. In
addition to speed, such diagnostic grids can be reused. Further, the high
throughput and methods can be utilized to rapidly generate information
derived from procedures which combine optical mapping and optical
sequencing methods.
The present invention is based on the development of techniques, including
high throughput techniques, which reproducibly and rapidly generate
populations of individual, elongated nucleic acid molecules that not only
retain biological function but are accessible to manipulation and make
possible rapid genome analysis.
4. BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. Schematic drawing of an electrophoretic microscopy chamber which is
specifically adapted to fluorescence microscopy studies.
FIG. 2. Partly schematic and partly block diagram showing an
interconnection of exemplary chamber electrodes in an electrophoresis
chamber which may be used in the present invention.
FIGS. 3A-3B. Schematic illustration of the instrumentation used in the
microscopic study of DNA molecules in a medium according to this
invention, and a more detailed diagram showing the instrumentation for
measuring birefringence.
FIGS. 4A-4I. Depicted herein are the DNA molecular conformational and
positional changes when G bacteriophage molecules are subject to two
sequential electric fields in different directions.
FIGS. 5A-5J. Depicted herein are the DNA molecular conformational and
positional changes during relaxation of G bacteriophage DNA molecules
after electrophoresis for 600 seconds, as revealed by the fluorescence
microscopy experiments described in Example 4.
FIG. 6. Optical mapping. DNA molecules and restriction enzyme are dissolved
in molten agarose without magnesium ions. The DNA molecules are elongated
by the flow generated when the mixture is sandwiched between a slide and
coverslip. Stretched molecules are fixed in place by agarose gelation.
Magnesium ion diffusion into the gel triggers digestion and cleavage sites
appear as growing gaps as the molecular fragments relax.
FIGS. 7A-7D. Histograms of optical mapping. Not 1 cut frequencies, showing
variation with molecule size and number of cut sites, are indicated.
Cutting frequencies were scored by counting the number of Not 1 cuts in
nucleic acid molecules present in microscope fields. Such fields typically
contain approximately 3-5 molecules. Because approximately half the fields
showed no Not 1 cutting and were, therefore, not scored, this
underestimates the number of uncut molecules. The expected number of cut
sites and chromosome sizes: 7A: Ch. 1(240 kb) 1; 7B.: Ch. V and VIII(595
kb) 3 and 2; 7C: Ch. XI(675 kb) 2; and 7D: Ch. XIII and XVI(950 and 975
kb) 1. Chromosome pairs V and VIII, and XIII and XVI were present on the
same mount.
FIGS. 8A-8H. Depicted are some restriction fragment relaxation modes for a
singly cleaved, gel-fixed, elongated molecule. Horizontal arrows indicate
direction of relaxation. Relaxation modes illustrated: 8A depicts fixed
molecule before cleavage, 8B-8E depict possible relaxation modes producing
detectably cleaved molecules, and 8F-8H depict relaxation modes producing
undetectably cleaved molecules.
FIG. 9. Schematic representation depicting possible relaxation events to
form pools of segments or "balls" at coil ends. Agarose gel is illustrated
as a series of pegs with free spaces available for molecules. Gel pegs
might intersect the embedded DNA molecule during gelation and possibly
entrap it. The coil segments positioned in the pool region comprise a
relaxed sub-coil region and have higher entropy than the coil stretched
out between them. These pools may act as molecular rivets in some
circumstances, particularly if the segment pool mass approaches that of
the intervening coil.
FIGS. 10A-10D. Optical mapping sizing results for Not I endonuclease
restriction fragments from S. cerevisiae chromosomes I, V, VIII, XI, XIII,
and XVI calculated as described, plotted against published results. The
diagonal line is for reference. Typical fragment images are shown in this
figure. (See example 13). The inset shows the estimate of population
standard deviation (kb). Error bars represent 90% confidence (7) on means
(main graph) or standard deviation (inset). 10A and 10B: the relative
intensity determination of fragment sizes. 10C and 10D: the relative
apparent length determination of fragment sizes.
FIGS. 11A-11C. Scatter plot of normalized absolute intensity vs. apparent
length. Absolute intensities from six individual images were calculated
and plotted against apparent length over a time interval typically used in
optical mapping (10-15 minutes). For each sample, the initial intensity
was found by averaging absolute intensity values from groups of 5 adjacent
images and taking the largest value. The values from several samples were
normalized by dividing values from each image by the initial intensity for
the sample. 11A: chromosome I 120 kb Not I fragment, 7 samples. 11B:
chromosome XI 285 kb Not I fragment, 4 samples. 11C: chromosome XI 360 kb
Not I fragment, 4 samples.
FIG. 12. Comparison of Not I endonuclease restriction maps of optical
mapping results of S. cerevisiae chromosomal DNA molecules with published
restriction maps (L&O). Maps were constructed from length (Len), intensity
(Int) or a combination of both (Com). Bar lengths for the optical mapping
data are proportional to the means plotted in FIGS. 10A-10D, and typical
images are shown in FIGS. 13A-13F.
FIGS. 13A-13F. Typical fluorescence microscopy images of S. cerevisiae
chromosomal DNA molecules stained with DAPI and embedded in agarose gel
during Not I restriction endonuclease cleavage. Chromosomal DNA molecules
were prepared and fixed as described in Example 13 and cited references.
Images were background corrected using a smoothed and attenuated
background image, smoothed, and stretched, using 16-bit precision. Images
show Not I restriction digestion evolution, with arrows highlighting cut
sites. Intervals are timed after addition of Mg.sup.2+. 13A: Ch. I (240
kb), 20 and 60 sec; 13B: Ch. XI (675 kb), 500, 880 and 1160 sec; 13C: Ch.
V (595 kb), 200, 240, 520 sec; 13D: Ch. VIII (595 kb), 440, 1220 and 1360
sec; 13E: Ch. XIII (950 kb), 100 and 560 sec; 13F: Ch. XVI (975 kb), 460
and 560 sec. Bars, 5 .mu.m. A 100.times. objective was used to image
results in FIGS. 13A-13D and a 63.times. objective was used for FIGS. 13E
and 13F.
FIG. 14. Optical mapping results from Rsr II and Asc I endonuclease
restriction digest of S. cerevisiae chromosomes III and XI. Maps were
constructed from fully cut length (Len) or intensity (Int) data, and
refined using partial cut length. Bar lengths are proportional to the
calculated means, and typical images are shown in FIGS. 15A-15C. Number of
cuts was determined as in FIGS. 7A-7D.
FIGS. 15A-15C. Fluorescence microscopy images of S. cerevisiae chromosomal
DNA molecules stained with DAPI and embedded in agarose gel during Rsr II
or Asc I restriction endonuclease cleavage. Chromosomal DNA molecules were
digested and analyzed as in FIGS. 13A-13F. Images show restriction
digestion evolution, with arrows highlighting cut sites. 15A: Ch. III, Rsr
II, 1100 and 1820 sec; 15B: Ch. XI, Rsr II, 20, 600, 920, 1060 sec; 15C:
Ch. XI, Asc I, 1160, 1500, 1780, 1940 sec. An isoschizomer to Rsr II, Csp
I, was also used and gave identical results. Bar, 5 .mu.m.
FIG. 16. Glass surface properties as a function of polylysine treatment.
Glass surfaces were incubated for 16 hours in different concentrations of
poly-D-lysine, MW=350,500. Lambda bacteriophage DNA molecules in EcoRI
restriction buffer and ethidium homodimer, minus magnesium ions, were
mounted onto the treated glass surfaces. Square and circle show ratio of
absorbed DNA and average length of absorbed DNA, respectively. Each point
represents roughly 50 molecules measured and bars show the standard
deviation about a mean. Sample preparation, imaging techniques and
analysis are given in Methodology.
FIGS. 17A-17W. Gallery of fluorescence microscopy images of lambda clones
from Optical Mapping results. Clones from a mouse yeast artificial
chromosome (YAC) (Burke et al., Science 236:806-812, 1987; Murray and
Szostak, Nature 305:189-193, 1983) spanning the Pygmy locus were subcloned
into Lambda FIX II and digested with EcoRI and BamHI. Maps for these and
other molecules (not shown) were constructed by Optical Mapping techniques
(Methodology) and shown in FIG. 19. Images show typical molecules used for
map construction. Bars: 5 microns. FIG. 17V is an enlargement of FIG. 17T
and FIG. 17W is at the same scale as FIG. 17V. The enzymes used for map
construction are indicated as (E) for EcoRI and (B) for BamH I. FIG. 17A
uncut lambda DNA; FIG. 17B, B3 (E); FIG. 17C, F (B); FIG. 17D, B (s); FIG.
17E, D (S); FIG. 17F, E (S); FIG. 17G, 914 (S); FIG. 17H, S(E); FIG. 17I,
G (S); FIG. 17J, C (E); FIG. 17K, B4 (E); FIG. 17L, Y11 (E); FIG. 17M, 618
(E); FIG. 17N, 617 (E); FIG. 17O, 305 (E); FIG. 17P, A (B); FIG. 17Q, 1004
(B); FIG. 17R, E (E); FIG. 17S, B6 (E); FIG. 17T, A2 (E); FIG. 17U, C3
(E); FIG. 17V, A2 (E); FIG. 17W, F (E).
FIGS. 18A-18D. EcoRI and BamH I endonuclease restriction fragment sizing
results for Lambda FIX II clones, calculated as described and plotted
against gel electrophoresis data. FIG. 18A, Relative fluorescence
intensity results. The diagonal line is for reference. Typical fragment
images are shown in FIGS. 17A-17W. Inset: estimate of population standard
deviation (kb). Error bars represent 90% confidence on means (main graph)
or standard deviation (inset). The size of the whole molecule was
determined by gel electrophoresis. b, results for small fragments. The
best fit line through the origin (slope 0.665) was used to calibrate
fragment originally estimated at less than 6.5 kb prior to incorporation
into maps. c, results after correction. d, Relative apparent length sizing
results from the same images. The diagonal line is for reference.
FIG. 19. EcoRI and BamH I restriction maps constructed by Optical Mapping.
Clones are labeled on the left side. The upper ticks are EcoRI restriction
sites and lower ticks are BamH I sites. Table 1 shows the fragment sizes.
FIGS. 20A-20H. Optically sizing insert DNA of lambda FIX II clones. Lambda
clones mounted on the surface were digested by an enzyme which cut at the
polylinker sites, as described in Methodology. The 20 kb and 9 kb vector
arms of FIX II cloning system were used as internal size standards to
convert relative sizes to absolute sizes. The results of fluorescence
intensity and length were shown in Table 2, together with sizes from PFGE.
Cases where the enzyme also cut the insert were easily interpreted. Scale
bar is 5 microns. FIGS. 20A-20B, Clone F (Sal I): 20 kb, 7.5 kb, 9.5 kb, 9
kb. FIGS. 20B-20D, Clone G (Sal I): 20 kb, 10.1 kb, 4.1 kb, 9 kb. FIGS.
20E-20F, clone B (NotI): 20 kb, 17.6 kb, 9 kb. FIGS. 20G-20H, B3 (SstI):
20 kb, 13.8 kb, 9 kb.
FIG. 21 DNA binding properties of glass surfaces as a function of APTES
deposition. Yeast (AB972) chromosome I molecules (240 kb, 72 mm contour
length, assuming B-DNA) in (10 mM Tris pH 7.6, 1 mM EDTA, 50 mM NaCl) were
applied in molten agarose to glass surfaces previously treated with APTES
for the indicated time. The number and length of molecules was measured by
fluorescence microscopy after staining with ethidium homodimer. The plot
shows the average number of molecules deposited per 100 m.sup.2 field
viewed (square) and the average molecule length (circle), plotted against
the time of prior APTES derivatization. Each point represents
.sup..about.b 60 molecules imaged. Bars indicate the standard deviation
about the means. Sample preparation, imaging techniques and analysis are
given in Materials and Methods.
FIGS. 22A-22D. Optical mapping sizing results for NotI endonuclease
restriction fragments of S. cerevisiae chromosomes I, V, VIII, and XI
calculated as described (Example 13) plotted against published results
(Link and Olson, Genetics 127:681, 1991). The diagonal line is for
reference. Each point represents 20 to 40 imaged fragments. FIGS. 22B and
22D: estimate of population standard deviation (kb). Error bars represent
the 90% confidence intervals. FIGS. 22A and 22B Relative apparent length
determination of restriction fragment sizes. FIGS. 22C and 22D Relative
fluorescence intensity determination of restriction fragment sizes.
FIGS. 23A-23D. 23A-23C: Typical fluorescence micrographs of S. cerevisiae
chromosomal DNA molecules digested with NotI restriction endonuclease.
Molecules were stained with ethidium homodimer after digestion. Arrows
indicate cleavage sites, bars 10 microns. FIG. 23A, chromosome XI, two
cuts; FIG. 23B, Chromosome V, three cuts; and FIG, 23C, chromosome VIII,
two cuts. FIG. 23D, graphical comparison of optical mapping results and
published PFGE restriction maps of yeast chromosomes digested with NotI.
Bar lengths for the optical mapping data are proportional to the means
based on the fluorescence intensity measurements plotted in FIGS. 22C-22D.
FIGS. 24A-24I. FIGS. 24A-G: Typical fluorescence micrographs of yeast
artificial chromosomes digested with NotI, MluI, EagI and NruI restriction
endonucleases and stained with ethidium homodimer. Arrows indicate
Cleavage sites, bars 10 microns. YAC 7H6 was digested with: FIG. 24A,
NruI; FIG. 24B EagI. YAC 3I4 was digested with: FIG. 24C, NotI; FIG. 24D,
MluI; FIG. 24E, EagI; FIG. 24F, NotI and MluI; FIG. 24G, MluI and EagI.
Graphical comparison of optical mapping results with PFGE mapping results
for YACs: FIG. 24H, 7H6; FIG. 24I, 3I4. Double digestion results are
included. Bar lengths for the optical mapping data are proportional to the
means based on fluorescence intensity measurements.
FIG. 25 is a diagram depicting a laminar flow elongation device.
FIGS. 26 A, B, and C illustrate the characteristic "sunburst" pattern of
fixation of elongated molecules using the spotting technique of the
present invention.
FIGS. 27 A and B show relaxation measurements as a function of molecular
size.
FIGS. 28 A and B are logarithmic plots of relaxation versus size.
FIG. 29 shows a enlarged view of a DNA spot and one method of spreading
molecules onto a derivatized surface.
FIG. 30 is a block diagram of a method for high throughput optical mapping
of lambda or cosmid clones.
FIG. 31 is a block diagram of the system used for high throughput optical
mapping of gridded YAC DNA.
FIG. 32 is a block diagram of one embodiment of the automated system for
high throughput optical mapping.
FIG. 33 illustrates a method of optimizing the image collection process and
maximizing the signal-to-noise ratio.
FIG. 34 is a block diagram of the image processing method in accordance
with a preferred embodiment of the present invention.
5. DETAILED DESCRIPTION OF THE INVENTION
Described herein are methods and compositions for characterizing and
manipulating individual nucleic acid molecules, including mammalian
chromosome-sized individual nucleic acid molecules. The methods and
compositions described herein can be utilized for optical mapping and
optical sequencing purpose to generate accurate, rapid, high throughput
analyses of nucleic acid molecules at the genome level.
Specifically, Section 5.1 describes methods for the elongation and fixation
of single nucleic acid molecules. Such methods include both agarose-based
(Section 5.1.1) and solid surface-based (Section 5.1.2) techniques.
Section 5.1 also describes assays for the optimization of parameters
important to the production of the solid, planar surfaces used-herein.
Further, Section 5.1 also describes flow-based elongation techniques
(Section 5.1.3) in which a single nucleic acid molecule is elongated,
manipulated and/or analyzed in a laminar flow elongation device.
Section 5.2 describes methods for the imaging and sizing of single nucleic
acid molecules. The Section includes, for example, nucleic acid staining,
microscopy and photography techniques useful for imaging single nucleic
acid molecules. Further, the Section describes methods for the sizing of
single nucleic acid molecules including both static and dynamic
measurement techniques. Section 5.3 describes genome analysis applications
to which the single nucleic acid molecule techniques of the invention may
be put. Such applications include, for example, optical mapping and
optical sequencing techniques. Finally, Section 5.4 discusses methods for
rapid, high throughput utilization of the single nucleic acid techniques
of the invention.
5.1. SINGLE NUCLEIC ACID MOLECULE ELONGATION TECHNIQUES
A variety of methods can be utilized for the rapid, controllable and
reproducible elongation of single nucleic acid molecules in such a manner
that allows rapid, efficient analysis and/or manipulation of the
molecules. These techniques can include, for example, gel-based (Section
5.1.1), solid surface-based (Section 5.1.2) and flow-based techniques
(Section 5.1.3), each of which will be separately described below.
5.1.1. GEL-BASED TECHNIQUES
Gel-based techniques can be utilized for the elongation of single nucleic
acid molecules. The gel-based techniques described herein maintain the
biological function of the nucleic acid molecules and, further, allow for
the manipulation and/or accurate analysis of the elongated single nucleic
acid molecules. Nucleic acid molecules which can be rapidly, efficiently
analyzed via such gel-based techniary include nucleic acid molecules which
range in length from about 20 kb up to mammalian chromosome-sized lengths
(i.e. greater than 1000 kb). Further, such gel-based techniques make
possible the utilization of dynamic measurement procedures, may generate a
lower level of nucleic acid shearing and make possible the utilization of
a wide range of biochemical activities with which the manipulate the
elongated nucleic acid molecules.
Briefly, gel-based techniques involve elongating single nucleic acid
molecules within a molten or nonpolymerized gel composition such that upon
cooling or polymerization, the elongated nucleic acid molecules are
maintained in a relatively stationary position, while remaining accessible
to, for example, enzymatic manipulation and/or hybridization to
complementary nucleic acid molecules or binding to sequence-specific
proteins or peptides. Further, the gelation process restrains elongated
nucleic acid molecules from appreciably relaxing to a random coil
conformation after, for example, their enzymatic cleavage.
For optimal imaging and manipulation potential, the amount which the single
nucleic acid molecules are elongated within the gel composition is
critical. Excessive elongation or stretching causes the molecule to become
difficult to visualize. For example, too much stretching presents too
little fluorochrome per imaging pixel, lending the intensities generated
by the measured molecular intensities to approach background values.
Insufficient stretching, however, generates too low a level of tension,
which can interfere with an analysis of single nucleic acid molecule
manipulations. For example, when restriction mapping, enough elongation
must occur such that, upon digestion, the newly formed nucleic acid
fragments pull away from each other, thus revealing restriction sites. An
additional requirement for optimal gel-based elongation requires that care
be taken to preserve the moisture within the gel, such that the maximum
biological function of the nucleic acid can be retained.
For optimal imaging/manipulation potential, the extent to which a nucleic
acid molecule is elongated within a gel must be great enough to generate a
sufficient level of intramolecular tension while not being so great that
the elongated molecule becomes difficult to image. In general, elongation
methods which produce single nucleic acid molecules that span
approximately 20% to 60% of their curvilnear contour lengths are
preferred.
Further, the elongated nucleic acid molecules within the gel must lie
within a shallow plane of focus for successful imaging. With respect to
larger nucleic acid molecules, for example, it is additionally important
for the molecules to lie within a plane approximately 0.2 .mu.m in
thickness for focused visualization.
Because gelation or polymerization fixes embedded molecules, systematically
varying parameters which affect the rate at which the gelation or
polymerization can modulate the degree of fixation and, ultimately, the
rate of molecule relaxation. Smaller nucleic acid molecules (i.e.,
molecules less than about 350 kb) relax quickly. Thus, it is preferred
that elongation take place under conditions which hasten
gelation/polymerization so that the nucleic acid molecules become trapped
in an extended conformation before substantial relaxation takes place.
Larger nucleic acid molecules relax at a slower rate, and, therefore, can
be elongated under conditions which allow for a slower rate of
gelation/polymerization.
With respect to agarose gels, parameters which affect the rate of gelation
include, for example, the gel concentration and/or temperature at which
the gel is formed. A higher gel concentration or gelation at a low
temperature hastens gel formation. With respect to polyacrylamide gels,
parameters which affect the rate of polymerization include, for example,
the acrylamide/bisacrylamide concentration and ratio, the temperature at
which polymerization takes place, and the ammonium sulfate and TEMED
concentrations used.
While any gel composition may be used for such elongation techniques, an
agarose gel composition is preferred, with an agarose composition
exhibiting a low gelling temperature being especially preferred. Such low
gelling temperature agarose compositions are the most optically clear
agarose compositions available and, further, because such compositions can
remain molten at 37.degree. C., the biological activity of enzymes, such
as restriction enzymes, within the molten agarose can easily be
maintained. Additionally, such agarose compositions are useful in that
rapid gelation is often desired for fixation of the elongated nucleic acid
molecules. For agarose gel compositions, a gel composition comprising from
about 0.1% to about 3.0%, with 0.1-1.5% being preferred.
Any number of techniques can be used to apply an external force which will
cause the nucleic acid molecules within the gel composition to become
elongated. For example, an elongating external force may include an
electrical or mechanical force. While the exact amount of external force
required for optimal elongation may vary according to, for example, the
specific gel composition and nucleic acid molecules being elongated, the
optimization of gel parameters can easily and without undue
experimentation be assayed by, for example, utilizing the visualization
and measurement techniques described in Section 5.2, below.
Elongation may, for example, be accomplished by generating a flow force
within a molten agarose gel containing single nucleic acid molecules. Such
a flow force may be set up by placing the nucleic acid/molten gel
composition between two solid surfaces, such as, for example, between a
slide and a coverslip. In such an embodiment, a hole preferably exists in
the slide through which reagents for the manipulation of the elongated
nucleic | | |
