Rapid genotyping of 25 autosomal STRs in a Japanese population using fluorescent universal primers containing locked nucleic acids.
Amplification of fluorescently labeled products is one of the most popular methods for genotyping genetic variations. Two-step amplification using fluorescent universal primers simultaneously produces multiple targeted fragments labeled with fluorescent dyes, and this strategy is applicable to large-scale, cost-effective genotyping. In this study, we developed a fast PCR-based, multiple short tandem repeat (STR) genotyping method using fluorescent universal primers containing locked nucleic acids (LNAs). Four amplification reactions, each assaying six or seven markers and using 0.5-1.0 ng of genomic DNA, produced obvious Fam-labeled peaks in all 26 loci tested (25 autosomal STRs and amelogenin). The overall amplification time was 37 min. Moreover, fluorescent signals for the 25 STRs obtained from LNA-containing primers were 1.5-9.0 fold higher compared to those from non-LNA primers. Using genomic DNA from 120 Japanese individuals, 16 out of the 25 STRs had observed heterozygosity greater than 0.7. Some of these 25 STRs also had high discriminatory power, similar to that of the 13 core STRs in the Combined DNA Index System dataset. The probability of incorrectly assigning a match based on the accumulated matching probability for these 25 STRs is 1.2 × 10(-22), and their combined use can provide robust information for Japanese forensics.
Department of Legal Medicine, Asahikawa Medical University, Asahikawa 078-8510, Japan. Electronic address: asari@asahikawa-med.ac.jp.
Masaru
M
Asari
Department of Legal Medicine, Asahikawa Medical University, Asahikawa 078-8510, Japan.
Katsuhiro
K
Okuda
Department of Legal Medicine, Asahikawa Medical University, Asahikawa 078-8510, Japan.
Daisuke
D
Yajima
Department of Legal Medicine, Asahikawa Medical University, Asahikawa 078-8510, Japan.
Chikatoshi
C
Maseda
Department of Legal Medicine, Asahikawa Medical University, Asahikawa 078-8510, Japan.
Chisato
C
Hoshina
Department of Clinical Pharmacology and Therapeutics, Kyoto University Hospital, Kyoto 606-8507, Japan.
Tomohiro
T
Omura
Department of Legal Medicine, Asahikawa Medical University, Asahikawa 078-8510, Japan.
Hiroshi
H
Shiono
Department of Clinical Pharmacology and Therapeutics, Kyoto University Hospital, Kyoto 606-8507, Japan.
Kazuo
K
Matsubara
Department of Legal Medicine, Asahikawa Medical University, Asahikawa 078-8510, Japan.
Keiko
K
Shimizu
D044466:Asian People / Q000235:genetics; D016172:DNA Fingerprinting / Q000379:methods; D017931:DNA Primers / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D060005:Genotyping Techniques / Q000379:methods*; D006579:Heterozygote; D006801:Humans; D007564:Japan; D018895:Microsatellite Repeats; D009841:Oligonucleotides / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D005456:Fluorescent Dyes; D009841:Oligonucleotides; C477371:locked nucleic acid
Fast PCR; Forensic DNA; Genotyping; Locked nucleic acid (LNA); STR; Universal primer