Multicolor-based discrimination of 21 short tandem repeats and amelogenin using four fluorescent universal primers.
The aim of this study was to develop a cost-effective genotyping method using high-quality DNA for human identification. A total of 21 short tandem repeats (STRs) and amelogenin were selected, and fluorescent fragments at 22 loci were simultaneously amplified in a single-tube reaction using locus-specific primers with 24-base universal tails and four fluorescent universal primers. Several nucleotide substitutions in universal tails and fluorescent universal primers enabled the detection of specific fluorescent fragments from the 22 loci. Multiplex polymerase chain reaction (PCR) produced intense FAM-, VIC-, NED-, and PET-labeled fragments ranging from 90 to 400 bp, and these fragments were discriminated using standard capillary electrophoretic analysis. The selected 22 loci were also analyzed using two commercial kits (the AmpFLSTR Identifiler Kit and the PowerPlex ESX 17 System), and results for two loci (D19S433 and D16S539) were discordant between these kits due to mutations at the primer binding sites. All genotypes from the 100 samples were determined using 2.5 ng of DNA by our method, and the expected alleles were completely recovered. Multiplex 22-locus genotyping using four fluorescent universal primers effectively reduces the costs to less than 20% of genotyping using commercial kits, and our method would be useful to detect silent alleles from commercial kit analysis.
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.
Chisato
C
Hoshina
Department of Clinical Pharmacology and Therapeutics, Kyoto University Hospital, Kyoto 606-8507, Japan.
Tomohiro
T
Omura
Department of Hospital Pharmacy and Pharmacology, Asahikawa Medical University, Asahikawa 078-8510, Japan.
Yoshikazu
Y
Tasaki
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
D000483:Alleles; D053523:Amelogenin / Q000032:analysis / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry / Q000378:metabolism*; D005260:Female; D005456:Fluorescent Dyes / Q000737:chemistry; D056426:Genetic Loci; D005838:Genotype; D060005:Genotyping Techniques; D006801:Humans; D008297:Male; D018895:Microsatellite Repeats / Q000235:genetics*; D009061:Mouth Mucosa / Q000378:metabolism; D060885:Multiplex Polymerase Chain Reaction
D053523:Amelogenin; D017931:DNA Primers; D005456:Fluorescent Dyes
Capillary electrophoresis; Fluorescent labeling; Forensic DNA; Multicolor genotyping; Short tandem repeat; Universal primer