US 5,843,649 A · Method of identifying clonal cell samples using heteroduplex generators

Abstract

Methods of distinguishing between neoplasms and pseudoneoplastic or hyperplastic processes are disclosed. The methods comprise first contacting DNA from cells of a tissue sample from a female individual with a cytosine-methylation specific endonuclease, generating amplified fragments of a cytosine-methylation regulatable polymorphic X chromosome gene using primers that bracket a restriction site of said cytosine-methylation specific endonuclease and a polymorphic sequence in said nucleotide sequence of the gene; generating heteroduplexes between amplified fragments and heteroduplex generators which consist of 1-5 nucleotide differences from the amplified fragments; and detecting the presence of a single species of heteroduplex or two species of heteroduplexes. The presence of a single species of heteroduplex which indicates amplified fragments from a clonal population of cells which is indicative a neoplasm and the presence of two species of heteroduplexes indicates amplified fragments from a mosaic population of cells is indicative a pseudoneoplastic or hyperplastic process. Reagents and kits for performing the methods are disclosed.

Claims16

Dependent claims

A method of determining whether a sample from a female individual contains cells of a neoplasm or cells of pseudoneoplastic or hyperplastic processes, said method comprising the steps of:

a) contacting DNA from said cells of said sample from a female individual with a cytosine-methylation specific endonuclease under digest conditions;
b) generating amplified fragments of a cytosine-methylation regulatable, polymorphic X chromosome gene of said DNA using primers that bracket a restriction site of said cytosine-methylation specific endonuclease and a polymorphic sequence in said gene;
c) generating heteroduplexes between said amplified fragments and a heteroduplex generator, wherein the sequence of said heteroduplex generator differs from the sequence of said amplified fragments by 1-5 nucleotide differences;
and
d) detecting the presence of a single species of heteroduplex or at least two species of heteroduplexes,
wherein the presence of a single species of heteroduplex indicates amplified fragments from a clonal population of cells indicative of a neoplasm and the presence of at least two species of heteroduplexes indicates amplified fragments from a mosaic population of cells indicative of a non-neoplastic, pseudoneoplastic, or hyperplastic process.

3 more elements

A kit comprising:

1) primers which bracket a methylation sensitive restriction site on a methylation regulated polymorphic X chromosome gene, wherein an amplified fragment is generated when DNA from a female individual is used as a substrate for said primer in a polymerase chain reactions and
2) a heteroduplex generator that forms heteroduplexes with said amplified fragment, said heteroduplex generator having a sequence which differs from the sequence of said amplified fragment by no more than 5 nucleotide sequence differences.

Dependencies are inferred from the claim text.

Method of identifying clonal cell samples using heteroduplex generators

Life of this patent

Method of identifying clonal cell samples using heteroduplex generators

Life of this patent

Abstract

Claims16