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TECHNICAL FIELD
The field of this invention is the sequencing of nucleic acids through the
use of microfabricated substrates and scanning tunneling microscopy.
BACKGROUND
A commitment has been made in recent years by several governments to
support the sequencing of the entire human genome. The human genome
contains roughly three billion base pairs of DNA, and consequently the
project has generated immense interest in large-scale DNA sequencing. Even
after the complete human genome has been sequenced, the determination of
individual genotypes in clinical screening remains an important
consideration for DNA sequencing. Genotyping performed using techniques
such as Southern blotting will suffer from imprecision, and even at best
cannot provide detailed sequence information. A simplified method for
rapid DNA sequencing would allow routine clinical determination of
medically important genes.
Traditional methods for obtaining a DNA sequence all share a fundamental
approach. A DNA species is isolated, and a complete nested set of DNA
fragments is generated, each fragment having a common starting point and
being one base longer than the preceding fragment. The set of fragments is
commonly produced by either limited chemical cleavage (Maxam and Gilbert
method), or enzymatically, by DNA synthesis in the presence of a small
amount of a chain terminating nucleotide (Sanger method). Both methods
utilize recognition of the specific bases that comprise DNA to generate
sets of fragments, each corresponding to termination at a single base
species.
The nested fragments are then size fractionated to determine the order of
bases. Polyacrylamide gel electrophoresis has been the method of choice
for a number of years, however it suffers from limitations in the length
of fragments that can be resolved, and in the length of time required for
good separation. In order to determine the nucleotide sequence, the
fragments must be labeled with a detectable label, such as a radioisotope
or fluorochrome. The label is used to visualize the separation pattern.
The length of sequence data available from a single reaction set as
described above is limited to perhaps one thousand bases. The reactions
and separations are time-consuming, expensive, and require a skilled
technician. Resolution by gel electrophoresis may lead to inaccuracies,
and limit the length of sequence which is obtained from a given set of
reactions. It is therefore of interest to devise means by which DNA
sequencing can be simplified, particularly utilizing methods which lend
themselves to automation.
RELEVANT LITERATURE
A review of current methods used in large scale and automated DNA
sequencing may be found in Hunkapillar et al. (1991) Science 254:59-74.
An overview of the use of scanning tunneling microscopy with biological
molecules may be found in STM and SFM in Biology, ed. O. Marti and M.
Amrein, 1993, Academic Press. The use of STM to visualize DNA is described
in Lindsay and Phillip (1991) Genet. Anal. Tech. Appl. 8:8; Allison et al.
(1990) Scanning Microsc. 4:517; Driscoll et al. (1990) Nature 346:294; M.
Salmeron et al. (1990) J. Vac. Sci. Tech. 8:635. The use of AFM to
visualize DNA is described in Weisenhorn et al. (1990) Scanning Microsc.
4:511.
The use of STM in microfabrication is discussed in Snow et al. (1993) Appl
Phys Lett 63:749-751.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention will be more fully understood from the following description,
read in connection with the accompanying drawings, of which:
FIG. 1 is a diagram of the sequencing apparatus.
FIG. 2A is a longtitudinal view of a trench substrate, including DNA. FIG.
2B is a cross-section of a trench substrate.
FIG. 3A is a longtitudinal view of a raised path substrate, including DNA.
FIG. 3B is a cross-section of a raised path substrate.
FIGS. 4A-D illustrate the compilation of scanning data to provide a
complete nucleotide sequence.
FIGS. 5A-H are cross-sections showing the substrate during fabrication for
a silanol coating.
FIGS. 6A-F are cross-sections showing the substrate during fabrication for
a gold coating.
FIGS. 7A-E are cross-sections showing a raised path substrate during
fabrication.
FIGS. 8A-E are a top view showing a substrate during fabrication of
initiation sites.
SUMMARY OF THE INVENTION
Apparatuses and methods are provided for determining the sequence of
nucleotides in a nucleic acid, i.e. DNA or RNA. The molecule, modified
with a base specific label by analog incorporation during synthesis or by
complementation, is linearly oriented on a planar surface, and the
position of each labeled base is determined by scanning the length of the
molecule with scanning microscopy. The process is repeated for each of the
four bases, and the data sets combined to provide the full nucleotide
sequence. The process is useful for determining any DNA sequence, i.e.
chromosomal DNA, DNA episomes, fragments and oligonucleotides, RNA, etc.
The apparatus includes fabricated substrates having an alignment path for
nucleic acid sequencing. Such substrates can have a plurality of alignment
paths to facilitate multiple molecule scanning. The alignment paths have a
binding surface to which nucleic acids adhere. A system is provided for
use in determining the sequence of nucleic acids; having a scanning
microscope, fabricated substrate, and data analysis computer.
Methods for determining the sequence of a nucleic acid using fabricated
substrates and scanning microscopy are provided. Methods for the
preparation, labeling and alignment of nucleic acids are also provided.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Methods and apparatuses are provided for determining the sequence of bases
in a nucleic acid molecule. The nucleotide sequence is determined by
orienting single-stranded nucleic acid molecules on a surface having one
or more linear alignment paths, and determining the presence of base
specific labels along the length of the molecule by scanning microscopy.
The process is repeated with labels specific for each base, and the data
sets thus provided are combined to give the complete nucleotide sequence.
Nucleic acids are polymers having a phosphate backbone, attached to which
are sugars and heterocyclic nitrogenous bases. Naturally occurring nucleic
acids are synthesized from nucleotide triphosphates comprising a
combination of four species of bases, purines: adenine (A) and guanosine
(G), and pyrimidines: cytosine (C) and one of thymidine (T) or uracil (U).
A nucleic acid can be chemically or enzymatically modified so that one of
the four base species is labeled. Such a modification may be the result of
synthesizing the molecule from a nucleotide triphosphate pool where one of
the naturally occurring base species is substituted with an analog.
Alternatively, the complementary binding specificity of the bases can be
exploited, where a nucleotide or analog is added post-synthetically which
will base pair with only one of the bases.
In the subject method, a nucleic acid molecule is isolated and adhered
through its phosphate backbone to the alignment path of a fabricated
substrate 11, as shown in FIGS. 2 and 3. A base specific label alters the
selected base and causes it to be distinguishable from the adjacent bases
by scanning microscopy. The label may be differentiated by size or
conductivity. For conductivity, a label comprising a single metal atom is
generally sufficient for detection. For topographic detection, a label
consisting of a group of at least about 10 .ANG., preferably at least
about 15 .ANG. in size, is sufficient for detection. The nucleic acid will
generally also contain an initiation marker distinguishable by scanning
microscopy, providing a reference for data analysis.
Analogs of bases with various groups are useful as a base specific label,
e.g. biotin, FITC, coumarin, digoxigenin, hydroquinone/quinone, etc.
covalently attached to the base through a linking group, as known in the
art. Other labels of interest include complexed metals, i.e. metallocenes,
e.g. ferrocene, dibenzenechromium, uranocene, bis(pentalenylnickel), etc.
attached to the base through any convenient covalent linkage, as known in
the art. It is preferable to use a linking group which is less than 5
.ANG. in length, such that the label will be situated over a single base.
The substrate is then positioned for analysis of the nucleic acid by a
scanning microscope. The scanning microscope may be scanning tunneling
(STM) or scanning force (SFM), collectively, (SXM). For most purposes, STM
will be used, because of its superior resolution. The microscope will have
at least one probe, which will scan along the alignment path. The
substrate may be analyzed in a wet cell, a controlled humidity cell, or a
dry cell. A wet cell may further include a power source for generating
electric potential, when used with a conductive binding surface as
described below.
The substrate will be formed from a base comprising a material amenable to
microfabrication, e.g. crystalline silicon; germanium; III-V compounds,
e.g. indium antimonide, cadmium telluride, silver iodide, gallium
arsenide, etc. The binding surface will usually be physically smooth, i.e.
a scanning microscope will distinguish between the topography of a nucleic
acid and the binding surface. Generally the base material will have a root
mean surface number (RMS no.) of less than about 25% of the height of a
DNA molecule, usually less than about 5 angstroms. Silicon is a convenient
and inexpensive base material.
The substrate will comprise one or more alignment paths having a nucleic
acid binding surface. The alignment paths may be of several different
structures and there may be one or a plurality of paths. The substrate may
vary in size, and will usually be large enough for convenient handling, at
least about 100 mm on a side, and not more than about 10 cm on a side. The
paths will usually be spaced at least about 1000 .ANG. apart and not more
than about 10,000 .ANG. apart. The substrate size and spacing allows a
very large number of paths to be fabricated relative to the number of
molecules that will be scanned. Therefore, the number of paths may be any
convenient number. Generally, the number of paths, multiplied by the
binding efficiency of DNA on the paths, will provide aligned molecules in
excess of the number that will be scanned. Generally the number of paths
will be at least about 100 paths, more usually at least about 1000 paths,
and may be as many as 10.sup.6 paths.
In one preferred embodiment, FIGS. 2A, 2B, the alignment path will be one
or more trenches which are at least about 20 .ANG. and not more than about
50 .ANG. wide. The trenches will at least be about 5 .ANG. and not more
than about 50 .ANG. deep, and will be at least about 1 .mu.m, usually at
least about 10 .mu.m long, and may be about 100 .mu.m or longer. The
nucleic acid binding surface 7 will be at the bottom of the trench and may
be an oxide surface covered with a film of highly polarized binding
material, such as 3-aminopropyltriethoxysilane; or a conductive metal
coating, e.g. an inert noble metal, such as gold, platinum, palladium,
etc. Alternatively, the alignment path may comprise a path 8, either level
with the surface of the substrate or, optionally, raised above the
surface. The path will be sufficiently wide to bind the phosphate backbone
of a nucleic acid, generally being at least about the width of the
phosphate backbone. Usually the path will be at least about 5 .ANG. and
not more than about 50 .ANG. wide. The height of the path may vary from a
level surface to about 50 .ANG. high, with the binding surface 9 at the
top of the path. A substrate with a conductive surface will usually be
employed in a wet cell, while the polar surface may be employed with
either wet or dry cells.
The substrate with the aligned nucleic acid molecule(s) is placed in a
scanning tunneling or atomic force microscope and the paths are scanned.
The scanning may be sequential by a single tip or parallel with multiple
tips. Scanning tunneling and force microscopes are well known and
commercially available. Tunneling and atomic force microscopy for the
surface characterization of molecules is described in Magonov (1993)
Applied Spectroscopy Reviews 28:1-121.
Turning now to FIG. 1, the substrate 11 with the aligned nucleic acid
molecule(s) is placed in the microscope and paths 12 are scanned. The
scanning may be sequential or simultaneous, if multiple tips (not shown)
are used. The scanning tunneling microscope includes a fine metal tip 13,
which may be tungsten, Pt-Ir, Pt-Rh, pure Pt, Au, and the like. For use in
a wet cell the tip will be insulated, with glass, silicon, wax, etc. The
tip is brought to within a few tenths of a nanometer of the surface of the
molecule. This is accomplished with piezoelectric transducers arranged to
form an orthogonal coordinate system: one transducer 14 is used to move
the tip toward and away from the sample, and the other two 16, 17 are used
to translate the tip laterally over the sample surface. A small bias
voltage 18 ranging from a few millivolts to a few volts is applied across
the gap between the tip and the sample. The tip is brought up to the
sample until a small current begins to flow, typically in the order of a
nanoampere. In a wet cell, it is desirable to keep the current below about
0.4 nA, for optimum resolution. A feedback loop 19 is then employed to
measure the tunneling current compared with a fixed reference value. The
tip is moved to maintain a constant current. Since the tunneling current
varies by about a factor of 10 for every 0.1 nanometer change in spacing
between the tip and the sample, the scanning tunneling microscope can
resolve height differences of less than 0.01 nanometers. It has a lateral
resolution of about 0.2 nanometers, roughly corresponding to the size
scale of the atoms comprising the tip and the sample.
An image of each aligned nucleic acid is obtained by applying a signal to
the Y piezo-transducer 17, thereby moving the tip along a selected path.
The error signal V from the feedback circuit corresponds to the height. An
increased signal represents a labeled base. This signal is held in the
processor's memory for each position along the path as a molecule is
scanned. Data collection is initiated when the initiation marker is
encountered, and completed when the path no longer contains detectable
nucleic acid. A signal is then applied to the X piezo-transducer 14 to
shift the tip to the next path. The path is then scanned and the signal is
recorded. This is repeated until all the paths are scanned. FIG. 4A shows
three scans of a molecule having labeled adenine bases, A. A plurality of
scans are preferable in view of the fact that during the labeling of the
polynucleotide all bases of the same kind are not labeled; therefore,
multiple samples are required to assure that all the bases of a particular
type are detected.
The processor compares the signals and provides a representation of all the
tagged bases, FIG. 4B. In compiling the sequence for each base, only paths
which are informative will be used. An informative path will generally
start with an initiation marker, and preferably match a known sequence
which immediately precedes or follows the initiation marker. Paths in
which a loop is detected, either physically or by sequence comparison will
be considered not informative. A consensus sequence for each base will be
compiled. A path which differs from the consensus at more than about 25%
of the positions will be considered not informative. As judged by the
above criteria, only informative paths will be considered. The number of
informative paths required for a complete data set, i.e. a data set in
which 100% of the labeled bases are recorded, will depend on the
efficiency of the labeling and alignment process. The number of paths
required can be calculated statistically. A base sequence data set is
compiled showing the position of each labeled base along the nucleic acid.
The data sets from the four bases which comprise either DNA or RNA are
then compiled as shown in FIG. 4C to provide a complete data set, showing
the position of each of the four nucleotide bases in the nucleic acid
molecule, FIG. 4D.
The fabrication of a substrate with trench alignment paths is illustrated
in FIGS. 5A-5H. A silicon substrate 21, FIG. 5A, is provided with a thin,
100 .ANG. nitride coating 22, FIG. 5B. A coating 23 of e-beam resist is
applied by spinning the coating onto the surface of the silicon nitride
film, FIG. 5C. A 500 .ANG. line is developed using a low energy e-beam.
The line is approximately 100 .mu.m long, although any length suitable for
retaining the molecule may be used. The resist is developed using readily
available microelectronic photolithography techniques, e.g. direct write
E-beam lithography, leaving a plurality of adjacent wenches 24, FIG. 5D.
The substrate with resist mask is anisotropically etched to a depth of
about 200 .ANG., removing the 100 .ANG. of nitride and 100 .ANG. of the
underlying silicon 21, FIG. 5E. A nitride film 26 is deposited on the
substrate to a thickness of 230 .ANG., FIG. 5F. The nitride is
anisotropically etched, leaving a 50 .ANG.-wide trench 27 with the silicon
exposed at the bottom, FIG. 5G. The wafer is then placed in an oxidizing
atmosphere and a 50 .ANG.-thick coating of oxide 28 is grown onto the
exposed silicon area, FIG. 5H. A shallower trench may be created by
stripping nitride from the top after oxidation. The surface of the oxide
is treated with a hydroxylated derivative of the base material, e.g.
silanol with silicon, hydroxylated gallium with gallium arsenide, etc. The
surface is then treated with a highly polar compound, such as
3-aminopropyltriethoxysilane, to form a nucleotide binding surface, as
described with reference to FIGS. 2A and 2B.
When the nucleic acid molecule is to be electrostatically bound, then the
alignment paths will have a conductive layer at the base of the trench.
FIGS. 6A-6F show the steps of a process for forming trenches with a gold
base. A silicon wafer 31, FIG. 6A, is provided with a gold layer 32, which
is approximately 750 .ANG. thick, FIG. 6B. A thin nitride film 33 is grown
onto the layer, FIG. 6C; a coating of resist 35 is spun onto the nitride
layer, FIG. 6D; a 500 .ANG. line is developed using a low energy e-beam;
the resist is developed using readily available microelectronic
photolithography techniques; and the substrate is anisotropically etched
stopping at the gold surface. The resist is then removed; a 230 .ANG.
nitride film 37 is deposited on the substrate, and the nitride is masked
and etched, leaving a 500 .ANG.-wide trench 38 with gold exposed on the
bottom of the trench as shown in FIG. 6F. The polynucleotide is held in
the groove by applying a voltage between the gold and polynucleotide and
maintaining a positive polarized surface, causing the phosphate ions to
adhere to the gold surface, providing a structure of the type as described
with reference to FIGS. 2A and 2B. While gold has been discussed as a
conductive surface, it shall be understood that other conductive
materials, as previously described, may also be used.
In FIGS. 7A-7E is shown a process for forming a flat or raised silicon path
on the surface of a substrate, illustrated in FIGS. 3A and 3B. A silicon
crystal base 41, FIG. 7A, is covered with a layer 42 of atomic hydrogen by
dipping the substrate in a 10 percent hydrochloric acid solution, FIG. 7B.
The substrate is then removed and dried by readily available
microelectronic drying techniques. The hydrogen film is removed along
paths 43 by the use of a scanning tunneling microscope, FIG. 7C. The
current associated with the scanning tunneling microscope will
simultaneously oxidize the silicon 44. The remaining hydrogen film is then
removed, FIG. 7D. Optionally, the sample can then be dipped in hydrozene
solution that will etch the silicon except for the areas covered by the
oxide 44, FIG. 7E, thus generating plateaus 45 of at least about 5 .ANG.
with an oxide layer on top. The paths are usually at least about 5 .ANG.
wide and usually not more than about 50 .ANG. wide. The height of the
paths is usually not more than about 50 .ANG.. The length of the path will
usually be at least about 1 .mu.m long, and may be about 100 or more .mu.m
long.
For all embodiments, the substrate may optionally include an initiation
site, as shown in FIG. 8A-D. The initiation site will provide an
initiation marker binding surface (IMBS), and will be positioned at one
end of the alignment path. The initiation marker will be distinguishable
by SXM, and may be a nucleic acid, protein, paramagnetic particle, etc.
Preferably, the IBMS will differ from the DNA binding surface, e.g. a
conductive DNA binding surface will be paired with a magnetic, or a polar
IBMS; a polar DNA binding surface will be paired with a conductive, or a
magnetic IMBS. The initiation site may be the width of the alignment path,
or may be substantially wider, from about 1.0 nm to about 100 nm in width.
In particular, a magnetic IMBS will require a larger initiation site to
accomodate ferromagnetic particles, where the particles are from about 10
nm to about 100 nm in diameter.
The substrate with initiation paths 70, FIG. 8A is coated with a resist 71,
FIG. 8B. An opening 72 is developed at one end of each alignment path
using readily available photolithography techniques, FIG. 8C. The opening
72 is etched, leaving a hole 73. The hole is plated with a conductive or
ferromagnetic surface, or a coating of oxide is grown on the surface. The
resist is then removed, FIG. 8E.
The DNA is prepared so that the sample contains a purified, linear
molecular species, that is, substantially all of the DNA molecules in the
sample will have the same sequence of nucleotides, or will be a mixture of
a single stranded DNA and its complementary strand. A number of methods
for generating purified DNA are known in the art. Of particular interest
is the use of the polymerase chain reaction (PCR) to amplify the DNA which
lies between two specific primers. The use of the polymerase chain
reaction is described in Saiki, et al. (1985) Science 239:487. A review of
current techniques may be found in Sambrook, et al. Molecular Cloning: A
Laboratory Manual, CSH Press 1989, pp. 14.2-14.33. The use of PCR allows
incorporation during DNA synthesis of base specific labels, and of labels
which provide for an initiation marker. PCR can be used to amplify DNA up
to about 35,000 nucleotides in length, usually DNA up to about 10,000
nucleotides in length (see J. Cohen [1994] Science 263:1564-1565). There
is no theoretical limit to how small a fragment may be amplified, however
in most cases the DNA will be at least about 18 nucleotides in length.
Use of PCR requires that there be a stretch of known sequence of at least
about 12 nucleotides, more usually at least about 18 nucleotides, from at
least one, and preferably both ends of the desired DNA molecule. The known
sequence is used to generate the specific primers used for the
amplification reaction. The reaction can proceed with a single primer, but
in that case will not provide exponential amplification. For sequencing
known genes in order to determine allelic or recombinational
polymorphisms, it is convenient to chose primer sequence(s) from known
conserved regions in the gene.
In sequencing DNA of unknown sequence one may ligate the unknown DNA to a
DNA of known sequence in order to generate primer sites. For example, a
fragment of DNA may be cloned into a number of commercially available
vectors, many of which have defined insertion sites, and for which PCR
amplification primers are sold. Most plasmid vectors can accomodate
fragments of about 10,000 nucleotides or less, vectors based on lambda
phage can accomodate fragments of up to about 24,000 nucleotides, and
cosmids can accept 30,000 to 40,000 nucleotide inserts. Larger inserts can
be cloned into artificial chromosomes, e.g. YACs.
Alternatively, amplification sites can be obtained by ligating
oligonucleotide linkers having a known sequence to the desired DNA
fragment. DNA fragments are obtained using any one of a variety of
chemical and enzymatic cleavages, e.g. restriction endonucleases, single
or double stranded DNAses, .sup.32 P scission, shear force, Fenton
reagent, EtBr cleavage, etc. The fragment is then ligated to single or
double stranded oligonucleotides with ligase, preferably T4 ligase. If the
original fragment population contained a mixture of DNA species, then it
is desirable to dilute the ligated fragments to an average of one molecule
per PCR reaction before amplification, in order to amplify a single DNA
species.
Samples containing a single DNA species can also be prepared by
conventional methods, e.g. cloning into vector, preferably a high copy
number plasmid or phage, and/or isolation of the DNA by density gradient,
size fractionation, e.g. sucrose gradient, gel electrophoresis, affinity
chromatography, molecular exclusion chromatography, etc. In order to have
a sample containing linear molecules with defined ends, the DNA will
usually be cleaved with a restriction endonuclease before the final
purification step.
RNA may also be sequenced by the subject methods. It is most convenient,
however, to convert the RNA into cDNA before sequencing, using
conventional methods of oligo dT, hexamer priming, etc., and reverse
transcriptase synthesis. If the RNA molecule itself is to be sequenced,
then it may be purified by affinity chromatography, size fractionation,
etc. Where the methods for alignment, complementation labeling, and
scanning refer to DNA, it should be understood that RNA may also be used.
The DNA is made single stranded before scanning. It is denatured by any
convenient means, such as heating to >95.degree. C., treatment with NaOH,
etc. The denatured DNA may be applied directly to the substrate, in which
case both strands will be scanned. In a preferred embodiment, the
complementary strands will be separated by strand separation gel
electrophoresis, affinity chromatography, etc. and one of the
complementary strands will be scanned.
If the DNA sample has been prepared by PCR, affinity chromatography
provides a convenient method for strand separation. One of the
amplification primers is end labeled with a group having a convenient
reciprocal binding member, i.e. biotin, digoxigenin, fluorescein
isothiocyanate (FITC), coumarin, etc. Such labeling methods are well known
in the art, and can be performed with commercially available kits. In this
way, one strand of the amplified DNA will have a label. After
denaturation, the DNA is applied to an immobilized specific binding member
for the end label, e.g. avidin or streptavidin for biotin, specific
antibodies, etc., immobilized on a column, beads, microtiter plate, etc.
The labeled DNA will bind to the immobilized binding member, and the
unbound DNA is washed away. The bound DNA is eluted by any convenient
means, such as a competitor molecule, e.g. free label, free binding
member, etc.; with an increased ionic strength buffer, etc.
An initiation marker is not required for the practice of the invention, but
it is convenient in providing a starting point for data analysis. An
initiation marker is any group which is attached to the DNA, and which is
distinguishable by SXM, as previously described. The initiation marker may
be attached to the DNA prior to the DNA being bound to the alignment
substrate, or may be present on the substrate before the DNA is added.
Suitable initiation markers are proteins, e.g. DNA binding proteins such
as helix-turn-helix specific DNA binding proteins and zinc finger specific
binding proteins; DNA binding transcription factors, restriction
endonucleases having an inactivated cleavage function; proteins which
specifically bind labeled DNA, such as avidin and streptavidin to
biotinylated DNA, antibodies to haptenated DNA, e.g. DNA conjugated to
digoxigenin, FITC, coumarin, etc.; oligonucleotides which will
specifically hybridize to a portion of the DNA, particularly
oligonucleotides with chemically modified bases; or direct detection of
end-labeled DNA. The binding group may conveniently be conjugated to a
superparamagnetic particle, as described is U.S. Pat. No. 4,452,773, where
the particle provides a large topographic marker of about 10-100 nm
diameter.
If PCR has been used to generate the DNA sample, then the known primer
sequence may be conjugated to the initiation marker. Preferably the
inition marker will be used to end-label the amplification primer. During
the final compilation of the sequence, the sequence of the amplification
primer will be used to verify that a scan is informative.
When the DNA is linearly aligned on the substrate it may be useful,
particularly with large fragments, to anchor one end of the DNA to one end
of the alignment path. This is conveniently accomplished by using a
substrate having an initiation marker binding site, and using the
initiation marker as an anchor. The initiation marker may be bound to the
substrate by one of several methods. A marker conjugated to a
superparamagnetic particle may be bound to a ferromagnetic initiation
site. A DNA or protein marker may be bound to the initiation site through
electrostatic interaction with a polar coating, or to a conductive surface
by application of a positive potential.
For sequencing, the DNA will be labeled with a base-specific marker that is
distinguishable by SXM either by size (topography) or by conductivity, as
previously discussed. In general, for reading one strand, four samples
will be prepared, each corresponding to a different labeled base species.
If both strands of a DNA will be scanned, then it is sufficient to label
only two of the bases species, either both purines or both pyrimidines,
where the complementary strand will provide the missing information. The
base-specific label can be provided by one of two general techniques:
synthetic labeling and complementation labeling.
Synthetic labeling exchanges one of the normal base species for a
chemically altered analog during synthesis of the DNA. The method is
appropriate when the DNA will be synthesized in vitro, particularly in
combination with PCR. Because the label is incorporated during synthesis,
it must be tolerated by the DNA polymerase. Generally the label will be
less than about 1 kd, usually less than about 0.5 kd. Polymerases which
lack proofreading functions are generally more tolerant of substituents,
e.g. Klenow fragment, Taq polymerase, etc. In order to amplify long
stretches of DNA, however, it may be necessary to use a mixture of
polymerases, where at least one polymerase has a proof-reading function.
The purity, and efficiency of incorporation of the base analog will
determine the percent of bases which will be labeled. In the final
synthesized DNA product, usually at least about 10% of the targeted base,
i.e. adenine for adenine derivatives, etc., will be labeled, more usually
at least about 25%, preferably at least about 50%, and may be as high as
90%. The percent of labeled bases can be determined by various physical
and enzymatic parameters. For example, a number of substituents are also
fluorochromes, i.e. FITC, coumarin, etc., and the density can be
calculated by fluorimetry. As discussed previously, the efficiency of
labeling will determine the number of scans which must be performed.
Labeling by complementation relies on specific base pairing interactions.
The label will be a free nucleotide or dinucleotide. It may be the
naturally occurring base species, or an analog, preferably having a metal
containing substituent group. It does not require a separate synthetic
reaction, and can comprise substituents which are not tolerated by
polymerases. The strength of the hydrogen bonding between bases is weaker
than the covalent bonds used in synthetic labeling, and is an equilibrium
reaction, so the conditions for scanning must be chosen to optimize
pairing.
A single stranded DNA sample is combined with the complementation label in
solution. The labeling may be performed while the DNA is aligned on a
substrate, or prior to alignment. The hybridization conditions will
maximize the interaction between base pairs, but will not allow
non-specific pairing. Usually at least about 50% of the possible base
pairs will be formed at a given time, more usually at least about 75% will
be formed.
Suitable complementation labels include the four naturally occurring
deoxyribonucleotides and ribonucleotides, and nucleotide analogs,
including ferrocene conjugated nucleotides, hydroquinone/quinone,
nitronyl, nitroxide, porphyrins, psoralens, coumarin, etc. Dinucleotides
may be used to increase the strength of the bonding, using natural
nucleotides or analogs. The use of dinucleotides will require that 16 sets
of scans be performed, in order to account for all possible dinucleotides.
The labeled single stranded DNA will be aligned on the substrate for
scanning. The substrate may have an initiation marker already bound, or
the DNA and initiation marker may be added simultaneously. If the
initiation marker is a large species, i.e. a protein or ferromagnetic
particle, then it is preferable to bind the initiation marker to the
substrate prior to adding the DNA. After alignment, the distance that the
DNA will extend along the alignment path will be proportional to the
number of base pairs of DNA. Usually the DNA will extend not more than
about 10 .mu.m, more usually the DNA will extend not more than about 5
.mu.m, and may only extend about 100 to 1000 nm.
The DNA will be applied to the substrate for alignment in a dilute
solution. It has been found that at concentrations greater than 100
.mu.g/ml, aggregates predominate. Generally the DNA concentration will be
greater than about 0.1 nM and not more than about 1 .mu.M. The optimum
concentration may be determined by titration, followed by scanning for the
presence of isolated molecules. Conveniently, an aqueous solution will be
used, although various other solvents, e.g. alcohols, ethers, toluene,
benzene, ammonia, alkanes, etc. may find use. The salt and buffer
composition will vary depending on whether the scan will be performed in a
wet or a dry cell. A phosphate buffer is preferred in a wet cell when
current will be applied, as most other common buffer salts are easily
oxidized. The phosphate buffer will be usually at least about 0.5 mM
NaPO.sub.4, more usually at least about 1 mM NaPO.sub.4, and usually not
more than about 50 mM NaPO.sub.4. When a dry cell scan is to be performed,
low salt solutions are used, in order to minimize salt crystals forming
after drying. Usually at least about 0.1 mM NaCl or other suitable salt,
i.e. KCl, MgCl.sub.2, CaCl.sub.2, etc., more usually at least about 1 mM,
and not more than about 50 mM, more usually not more than about 10 mM
salt, will be used.
The substrate will be immersed in the DNA solution for at least about 10
minutes, usually at least about 1 hour, and may be immersed for as long as
one day, or longer. The solution may be gently circulated, by rocking,
rotation, etc. In one preferred embodiment, after the DNA is bound to the
initiation marker, the solution is circulated directionally by peristaltic
pump, etc., moving from the position of the anchor molecule and following
the alignment paths across the substrate.
The DNA is then bound to the substrate surface. In a dry cell scan, the
substrate will be removed from the DNA solution, optionally washed with a
DNA-free solution, and allowed to dry. In a wet cell scan, the DNA will
generally be bound by applying current to the solution, usually at least
about +10 mV, more usually at least about +100 mV, and usually not more
than about +250 mV. The current will usually be applied for at least about
10 minutes, and usually for not more than about 60 minutes. The DNA will
remain adhered in the absence of a positive voltage potential, and the
substrate may be rinsed in DNA-free solution after adherence.
The scanning tunneling microscope is used to characterize and quantify
topographic and electronic changes along the leng | | |
