Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-05-06
2001-02-06
Schwartzman, Robert A. (Department: 1635)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069100, C536S023100, C536S024300
Reexamination Certificate
active
06183958
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to methods and reagents for detecting mispaired nucleotides in duplex nucleic acids for use, for example, in identifying genetic variations in nucleic acid sequences for research, therapeutic, and diagnostic applications.
Genetic variation occurs at approximately 1 out of every 100 bases within the genome. Research aimed at discovering genetic variation associated with diseases or disease therapies, as well as diagnostic tests aimed at using genetic information to manage patient care, requires efficient methods for detecting and typing genetic variance in various test sequences. Variances may be detected by a variety of methods. Many of these methods require the use of a probe with a unique sequence (representing a single allelic form of the sequence) as a reference by which to identify differences in the sequences of homologous DNA segments in patient test samples. Probes with a unique sequence are commonly produced from cloned DNA or cDNA. However, the use of probes from cloned DNA limits the ability to identify variances to DNA segments for which such clones are readily available, or alternatively requires the cloning of each DNA segment to be analyzed.
SUMMARY OF THE INVENTION
The present invention involves a general method for obtaining and using probes with unique sequences (monoallelic probes) from certain cells or tissues that are hemizygous for genes, chromosomal segments, or chromosomes that are the object of the analysis. Such probes are useful for the analysis of sequence variation, for example, by heteroduplex formation.
Accordingly, in a first aspect, the invention features a method for detecting a nucleotide mismatch in a nucleic acid sample that includes the steps of: (a) providing a nucleic acid probe derived from a hemizygous cell, the probe being complementary to a hemizygous chromosome or segment thereof present in the hemizygous cell; (b) forming a duplex between the nucleic acid sample and the probe; and (c) determining if the duplex contains a nucleotide mismatch.
In various embodiments of this aspect of the invention, the determining step is carried out using a denaturing gradient gel electrophoresis technique; the nucleotide mismatch represents a sequence variance in a population; the probe has a known sequence, and may be detectably labeled; the hemizygous cell results from the loss of a chromosome or segment thereof; the hemizygous cell includes multiple copies of the hemizygous chromosome or segment thereof; the hemizygous cell may be human; the hemizygous cell may be an immortalized cell; the hemizygous cell may be derived from a complete hydatidiform mole, an ovarian teratoma, an acute lymphocytic leukemia, an acute myeloid leukemia, a solid tumor, a squamous cell lung cancer, an endometrial ovarian cancer, a malignant fibrous histiocytoma, or a renal oncocytoma; the hemizygous cell may be NALM-16 or KBM-7; and the hemizygous cell may be derived from a haploid germ cell.
In yet other embodiments of the first aspect of the invention, the presence of the nucleotide mismatch correlates with a level of therapeutic responsiveness to a drug or other therapeutic intervention; the presence of the nucleotide mismatch indicates a disease or condition, or a predisposition to develop the disease or condition; the nucleic acid probe is produced by amplifying at least a portion of the hemizygous chromosome or segment thereof to produce the probe; the determining step utilizes a protein that binds or cleaves the nucleotide mismatch, for example, MutS or a resolvase (e.g., T4 endonuclease VII), and the determining step utilizes a chemical agent that detects the nucleotide mismatch. This method of the first aspect of the invention may be used to determine the haplotype of the nucleic acid sample.
In a second aspect, the invention features a method for detecting a nucleotide mismatch in a nucleic acid sample that includes the steps of: (a) providing a nucleic acid probe derived from a sex chromosome; (b) forming a duplex between the nucleic acid sample and the probe; and (c) determining if the duplex contains a nucleotide mismatch.
In a third aspect, the invention features a method for detecting a nucleotide mismatch in a nucleic acid sample that includes the steps of: (a) providing a nucleic acid probe derived from a somatic cell hybrid, the probe being complementary to a chromosome or segment thereof, where only one allele of the chromosome or segment thereof is present in the somatic cell hybrid; (b) forming a duplex between the nucleic acid sample and the probe; and (c) determining if the duplex contains a nucleotide mismatch.
In a fourth aspect, the invention features a kit for detecting a nucleotide mismatch that includes: (a) a nucleic acid probe derived from a hemizygous cell, the probe being complementary to a hemizygous chromosome or segment thereof; and (b) a means for detecting a nucleotide mismatch. In preferred embodiments, the probe is detectably labeled; the detecting means is a protein that binds or cleaves the nucleotide mismatch, for example, MutS or a resolvase (e.g., T4 endonuclease VII); and the detecting means is a chemical agent that detects the nucleotide mismatch.
In a fifth aspect, the invention features a method for producing a nucleic acid probe for the detection of a nucleotide mismatch that includes the steps of: (a) providing a hemizygous cell having at least one hemizygous chromosome or segment thereof, and (b) amplifying at least a portion of the hemizygous chromosome or segment thereof to produce the probe.
In a sixth aspect, the invention features a method for producing a nucleic acid probe for the detection of a nucleotide mismatch that includes the steps of: (a) providing nucleic acid from a hemizygous cell having at least one hemizygous chromosome or segment thereof; and (b) using the nucleic acid to produce a probe, the probe being complementary to at least a portion of the hemizygous chromosome or segment thereof. In one preferred embodiment, the nucleic acid is amplified, where the amplified nucleic acid is a representation of the genomic DNA of the hemizygous cell. In another embodiment of this aspect, the nucleic acid is an RNA or DNA library.
In preferred embodiments of the fifth and sixth aspects of the invention, the probe has a known sequence; the method further includes detectably labeling the probe; the hemizygous cell may be human; the hemizygous cell may be an immortalized cell; the hemizygous cell may be derived from a complete hydatidiform mole, an ovarian teratoma, an acute lymphocytic leukemia, an acute myeloid leukemia, a solid tumor, a squamous cell lung cancer, an endometrial ovarian cancer, a malignant fibrous histiocytoma, or a renal oncocytoma; the hemizygous cell is NALM-16 or KBM-7; and the hemizygous cell may be derived from a haploid germ cell.
In a seventh aspect, the invention features a nucleic acid probe for the detection of a nucleotide mismatch, the probe being derived from a hemizygous cell and being complementary to a hemizygous chromosome or segment thereof. In a preferred embodiment of this aspect of the invention, the probe is detectably labeled.
In an eighth aspect, the invention features a nucleic acid probe derived from an autosomal chromosome of a mammalian cell, the probe having a unique sequence. In one preferred embodiment of this aspect of the invention, the probe is detectably labeled
In a final aspect, the invention features a method for determining if two nucleotide mismatches are located on the same strand of DNA in a nucleic acid sample that includes the steps of: (a) providing a first nucleic acid probe derived from a hemizygous cell, the first nucleic acid probe having a first unique sequence; (b) forming a first duplex between the nucleic acid sample and the first nucleic acid probe; (c) contacting the first duplex with a compound that cleaves a duplex containing a nucleotide mismatch under conditions which allow the compound to cleave the first duplex if the first duplex contains a nucleotide mismatch; (d) providing a sec
Clark & Elbing LLP
Schwartzman Robert A.
Variagenics Inc.
Wang Andrew
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