Multiplex dosage pyrophosphorolysis-activated polymerization: application to the detection of heterozygous deletions. (May 2006)

• Record Type: Journal Article
• Authors: Liu, Qiang; Nguyen, Vu Q.; Li, Xuemin; Sommer, Steve S.
• Is Part Of: Biotechniques. Volume 40:Number 5(2006)
• Page Start: 661
• Page End: 668
• Publication Date: 2006-05
• Subjects: Biology, Experimental -- Periodicals; Molecular biology -- Periodicals; Medical technology -- Periodicals; Biology, Experimental; Medical technology; Molecular biology; Clinical Laboratory Techniques -- Periodicals; Research -- Periodicals; Medical Laboratory Science -- Periodicals; Periodicals; Electronic journals; 570
• DOI: 10.2144/000112164
• Languages: English
• ISSNs: 0736-6205

Abstract

Large heterozygous chromosomal deletions and gene duplications are important classes of mutations that are generally missed by standard PCR amplification and sequencing. Multiplex dosage pyrophosphorolysis-activated polymerization (MD-PAP), a derivative of PAP, was utilized to detect these types of mutatiom. PAP is a method for nucleic acid amplification in which 3′ blocked oligonucleotides (P*) are activated by pyrophosphorolysis when annealed to the target template and subsequently extended. A key advantage to this technology is that PAP reactions produce little or no primer-dimer or false priming. As a result of this enhanced specificity, MD-PAP is easy to optimize. Herein, we utilize MD-PAP to determine gene dosage of each exon of the human factor IX gene by comparison with one endogenous internal control from the ATM gene. Estimated dosage is proportional to the actual template copy number over a minimum dynamic range from 1 to 16 copies. A blinded analysis detected 100% of 43 heterozygous deletions of exons in the human factor IX gene.