Mutation detection using denaturing gradients

Details Mutation detection using denaturing gradients Mutation detection using denaturing gradients

2001-03-12

2003-07-22

Whisenant, Ethan (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C204S450000, C204S456000, C536S023100, C536S024300, C536S024330

Reexamination Certificate

active

06596487

ABSTRACT:

FIELD
The present invention relates generally to the field of genetic analysis, particularly to the detection of single nucleotide polymorphisms.
BACKGROUND
The human genome is made up of approximately 30,000 unique genes, which control the development and characteristics of the mature human. Each gene is composed of a sequence of nucleotides which are generally conserved across the species and are referred to as wild type. Single nucleotide polymorphisms (SNPs) are nucleotide sequence variants from the predominant wild type. Typically, single nucleotide polymorphisms are associated with expressed mutations. There is considerable interest in the development of methods which permit the rapid, accurate identification of single nucleotide polymorphisms.
Singh and Ullman in WO/056925 (Aclara Biosciences, Sep. 28, 2000) describe methods for single nucleotide polymorphism detection using complementary coded primers and differentially labeled terminating nucleotides.
Arnold, Theriault and Bedilion in WO/050869 (Incyte Pharmaceuticals, Aug. 31, 2000) describe methods for detecting multiple single nucleotide polymorphisms in a sandwich assay employing SNP probes.
Nerenberg, Canter and Radtkey in WO/061805 (Nanogen/Becton Dickinson Partnership, Oct. 19, 2000) describe methods for the analysis of SNPs using an electronically bioactive microchip.
Takenaka in WO/111351 (Feb. 15, 2001) describes a method for detecting SNPs using gold electrodes formed on the bottom face of a chip.
Despite these advances, there remains a need for an accurate, simple method for the detection of single nucleotide polymorphisms.
SUMMARY
The present invention provides methods for detecting single nucleotide polymorphisms in a sample by applying an energy gradient to a mixture of probe-hybridized target wild type and mutant polynucleotides.
A nucleotide probe, having a nucleotide sequence complementary to at least a portion of one strand of either the target wild type or target mutant polynucleotide(s), is exposed to a sample containing target wild type polynucleotide(s) and target mutant polynucleotide(s) under hybridizing conditions. If a wild type probe is used, target wild type polynucleotide(s) will form homoduplexes with the wild type probe, while target mutant polynucleotide(s) will form heteroduplexes with the wild type probe. If a mutant probe is used, target mutant polynucleotide(s) will form homoduplexes with the mutant probe, while target wild type polynucleotide(s) will form heteroduplexes with the mutant probe. Either the probe or the target may be attached to a suitable solid support.
An energy gradient is applied to the duplex-containing sample to induce selective denaturation of the duplexes. The gradient may be a thermal or chemical one and is preferably temporally linear. The hetero- and homo-duplexes deanneal at different times so that the detection of more than one type of polynucleotide(s) is indicative of a single nucleotide polymorphism. Preferably, a reference homozygous wild type is used as a control. In those cases in which both alleles of a test sample are mutant, only a single eluent peak or signal will be detected. The presence of mutation may be confirmed by comparing the elution time to that of the control.
Capillary electropheresis, gel electropheresis, high performance liquid chromatography, or microfluidics are typically used for identification of the separated strands.


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M.L. Fontaneo, et al., Process of analyzing nucleic acids by Hybridization and a device for its implementation, FLS, inc. pp. 1-41.

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