Genotyping of 38 insertion/deletion polymorphisms for human identification using universal fluorescent PCR.
Short insertion/deletion (Indel) polymorphisms of approximately 2-6 bp are useful as biallelic markers for forensic analysis, and the application of Indel genotyping as a supplementary tool would improve human identification accuracy. We examined the allele frequencies of 37 autosomal Indels in the Japanese population and developed a novel dual-color genotyping method for human identification on the basis of universal fluorescent PCR, including the sex-typing amelogenin locus. Target genomic fragment sizes for 38 Indels were 49-143 bp. We analyzed these Indels in 100 Japanese individuals using the M13(-47) sequence as a universal primer. For dual-color genotyping, we designed a novel universal primer with high amplification efficiency and specificity. Using FAM-labeled M13(-47) and HEX-labeled modified M13(-47) primers, fluorescent signals at all loci were clearly distinguished in two independent multiplex PCRs. Average minor allele frequency was 0.39, and accumulated matching probability was 2.12 × 10(-15). Complete profiles were successfully amplified with as little as 0.25 ng of DNA. This method provides robust, sensitive, and cost-effective genotyping for human identification.
Department of Legal Medicine, Asahikawa Medical University, 2-1-1-1 Midorigaoka-higashi, Asahikawa 078-8510, Japan; Oral and Maxillofacial Surgery, Asahikawa Medical University, 2-1-1-1 Midorigaoka-higashi, Asahikawa 078-8510, Japan.
Kumiko
K
Oka
Masaru
M
Asari
Tomohiro
T
Omura
Masatsugu
M
Yoshida
Chikatoshi
C
Maseda
Daisuke
D
Yajima
Kazuo
K
Matsubara
Hiroshi
H
Shiono
Mitsuyoshi
M
Matsuda
Keiko
K
Shimizu
D053523:Amelogenin / Q000235:genetics; D017931:DNA Primers; D005456:Fluorescent Dyes; D018732:Forensic Anthropology / Q000379:methods*; D005787:Gene Frequency; D014644:Genetic Variation; D015894:Genome, Human; D005838:Genotype; D060005:Genotyping Techniques / Q000191:economics; D006801:Humans; D054643:INDEL Mutation; D007564:Japan; D060885:Multiplex Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D012680:Sensitivity and Specificity
D053523:Amelogenin; D017931:DNA Primers; D005456:Fluorescent Dyes
Capillary electrophoresis; Forensic genotyping; Human identification; Insertion/deletion polymorphism; Multiplex PCR; Universal fluorescent PCR