Rapid genotyping of 25 autosomal STRs in a Japanese population using fluorescent universal primers containing locked nucleic acids

Asari, Masaru; Okuda, Kunio; Yajima, Daisuke; Maseda, Chikatoshi; Hoshina, Chisato; Omura, Tomohiro; Shiono, Hiroshi; Matsubara, Kazuo; Shimizu, Keiko

doi:10.1016/j.jflm.2015.01.004

Cited by 5 publications

(7 citation statements)

References 18 publications

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“…Incorporation of LNAs raises the Tm value, enabling primers with greater stability to be designed. It is considered that stable binding to the DNA strand in PCR [17,19] increases the height of the peak for some genes and improves the accuracy of methylation level determination.…”

Section: Discussionmentioning

confidence: 99%

“…Reasons for these differences can be differences in the Tm value of the primers, the amplification size, and the number of CpG sites in the target gene. We previously showed that LNA primers can improve amplification efficiency of some low‐amplification‐efficiency genes, but not all genes [19]. Similarly, LNAs are not effective for methylation analysis of all genes.…”

Section: Discussionmentioning

confidence: 99%

“…The effect of LNAs on the efficiency of STR amplification from 1 ng or less of DNA has been investigated with the aim of constructing a highly sensitive analysis method for trace DNA [18]. In addition, we have shown that LNA-containing primers improve amplification efficiency when the amplification efficiency using multiplex PCR is extremely low [19].…”

Section: Introductionmentioning

confidence: 99%

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Age prediction by methylation analysis of small amounts of DNA using locked nucleic acids

Takahashi

Asari

Isozaki

et al. 2022

Journal of Forensic Sciences

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Age prediction based on methylation analysis has been reported in many populations, with 10 ng or more of DNA usually required for each determination. In this study, we designed thermostable locked nucleic acid (LNA) primers by replacing a small number of DNA bases in standard DNA primers with LNAs. We evaluated these primer sets by single-base extension analysis using 10, 5, or 2 ng of DNA that would be less than template DNA used in standard methylation testing, and determined sensitivity and accuracy. We analyzed EDARADD, SST, and KLF14 genes, targeting one CpG site in each gene. Melting temperature values of most LNA primers were 4°C higher than those of DNA primers. The intensities of signals from the EDARADD and SST genes were significantly improved by the LNA primers, by 3.3 times and 1.4 times, respectively, compared with the DNA primers using 2 ng of DNA. Coefficient of variation (CV) analysis was used to assess the accuracy of the determined methylation levels.CVs were increased using small amounts of DNA, but lower CVs were detected using LNA primers. We also showed high accuracy of age prediction for 51 individuals using LNA primers. The lowest mean absolute deviation was obtained using 10 ng of DNA and was 3.88 years with the LNA primers. Thermostable PCR primers were simply designed, and the LNAs improved the sensitivity and accuracy of methylation analysis for 10 ng or less of DNA.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Age prediction by methylation analysis of small amounts of DNA using locked nucleic acids

Takahashi

Asari

Isozaki

et al. 2022

Journal of Forensic Sciences

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“…32 LNAs have been incorporated in STR detection probes. 33,34 Owing to the bicyclic structure, LNA nucleotides have an N-(RNA) type ribose conformation (Figure 1B). 35−37 Incorporating LNA into synthetic oligonucleotide probes provides high affinity and specificity to DNA targets.…”

Section: ■ Introductionmentioning

confidence: 99%

“…35−37 Incorporating LNA into synthetic oligonucleotide probes provides high affinity and specificity to DNA targets. When used for PCR STR profiling, primers with LNAs for STR improve the limit of detection (LOD) values to 0.25 ng of DNA 33,34,38 while reducing amplification time to 37 min. 34 Ultraspecific fluorescent probes have been proposed to detect concentrations of nucleic acids in the picomolar range without the use of amplification methods.…”

Section: ■ Introductionmentioning

confidence: 99%

Short Tandem Repeat DNA Profiling Using Perylene-Oligonucleotide Fluorescence Assay

Hernández Bustos,

Martiny,

Bom Pedersen

et al. 2023

Anal. Chem.

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We report an amplification-free genotyping method to determine the number of human short tandem repeats (STRs). DNA-based STR profiling is a robust method for genetic identification purposes such as forensics and biobanking and for identifying specific molecular subtypes of cancer. STR detection requires polymerase amplification, which introduces errors that obscure the correct genotype. We developed a new method that requires no polymerase. First, we synthesized perylene-nucleoside reagents and incorporated them into oligonucleotide probes that recognize five common human STRs. Using these probes and a bead-based hybridization approach, accurate STR detection was achieved in only 1.5 h, including DNA preparation steps, with up to a 1000-fold target DNA enrichment. This method was comparable to PCR-based assays. Using standard fluorometry, the limit of detection was 2.00 ± 0.07 pM for a given target. We used this assay to accurately identify STRs from 50 human subjects, achieving >98% consensus with sequencing data for STR genotyping.

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Multicolor-based discrimination of 21 short tandem repeats and amelogenin using four fluorescent universal primers

Asari

Okuda

Hoshina

et al. 2016

Analytical Biochemistry

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Rapid genotyping of 25 autosomal STRs in a Japanese population using fluorescent universal primers containing locked nucleic acids

Cited by 5 publications

References 18 publications

Age prediction by methylation analysis of small amounts of DNA using locked nucleic acids

Age prediction by methylation analysis of small amounts of DNA using locked nucleic acids

Short Tandem Repeat DNA Profiling Using Perylene-Oligonucleotide Fluorescence Assay

Multicolor-based discrimination of 21 short tandem repeats and amelogenin using four fluorescent universal primers

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