Rapid mass spectrometric identification of human genomic polymorphisms using multiplexed photocleavable mass-tagged probes and solid phase capture.
A mass spectrometric approach for rapid and simultaneous detection of several single nucleotide polymorphisms (SNPs) is reported. Oligonucleotide single base extension (SBE) 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 (MALDI-TOF) MS, are detected at pmol levels. This method is applied to the analysis of mitochondrial DNA polymorphisms for the purpose of human identification.
Hammond
Naomi
N
School of Chemistry, University of Southampton, Southampton, UK. tb2@soton.ac.uk
Koumi
Peter
P
Langley
G John
GJ
Lowe
Alex
A
Brown
Tom
T
D019921:Biotinylation; D004247:DNA / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D015345:Oligonucleotide Probes / Q000138:chemical synthesis / Q000737:chemistry; D010777:Photochemistry; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D019809:Streptavidin; D014466:Ultraviolet Rays
D017931:DNA Primers; D015345:Oligonucleotide Probes; D004247:DNA; D019809:Streptavidin