Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1996-02-13
1998-08-04
Zitomer, Stephanie W.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
935 77, 935 78, 536 243, C12Q 168, C07H 2104, C07H 2102
Patent
active
057891672
DESCRIPTION:
BRIEF SUMMARY
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 hybridi
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GeneVue, Inc.
Haliday Emily M.
Terlizzi Laura
Zitomer Stephanie W.
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