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| | Reference | Relevancy | Comments | Brandiff et al., "DNA Sequence Mapping by Fluorescence In Situ Hybridization," Environmental and Molecular Mutagenesis, 18:259-262 (1991).
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[0 after 0 votes] | | Bustamante, "Direct Observation and Manipulation of Single DNA Molecules Using Fluorescence Microscopy," Ann. Rev. Biophys. Biophys. Chem., 20:415-446 (1991).
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[0 after 0 votes] | | Dunn et al. "A Novel Method to Map Transcripts: Evidence for Homology between an Adenovirus mRNA and Discrete Multiple Regions of the Viral Genome," 12:23-36 (1977).
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[0 after 0 votes] | | Hahnfeldt et al., "Polymer models for interphase chromosomes," Proc. Natl. Acad. Sci. USA, 90:7854-7858 (1993).
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[0 after 0 votes] | | Lawrence et al., "Interphase and Metaphase Resolution of Different Distances Within the Human Dystrophin Gene," Science, 249:928-932 (1990).
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[0 after 0 votes] | | Leversha, "FISH and the technicolour revolution," The Medical Journal of Australia, 158:545-551 (1993).
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[0 after 0 votes] | | Maniatis et al., "Molecular Cloning, A Laboratory Manual," Cold Spring Harbor Laboratory (N.Y.) 181, paragraph 4 (1982).
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[0 after 0 votes] | | Matthews et al., "Analytical Strategies for the Use of DNA Probes," Analytical Biochemistry, 169:1-25 (1988).
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[0 after 0 votes] | | Nicholls et al., "Nucleic Acid Analysis by Sandwich Hybridization," Journal of Clinical Laboratory Analysis, 3:122-135 (1989).
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[0 after 0 votes] | | Ried et al., "Simultaneous visualization of seven different DNA probes by in situ hybridization using combinatorial fluorescence and digital imaging microscopy," Proc. Natl. Acad. Sci. USA, 89:1388-1392 (1992).
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[0 after 0 votes] | | Saiki et al., "Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes," Proc. Natl. Acad. Sci. USA, 86:6230-6234 (1989).
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[0 after 0 votes] | | Smith et al., "Observation of Individual DNA Molecules Undergoing Gel Electrophoresis," Science, 243:203-206 (1989).
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[0 after 0 votes] | | Smith et al., "Direct Mechanical Measurements of the Elasticity of Single DNA Molecules by Using Magnetic Beads," Science, 258:1122-1126 (1992).
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[0 after 0 votes] | | Smith et al., "Electrophoretic Charge Density and Persistence Length of DNA as Measured by Fluorescence Microscopy," Biopolymers, 29:1167-1173 (1990).
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[0 after 0 votes] | | Southern, "Detection of Specific Sequences Among DNA Fragments Separated by Gel Electrophoresis," J. Mol. Biol., 98:503-517 (1975).
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[0 after 0 votes] | | Trask, "Fluorescence in situ hybridization," TIG, 7:149-154 (1991).
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[0 after 0 votes] | | van den Engh et al., "Estimating Genomic Distance from DNA Sequence Location in Cell Nuclei by a Random Walk Model," Science, 257:1410-1412 (1992)..
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Description |
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FIELD OF THE INVENTION
The present invention relates to analysis of DNA sequences and, in particular, to optically determining the position of an oligonucleotide sequence on a large DNA molecule.
BACKGROUND OF THE INVENTION
There have been significant advances in DNA analysis methods in recent years. At present, there are several distinct methods for DNA analysis. There are several methods which involve use of an oligonucleotide sequence as a probe to bind to a
complementary oligonucleotide sequence which may be present in the sample. This method takes many embodiments from fluorescent in situ hybridization of chromosomes in tissue sections to dot blot analysis of DNA fragments.
The are several DNA analysis methods which involve amplification, usually using the polymerase chain reaction method. In that method, primer, usually on the order of 20 to 30 nucleotides, are used to amplify a region of DNA as large as a few
kilobases. The amplified region can be analyzed by a number of methods, including use of probes or sequencing. Alternatively, analyses can be based on the pattern of fragments produced in the amplification.
In addition, DNA can be digested with a restriction endonuclease and the fragment patterns produced can be analyzed to determine alleles present in the sample DNA. Numerous additional methods are used in mapping and cloning to try to identity
the location of a gene or to determine which variant of a known gene is present in a sample. All of these analysis methods have advantages and disadvantages and additional, more informative methods are still being sought.
SUMMARY OF THE INVENTION
The present invention provides a method for analyzing a sample oligonucleotide sequence. The method comprises contacting the sample oligonucleotide sequence with an anchor sequence which comprises an oligonucleotide sequence which is immobilized
to a support and which hybridizes with the sample oligonucleotide sequence. The sample oligonucleotide sequence is also contacted with a probe comprising an oligonucleotide sequence which hybridizes to a target oligonucleotide sequence to be detected.
The contacting is performed in a suitable buffer to form a complex. The resultant complex is subjected to a field which moves unbound oligonucleotide sequences away from the anchor sequence in the direction of the field. Whether the probe is bound to
the sample oligonucleotide sequence is determined to determine whether the target oligonucleotide sequence is present in the sample oligonucleotide sequence. Preferably, the complex is subjected to the field for a time sufficient to extend the sample
oligonucleotide sequence and the position of the probe in relation to the anchor sequence is also determined.
The field can be a magnetic or, preferably, an electric field in which the oligonucleotide sequences migrate based on their inherent charges. Preferably, the probe is labeled with a fluorochrome, preferably a fluorochrome which is present in a
bead, which facilitates use of a fluorescent microscope to determine the presence or position of the probe on the sample oligonucleotide sequence.
In one embodiment, the method is used for mapping. The method can determine whether a first and a second target are on a molecule of sample DNA by having the anchor sequence hybridize to the first oligonucleotide sequence and the probe hybridize
to the second oligonucleotide sequence. Alternatively, the nucleotide sequence near the end of the sample DNA molecule to be mapped can be determined using short probes of random sequences, identifying a pair of probes which hybridize toward the end of
the sample DNA molecule, and using one probe and the complement of the other to amplify the segment between the probes by the polymerase chain reaction. The nucleotide sequence of this segment can then be determined, and one of the strands from this
amplified segment can then be used as a new anchor sequence, to walk down the DNA molecule.
In another embodiment, sample DNA is characterized (fingerprinted) using an anchor sequence which hybridizes to a conserved region in the sample DNA and a plurality of probes of random or arbitrary sequence of from about 5 to about 15 bases.
Following hybridization, the sample DNA is extended and unbound probes are removed by the field. The positions of the probes in relation to the anchor sequence are determined. The method can be used in paternity and other forensic applications by using
the same anchor and probes on the mother and putative father's DNA or crime scene and suspect DNA.
In another embodiment, the method can be used to determine whether a test oligonucleotide sequence is present in the sample oligonucleotide sequence. In that embodiment, the anchor sequence comprises an immobilized, conserved oligonucleotide
sequence known to hybridize with an oligonucleotide sequence in the sample oligonucleotide sequence and the probe hybridizes with the test oligonucleotide sequence. The test oligonucleotide sequence can be any oligonucleotide sequence of interest, such
as, for example, a sequence characteristic of a disease gene allele. In a preferred embodiment, a plurality of different probes which bind to different test sequences are used. Each of the probes is labeled with a different fluorochrome or combination
of fluorochromes.
A device for performing the method and reagents are also described.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates a top view of one embodiment of a DNA analysis device of this invention, prior to attachment of a cover.
FIG. 2 illustrates a side view of another embodiment of a DNA analysis device of this invention having a composite electrode.
FIG. 3 illustrates a top view of another embodiment of a DNA analysis device of this invention.
FIG. 4 illustrates a side view of the embodiment of the device illustrated in FIG. 3.
DESCRIPTION OF THE INVENTION
The present invention provides a method of analyzing oligonucleotide sequences in which the sample oligonucleotide sequence is contacted with an anchor sequence and a probe in a suitable buffer for hybridization. The anchor sequence is an
oligonucleotide sequence immobilized to a support which hybridizes with a conserved oligonucleotide sequence present in the sample oligonucleotide sequence. The probe can be any oligonucleotide sequence of interest depending on the type of analysis to
be performed. Following formation of a complex by hybridization of the sample oligonucleotide sequence to the anchor sequence, the oligonucleotide sequences are subjected to a field which moves unbound probes away from the anchor sequence. The field
preferably also extends the complexed sample oligonucleotide sequence in the direction of the field, thereby facilitating determining the distance between the anchor sequence and the probe and thus the relative positions of the sequences to which the
probe and anchor sequence bind. In a preferred embodiment, the probes are labeled with a fluorochrome, facilitating determining distances to within about forty nucleotides using a fluorescent microscope.
In one embodiment, the method can be used for mapping by determining whether two oligonucleotide sequences are on the same molecule of DNA and the distance and direction of the sequences. In another embodiment, the method can be used to
characterize DNA sequences and to determine either whether sample and test DNA are from the same individual or whether a putative father can be the father of a child. In another embodiment, sample DNA can be analyzed to determine whether one or a
plurality of oligonucleotide sequences, such as oligonucleotide sequences characteristic of a disease gene, are present. A device and reagents facilitating the method are also described.
Reagents and Device for Practicing the Method
The reagents and device used to practice the method of this invention are described below, followed by a description of the method.
The Sample Oligonucleotide Sequence
The sample oligonucleotide sequence can be any oligonucleotide sequence. The present invention is particularly advantageous in analyzing long oligonucleotide sequences; e.g., greater than sizes which can be readily analyzed by polymerase chain
reaction methods (2-3 kilobases). The sample oligonucleotide sequence can be DNA or RNA and can be single-stranded or double-stranded. Conveniently, the sample is a single-stranded oligonucleotide sequence, preferably a DNA sequence. However, the
method can be performed wherein either the sample oligonucleotide sequence or the anchor sequence is double-stranded and the complex formed by hybridization is a triple helix. The binding rules for triple helix formation are well known and are described
in International Application No. PCT/US89/05769 (Publication No. WO 90/06934, published Jun. 28, 1990) to Hogan et al., for example. That application is incorporated herein by reference in its entirety.
Extraction of sample oligonucleotide sequences from cells and preparation of single-stranded oligonucleotide sequences are well known. All nucleated cells contain genomic DNA and RNA and, therefore, are potential sources of the sample
oligonucleotide sequence. For higher animals, peripheral blood cells are typically used rather than tissue samples. As little as 0.01 to 0.05 cc of peripheral blood provides sufficient DNA for analysis. Hair, semen and tissue can also be used as
samples. In the case of fetal analyses, placental cells or fetal cells present in amniotic fluid can be used. When the sample oligonucleotide sequence is DNA, the DNA is isolated from nucleated cells under conditions that minimize DNA degradation.
Typically, the isolation involves digesting the cells with a protease that does not attack DNA at a temperature and pH that reduces the likelihood of DNase activity. For peripheral blood cells, lysing the cells with a hypotonic solution (water) is
sufficient to release the DNA. Generally, red white blood cells which do not contain nuclei are separated from the nucleated white blood cells prior to extraction of DNA.
DNA isolation from nucleated cells is described by Kan et al., N. Engl. J. Med. 297:1080-1084 (1977); Kan et al., Nature 251:392--392 (1974); Kan et al., PNAS 75:5631-5635 (1978); and in Sections 2.1 and 2.2 of Ausubel, Short Protocols in
Molecular Biology, Second Edition, John Wiley and Sons, New York (1992). In addition, commercial DNA purification kits are available. (See page 190-191 in BioRad Laboratories: BioRad Laboratories Catalog: Life Science Research Products. Hercules,
Calif., Bio-Rad Laboratories (1993) which describes the InstaGene DNA purification kit.) Each of the above references is incorporated herein by reference in its entirety. Extraction procedures for samples such as blood, semen, hair follicles, semen,
mucous membrane epithelium and other sources of genomic DNA are well known. For plant cells, digestion of the cells with cellulase releases DNA. Thereafter DNA is purified as described above.
Procedures for isolating RNA from cells are described in Sections 4.1 and 4.2 of Ausubel, Short Protocols in Molecular Biology, Second Edition, John Wiley and Sons, New York (1992) and pages 38 to 50 of Pharmacia Corporation: Pharmacia Catalog:
Molecular and Cell Biology, Pharmacia Corporation, 1993.
The extracted oligonucleotide sequence can be purified by dialysis, chromatography, or other known methods for purifying oligonucleotides prior to analysis. Typically, the oligonucleotide sequence is not purified prior to analysis by the methods
of this invention. However, usually, RNA in the sample is digested when the sample oligonucleotide sequence is DNA, and DNA is digested when the sample oligonucleotide sequence is RNA.
The method can be performed on a sample oligonucleotide sequence which is less than about 200 kb using normal handling techniques in preparing the sample oligonucleotide sequence. However, when the sample oligonucleotide sequence is greater than
about the size of T4 DNA (greater than about 200 kb), special handling techniques are used in preparing the sample oligonucleotide sequence.
The techniques for handling long oligonucleotide sequences are well known and involve processing techniques which minimize forces which can shear oligonucleotide sequences. For example, cells containing the sample oligonucleotide sequence can be
lysed in a gel to preserve the integrity of the oligonucleotide sequences. The gel is usually a gel which is solid at room temperature and liquid at an elevated temperature such as 37.degree. C. For example, on page 84 in Promega Corporation:
Catalogue: Biological Research Products, Madison, Wis., Promega Corporation, (1992-3) describes preparation of intact yeast chromosomes from whole cells embedded in 0.8% low melt point agarose. The cells are lysed and deproteinized in situ as described
in McClelland et al., Nucl. Acid Res. 15:5985 (1987).
A fragment of the gel is removed for analysis to avoid pipetting, mixing, and other techniques which exert shear forces. The reagents for the analysis method are allowed to diffuse into the gel to achieve the desired effects on the cells
containing the DNA or the DNA itself. When using such samples, the analysis procedure is performed at an elevated temperature at which the gel is liquid.
When the sample oligonucleotide sequence is DNA, preferably, the DNA strands are separated into single stranded DNA prior to analysis. This strand separation can be accomplished by a number of methods including physical or chemical means. A
preferred method is the physical method of separating the strands by heating the DNA until it is substantially (approximately 93%) denatured. Heat denaturation involves temperatures ranging from about 80.degree. to 105.degree. C. for times ranging
from about 15 to 30 seconds. Typically, heating the DNA to a temperature of from 90.degree. to 93.degree. C. for about 30 seconds to about 1 minute is sufficient. Alternatively, sample oligonucleotide sequences can be denatured by heating at
75.degree. C. for 5 minutes in 70% formamide (final concentration), 0.3M NaCl, 0.01M Tris-HCl, pH 8.5, 0.001M EDTA.
The Anchor Sequence
The anchor sequence is an oligonucleotide sequence immobilized to a support which hybridizes with a conserved oligonucleotide sequence present in the sample oligonucleotide sequence. The anchor sequence is also unique in the total
oligonucleotide population the sample oligonucleotide has been obtained from; e.g. human genomic DNA. The anchor sequence can be any oligonucleotide sequence which hybridizes to the sample sequence, forming a complex which is sufficient to immobilize
the sample oligonucleotide sequence in the presence of an electric field that removes unbound probes. Preferably, the anchor sequence forms a complex which is sufficient to immobilize the sample oligonucleotide sequence in the presence of an electric or
magnetic field that extends or elongates the sample oligonucleotide sequence in the direction of the field, thereby facilitating determining the distance of the probe from the anchor sequence.
The anchor sequence can be a DNA sequence or an RNA sequence. The anchor sequence is preferably a single-stranded oligonucleotide sequence to provide sufficient binding to immobilize the sample oligonucleotide sequence in the electric field.
However, double-stranded oligonucleotide sequence which hybridizes to a single-stranded sample oligonucleotide sequence to form a triple helix are also contemplated. In addition, the use of a peptide nucleic acid oligomer sequence which can displace one
strand of a double helix to hybridize to the sequence on the second strand is contemplated (see Peffer et al., Proc. Natl. Acad. Sci. USA 90:10648-10652 (1993)).
The anchor sequence is preferably a conserved oligonucleotide sequence to ensure that the sample sequence contains a sequence which hybridizes to the anchor sequence. In addition, the anchor sequence is sufficiently long to ensure that the
sequence is unique. Such considerations are well known and are substantially the same considerations used in selecting a probe for various techniques, such as for in situ hybridization (ISH).
When distances between the anchor sequence and probe are to be determined, the anchor sequence is sufficiently long and sufficiently complementary to the target sequence in the sample oligonucleotide sequence to ensure that the sample
oligonucleotide sequence remains hybridized to the anchor sequence in the presence of the field used to extend the sample oligonucleotide sequence. Preferably the anchor sequence is from about 10 to about 100 bases, preferably from about 20 to about 40
bases, most preferably about 30 bases.
As the length of the anchor sequence increases, the degree of specificity for a unique sequence increases. In addition, the strength of the bonds formed by hybridization increases as the length of the anchor sequence increases, providing the
sample oligonucleotide sequence/anchor sequence complex with the ability to remain a complex and thus immobilized in a stronger field. However, longer oligonucleotide sequences are more difficult and expensive to prepare than shorter oligonucleotide
sequences. Therefore, these conflicting interests are balanced in selecting the length of the anchor sequence.
As in ISH applications, the anchor sequence need not be the Watson Crick complementary sequence to the target sequence on the sample oligonucleotide sequence so long as the binding between the anchor sequence and the sample oligonucleotide
sequence is sufficient to immobilize the sample oligonucleotide sequence, as stated above. However, the anchor sequence is preferably complementary to the target oligonucleotide sequence in the sample oligonucleotide sequence to optimize the ability of
the sample oligonucleotide sequence/anchor sequence complex to remain complexed when subjected to the field.
Methods for selecting and synthesizing such oligonucleotide sequences are well known. In addition, considerations for selecting anchor sequences for particular applications are described in detail hereinafter.
The anchor sequence can be bound to the support either directly or through a linking group by any of the methods described hereinafter. The linking group can be an irrelevant oligonucleotide sequence which allows the anchor sequence to extend
from the surface of the support to facilitate binding of the entire anchor sequence to a sample oligonucleotide sequence. Such irrelevant oligonucleotide sequences are preferably sequences which are flexible and can bend, such as for example, poly-T
having at least three nucleotides, preferably at least 5 nucleotides.
Alternatively, the linking group can be a polypeptide, preferably a relatively short polypeptide. Methods for linking peptides or proteins to nucleic acids and to the solid phase are discussed above. The peptide linker is preferably from four
to twenty, preferably from five to 10, amino acid residues in length. The peptide does not include with a large number of positively charged groups to avoid non-specific binding of oligonucleotide. The peptide preferably also does not have a large
number of negative charges to facilitate binding to the anchor sequence. Preferably the peptide does not include very hydrophobic amino acids, such as leucine, because leucine is relatively insoluble, thus difficult to work with, and might bind to the
surface of the polystyrene bead. A polypeptide with one amino group at one end and one carboxyl group at the other is preferred. Homopolymers are preferred.
Most preferred linkers are polyglycine, polyalanine, and polyproline. Polyalanine forms an alpha helix which would hold the anchor sequence away from the support surface or, when linking the probe to the label, would hold the probe
oligonucleotide sequence out from the bead. Polyproline forms a rigid coil (not an alpha helix), which can also be used to hold the oligonucleotide sequence out from bead or support. In addition, a large variety of other small organic molecules can
also be used.
The anchor can be linked to the solid support at an internal oligonucleotide in the anchor sequence, so that there are two "arms" of the anchor sequence hanging from the linker. A restriction site near the end of one of the arms then provides a
means for cutting that arm off. DNA with a linker site anywhere in the sequence is available commercially from; e.g., The Midland Certified Reagent Company (Midland, Tex.) and Peninsula Laboratories (Belmont, Calif.).
The support to which the anchor sequence is immobilized can be any solid phase on which hybridization can be performed. The solid phase is preferably a glass or plastic surface, conveniently a microscope slide. For use with an epifluorescent
microscope, the slide is preferably opaque to absorb light which may otherwise be reflected. A preferred support is described hereinafter in describing a preferred device of this invention.
The anchor sequence is bound to the support by conventional methods. The goal of the surface chemistry is to attach a few sample oligonucleotide sequence DNA to the support through hybridization with the anchor sequence, as it is necessary to
observe only a few molecules. Thus, the chemistry need not be very efficient. This is in contrast to the requirements in, for example, procedures for solid support DNA synthesis machines, where the goal is to produce as much product as possible.
Therefore, a number of techniques are suitable.
For example, to attach the anchor sequence to the support, a portion of the support is coated with a solution of gelatin, which is then allowed to dry into a film coating the surface of the support. The gelatin is then cr | | |
