Improved approaches to multiplexed PCR and to the genotyping of SNPs by mass spectrometry

Hammond, Naomi Rachel (2007) Improved approaches to multiplexed PCR and to the genotyping of SNPs by mass spectrometry. University of Southampton, Doctoral Thesis.

Abstract

PCR is used extensively in many areas of DNA testing. Primer dimers (PD) form when two primers hybridise together and are extended during PCR. The PD may then compete with primers for template annealing sites and markedly reduce the efficiency of the PCR.  PD is significant problem in multiplex PCR, in which  more than one primer pair is used simultaneously to create multiple PCR products.  To inhibit the amplification of PD, an approach using oligonucleotides containing 2’-O-methyl RNA substitutions as PCR primers has been investigated.  In a multiplex PCR, just one substitution positioned one base upstream of the 3’-end of each primer, yielded the desired PCR amplicons whilst inhibiting PD amplification.  2’-O-methyl RNA monomers can be incorporated into DNA PCR primers as a cost competitive and convenient method of eliminating undesired PD amplification in single and multiplex PCR.

A mass spectrometric approach for rapid and simultaneous detection of several single nucleotide polymorphisms has been developed.  Oligonucleotide single base extension primers, labelled at the 5’-end with photocleavable, quaternised and brominated peptidic mass tags, are extended by a mixture of the four dideoxynucleotides of which one is biotinylated.  The 3’-biotinylated extension products are captured by streptavidin-coated solid phase magnetic beads, whilst non-biotinylated extension products and unreacted primers are washed away.  Quaternised and brominated mass tags, cleaved from captured extension products during analysis by matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry, are detected at pmol levels.  This method is applied to the analysis of mitochondrial DNA polymorphisms for the purpose of human identification.

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