Pyrophosphorolysis-activatable oligonucleotides may facilitate detection of rare alleles, mutation scanning and analysis of chromatin structures.
Pyrophosphorolysis-activated polymerization (PAP) was initially developed to enhance the specificity of allele-specific 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 pyrophosphorolysis-mediated activation of a pyrophosphorolysis-activatable oligonucleotide (P*) followed by extension of the activated oligonucleotide. Herein, we demonstrate that genetically engineered DNA polymerases greatly improve the efficiency of PAP, making it a practical technique for detection of rare mutations. We also show that P* oligonucleotides have the novel and unexpected property of high sensitivity to mismatches throughout at least the 16 3'-terminal nucleotides. Thus, PAP constitutes a technology platform of potential utility whenever high specificity is required along the length of an oligonucleotide.
Liu
Qiang
Q
Departments of Molecular Genetics, City of Hope National Medical Center, 1500 East Duarte Road, Duarte, CA 91010-3000, USA.
Sommer
Steve S
SS
D000483:Alleles; D020137:Base Pair Mismatch / Q000235:genetics; D001483:Base Sequence; D002843:Chromatin / Q000737:chemistry* / Q000235:genetics; D011088:DNA Ligases / Q000378:metabolism; D004252:DNA Mutational Analysis / Q000379:methods*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D015224:Dideoxynucleosides / Q000378:metabolism; D011756:Diphosphates / Q000378:metabolism*; D005820:Genetic Testing / Q000379:methods*; D006868:Hydrolysis; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D020411:Oligonucleotide Array Sequence Analysis; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D015202:Protein Engineering; D012680:Sensitivity and Specificity; D019914:Taq Polymerase / Q000235:genetics / Q000378:metabolism; D013698:Templates, Genetic
D002843:Chromatin; D015224:Dideoxynucleosides; D011756:Diphosphates; D009841:Oligonucleotides; D019914:Taq Polymerase; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase; D011088:DNA Ligases