Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1996-07-23
1998-04-21
Jones, W. Gary
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435 6, 435 911, 435 9153, 435810, 536 2433, 935 77, 935 78, C12P 1934, C12Q 168, C07H 2104
Patent
active
057416788
ABSTRACT:
There is disclosed a quantitative sensitive method to enable the detection of point mutations at a known site to a diagnostic kit which uses a multi step (for example, four steps) or a single step reaction. The method uses selective polymerase chain reaction (PCR) amplification of mutant test gene sequences involving first stage amplification of both mutant and wild-type sequences, first stage restriction enzyme digestion of only wild-type sequences, second stage amplification of undigested amplified fragments enriched in mutant sequences and second stage digestion of previously undigested wild-type sequences. Long and short tail primers are used in the first and second stages of amplification respectively to enable selective amplification (in the second stage) of only previously amplified material and none of the original test genomic DNA. The short tail primers are labelled with biotin and fluorescence at their respective 5' and 3' ends to enable easy detection and quantitation of mutations in the test gene via automated fluorescence readers. The use of multi steps as well as a single step reaction is disclosed. The process is exemplified with respect to its use in detecting mutations in the human K-ras gene, yet it is applicable for any given mutation in a defined site.
REFERENCES:
patent: 4800159 (1989-01-01), Mullis et al.
Kahn et al. (1991) Oncogene 6: 1079-1083.
Landgraf et al. (1991) Analytical Biochem. 193: 231-235.
Innis et al. (1990) "PCR Protocols--A Guide to Methods and Applications," Academic Press, Inc., pp. 28-38.
American Health Foundation
Jones W. Gary
Tran Paul B.
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