Single nucleotide polymorphism genotyping by on-line liquid chromatography-mass spectrometry in forensic science of the Y-chromosomal locus M9.
A method is described for genotyping alleles of the Y-chromosomal locus M9, incorporating DNA extraction, amplification by polymerase chain reaction (PCR), sample purification by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC), and allele identification by on-line hyphenation to electrospray ionization mass spectrometry (ESI-MS). The alleles G and C were differentiated in 114 base pair amplicons on the basis of intact molecular mass measurements with a mass accuracy between 0.007 and 0.017%. The accuracy of mass determination was significantly reduced to less than 0.0036% upon amplification of a short, 61 bp fragment. The application of steep gradients of acetonitrile in 25 mM butyldimethylammonium bicarbonate not only enabled the efficient separation of non-target components from the PCR product in a monolithic, poly-(styrene-divinylbenzene)-based capillary column, but also allowed the high-throughput analysis of the PCR products with cycle times of 2 min. The new method was compared to a conventional restriction fragment length polymorphism assay with capillary gel electrophoretic analysis. In a blind study, 90 samples of unrelated individuals were genotyped. The high accuracy (<0.004%) and small relative standard deviation (<0.007%, n=20) of mass measurements, which enables even the differentiation of A and T alleles with a mass difference of 9 mass units, make IP-RP-HPLC-ESI-MS a potent tool for the routine characterization of SNPs in forensic science.
Berger
Burkhard
B
Institute of Legal Medicine, Leopold-Franzens University, Müllerstrasse 44, A-6020 Innsbruck, Austria.
Hölzl
Georg
G
Oberacher
Herbert
H
Niederstätter
Harald
H
Huber
Christian G
CG
Parson
Walther
W
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D041322:Chromosomes, Human, Y; D017931:DNA Primers; D005554:Forensic Medicine; D005838:Genotype; D006801:Humans; D020641:Polymorphism, Single Nucleotide; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D017931:DNA Primers