Multiplex PCR and minisequencing of SNPs--a model with 35 Y chromosome SNPs

TY - JOUR

T1 - Multiplex PCR and minisequencing of SNPs--a model with 35 Y chromosome SNPs

AU - Sanchez, Juan J

AU - Børsting, Claus

AU - Hallenberg, Charlotte

AU - Buchard, Anders

AU - Hernandez, Alexis

AU - Morling, Niels

PY - 2003/10/14

Y1 - 2003/10/14

N2 - We have developed a robust single nucleotide polymorphism (SNPs) typing assay with co-amplification of 25 DNA-fragments and the detection of 35 human Y chromosome SNPs. The sizes of the PCR products ranged from 79 to 186 base pairs. PCR primers were designed to have a theoretical Tm of 60 +/- 5 degrees C at a salt concentration of 180 mM. The sizes of the primers ranged from 19 to 34 nucleotides. The concentration of amplification primers was adjusted to obtain balanced amounts of PCR products in 8mM MgCl2. For routine purposes, 1 ng of genomic DNA was amplified and the lower limit was approximately 100 pg DNA. The minisequencing reactions were performed simultaneously for all 35 SNPs with fluorescently labelled dideoxynucleotides. The size of the minisequencing primers ranged from 19 to 106 nucleotides. The minisequencing reactions were analysed by capillary electrophoresis and multicolour fluorescence detection. Female DNA did not influence the results of Y chromosome SNP typing when added in concentrations more than 300 times the concentrations of male DNA. The frequencies of the 35 SNPs were determined in 194 male Danes. The gene diversity of the SNPs ranged from 0.01 to 0.5.

AB - We have developed a robust single nucleotide polymorphism (SNPs) typing assay with co-amplification of 25 DNA-fragments and the detection of 35 human Y chromosome SNPs. The sizes of the PCR products ranged from 79 to 186 base pairs. PCR primers were designed to have a theoretical Tm of 60 +/- 5 degrees C at a salt concentration of 180 mM. The sizes of the primers ranged from 19 to 34 nucleotides. The concentration of amplification primers was adjusted to obtain balanced amounts of PCR products in 8mM MgCl2. For routine purposes, 1 ng of genomic DNA was amplified and the lower limit was approximately 100 pg DNA. The minisequencing reactions were performed simultaneously for all 35 SNPs with fluorescently labelled dideoxynucleotides. The size of the minisequencing primers ranged from 19 to 106 nucleotides. The minisequencing reactions were analysed by capillary electrophoresis and multicolour fluorescence detection. Female DNA did not influence the results of Y chromosome SNP typing when added in concentrations more than 300 times the concentrations of male DNA. The frequencies of the 35 SNPs were determined in 194 male Danes. The gene diversity of the SNPs ranged from 0.01 to 0.5.

KW - Chromosomes, Human, Y

KW - DNA

KW - DNA Primers

KW - Denmark

KW - Electrophoresis, Capillary

KW - Female

KW - Fluorescence

KW - Humans

KW - Male

KW - Models, Genetic

KW - Polymerase Chain Reaction

KW - Polymorphism, Single Nucleotide

KW - Sequence Analysis, DNA

M3 - Journal article

C2 - 14550618

SN - 0379-0738

VL - 137

SP - 74

EP - 84

JO - Forensic Science International

JF - Forensic Science International

IS - 1

ER -