Analysis of the Allele‐specific PCR Method for the Detection of Neoplastic Disease
作者:
C.,
Rhodes Charles,
Honsinger Donna,
Porter George,
期刊:
Diagnostic Molecular Pathology
(OVID Available online 1997)
卷期:
Volume 6,
issue 1
页码: 49-57
ISSN:1052-9551
年代: 1997
出版商: OVID
关键词: Allele-specific PCR;Peptide nucleic acid
数据来源: OVID
摘要:
PCR assays for the presence of mutant K-rasor p53 sequences are potentially useful as sensitive tests for tumor diagnosis. The technical challenge is to design assays sensitive enough to detect a few molecules of mutant DNA yet sufficiently specific that a false positive signal is not produced by a 105- or 106-fold excess of normal DNA. We determined the detection limit of allele-specific PCR (ASA) as a function of the particular mismatch involved using all 12 possible mismatches in two different DNA sequence contexts (K-rascodon 12 and p53 codon 273). Depending on the identity of the mismatch, mismatched template was amplified 102-104-fold less than perfectly matched template. In other words, a mutant allele could be detected by ASA if it represented > 1–0.01 of the total DNA from that locus. Peptide nucleic acid (PNA) clamping was used to improve the K-rasASA assay. Selective amplification of mutant sequences was achieved using a PNA complementary to the normal sequence to inhibit the amplification of wild-type DNA. PNA clamping followed by ASA resulted in significant improvement in sensitivity and specificity, permitting the detection of tumor DNA diluted with a 300,000-fold excess of normal human DNA.
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