| 摘要 |
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. |
