PAP: Detection of ultra rare mutations depends on P* oligonucleotides: “Sleeping Beauties” awakened by the kiss of pyrophosphorolysis
✍ Scribed by Qiang Liu; Steve S. Sommer
- Book ID
- 102860950
- Publisher
- John Wiley and Sons
- Year
- 2004
- Tongue
- English
- Weight
- 618 KB
- Volume
- 23
- Category
- Article
- ISSN
- 1059-7794
No coin nor oath required. For personal study only.
✦ Synopsis
For the Mutation Detection 2003 Special Issue
Pyrophosphorolysis-activated polymerization (PAP) was initially developed to enhance the specificity of allelespecific PCR for detection of known mutations in the presence of a great excess of wild-type allele. The high specificity of PAP derives from the serial coupling of activation of a 3 0 blocked pyrophosphorolysis-activable oligonucleotide (P n ) with extension of the unblocked, activated P n . In theory, PAP can detect a copy of a single base mutation present in 3 Â 10 11 copies of the wild-type allele. In practice, the selectivity of detection is limited by polymerase extension errors, a bypass reaction, from the unblocked oligonucleotide annealed to the opposing strand. Bi-directional PAP allele-specific amplification (Bi-PAP-A) is a derivative of PAP that uses two opposing pyrophosphorolysis activable oligonucleotides (P n ) with one nucleotide overlap at their 3 0 termini. This eliminates the problematic bypass reaction. The selectivity of Bi-PAP-A was examined using k phage DNA as a model system. Bi-PAP-A selectively detected two copies of a rare mutated allele in the presence of at least 2 Â 10 9 copies of the wild-type k phage DNA. We then applied Bi-PAP-A to direct detection of spontaneous somatic mutations in the lacI transgene in BigBlue transgenic mice at a frequency as low as 3 Â 10 À9 . A 370fold variation in the frequency of a specific somatic mutation among different mouse samples was found, implying hyper-Poisson variance and clonal expansion of mutation occurring during early development.
Bi-PAP-A is a simple, rapid, and general method capable of automation and particularly suited to detection of ultra rare mutations. We also show that P n oligonucleotides have the novel and unexpected property of high specificity to mismatches with the template throughout lengths of the P n . Thus, PAP also can form the basis of microarray-based scanning or resequencing methods to detect virtually all mutations. Hum Mutat 23:426-436, 2004 r 2004 Wiley-Liss, Inc.