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Title:
HIGHLY SENSITIVE METHOD FOR DETECTING LOW FREQUENCY MUTATIONS
Document Type and Number:
WIPO Patent Application WO/2013/123031
Kind Code:
A3
Abstract:
The disclosed edge-blocker oligonucleotide based AS-NEPB-PCR method amplifies allele specific DNA (or RNA) while dramatically blocking amplification of wild type (WT) DNA (or RNA). The AS-NEPB-PCR design allows ready modification of an existing PCR reaction setup with an edge-blocker oligonucleotide together with an allele specific primer complementary to the mutant sequence to achieve allele specific amplification. The method simplifies assay optimization procedures and achieved sensitivity sufficient to detect a signal present at 0.1% level with close to 100% specificity, which is useful in detecting SNP or genetic mutations. The method was used to detect three different genetic mutations in cancer, in KRAS, BRAF, and EGFR, with three different types of modified edge-blocker oligonucleotides (phosphate, inverted dT and amino-C7). It was possible to detect one copy of mutant DNA in 1000-copy of normal DNA background of a heterogeneous sample, and was far more sensitive than the other blocking method.

Inventors:
WANG HAIYING (US)
JIANG YUQIU (US)
WANG YIXIN (US)
Application Number:
PCT/US2013/025913
Publication Date:
March 12, 2015
Filing Date:
February 13, 2013
Export Citation:
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Assignee:
JANSSEN DIAGNOSTICS LLC (US)
International Classes:
C12Q1/68; C07H21/04
Domestic Patent References:
WO2002068684A22002-09-06
Foreign References:
US20100285478A12010-11-11
Other References:
PARSONS, BL ET AL.: "Allel-Specific Competitive Blocker-PCR Detection Of Rare Base Substiution.", METHODS IN MOLECULAR BIOLOGY., vol. 291, 2005, pages 235 - 245, XP008061211
MORLAN, J ET AL.: "Mutation Detection By Real-Time PCR: A Simple, Robust And Highly Selective Method.", PLOS ONE., vol. 4, no. 2, 2009, pages 1 - 8, XP055111534
WANG, Y ET AL.: "Aristolochic Acid-Induced Carcinogenesis Examined By ACB-PCR Quantification Of H-Ras And K-Ras Mutant Fraction.", MUTAGENESIS., vol. 26, no. 5, 3 June 2011 (2011-06-03), pages 619 - 628, XP055184985
MCKINZIE, PB ET AL.: "Detection Of Rare K-Ras Codon 12 Mutations Using Allele-Specific Competitive Blocker PCR.", MUTATION RESEARCH., vol. 517, 21 March 2002 (2002-03-21), pages 209 - 220, XP002332580
Attorney, Agent or Firm:
JOHNSON, Philip, S. et al. (One Johnson & Johnson PlazaNew Brunswick, NJ, US)
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