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

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• 作者：Sanchez ; Juan J. ; B&oslash ; rsting ; Claus ; Hallenberg ; Charlotte ; Buchard ; Anders ; Hernandez ; Alexis ; et. al.
• 关键词：Y chromosome ; Single nucleotide polymorphism ; Multiplex PCR ; Minisequencing ; Genotyping
• 刊名：Forensic Science International
• 出版年：2003
• 期刊代码：104_03790738
• 类别：med
• 出版时间：October 14, 2003
• 卷：137
• 期：1
• 页码：74-84
• 文件大小：393 K

摘要

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 T_m of 60±5°C at a salt concentration of 180mM. 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, 1ng of genomic DNA was amplified and the lower limit was approximately 100pg 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.