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A post-labeling method for multiplexed and multicolored genotyping analysis of SSR, indel and SNP markers in single tube with bar-coded split tag (BStag)
BACKGROUND: Genotyping analysis using capillary DNA sequencing with fluorescently labeled primer pairs obtained by polymerase chain reaction (PCR) is widely used, but is expensive. The post-PCR labeling method using fluorescently labeled short oligonucleotides and nested PCR of the amplified product...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2011
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3126724/ https://www.ncbi.nlm.nih.gov/pubmed/21615927 http://dx.doi.org/10.1186/1756-0500-4-161 |
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author | Shimizu, Tokurou Yano, Kanako |
author_facet | Shimizu, Tokurou Yano, Kanako |
author_sort | Shimizu, Tokurou |
collection | PubMed |
description | BACKGROUND: Genotyping analysis using capillary DNA sequencing with fluorescently labeled primer pairs obtained by polymerase chain reaction (PCR) is widely used, but is expensive. The post-PCR labeling method using fluorescently labeled short oligonucleotides and nested PCR of the amplified product obtained from unlabeled primer pairs is a simple and inexpensive alternative. However, previously reported protocols often produced spurious peaks or inconsistent amplification under multiplexed analysis as a result of simultaneous progress of both the amplification and labeling reactions and local homology of the attached tag sequence. RESULTS: A set of 16 bp-long oligonucleotide sequences termed bar-coded split tag (BStag), comprising a common basal region, a three-nucleotide 'bar-code' sequence, and a mismatched nucleotide at the middle position were designed for selective post-PCR labeling. The BStag was attached at the 5' end of the forward primer of interest. The melting temperature of the BStag was low enough to separate the labeling reaction from initial PCR amplification, and each sequence was minimally divergent but maintained maximum selectivity. Post-PCR labeling of the amplified product was achieved by extending for three cycles at a lower annealing temperature after the conventional amplification program with the appropriate fluorescently labeled BStag primer. No amplification was confirmed with BStag primers for 12 plant species. The electropherogram of the labeled product obtained using this method was consistent with that of prelabeled primer, except for their apparent size. CONCLUSIONS: BStag enabled multiplexed post-PCR labeling of simple sequence repeat or insertion/deletion markers with different dyes in a single tube. BStag in conjunction with locus specific oligo and allele specific oligo was also useful for single nucleotide polymorphism analysis. The labeling protocol was simple and no additional operation was required. Single-tube multiplexed post-PCR labeling is useful for a wide variety of genotyping studies with maximal flexibility and minimal costs. |
format | Online Article Text |
id | pubmed-3126724 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-31267242011-06-30 A post-labeling method for multiplexed and multicolored genotyping analysis of SSR, indel and SNP markers in single tube with bar-coded split tag (BStag) Shimizu, Tokurou Yano, Kanako BMC Res Notes Research Article BACKGROUND: Genotyping analysis using capillary DNA sequencing with fluorescently labeled primer pairs obtained by polymerase chain reaction (PCR) is widely used, but is expensive. The post-PCR labeling method using fluorescently labeled short oligonucleotides and nested PCR of the amplified product obtained from unlabeled primer pairs is a simple and inexpensive alternative. However, previously reported protocols often produced spurious peaks or inconsistent amplification under multiplexed analysis as a result of simultaneous progress of both the amplification and labeling reactions and local homology of the attached tag sequence. RESULTS: A set of 16 bp-long oligonucleotide sequences termed bar-coded split tag (BStag), comprising a common basal region, a three-nucleotide 'bar-code' sequence, and a mismatched nucleotide at the middle position were designed for selective post-PCR labeling. The BStag was attached at the 5' end of the forward primer of interest. The melting temperature of the BStag was low enough to separate the labeling reaction from initial PCR amplification, and each sequence was minimally divergent but maintained maximum selectivity. Post-PCR labeling of the amplified product was achieved by extending for three cycles at a lower annealing temperature after the conventional amplification program with the appropriate fluorescently labeled BStag primer. No amplification was confirmed with BStag primers for 12 plant species. The electropherogram of the labeled product obtained using this method was consistent with that of prelabeled primer, except for their apparent size. CONCLUSIONS: BStag enabled multiplexed post-PCR labeling of simple sequence repeat or insertion/deletion markers with different dyes in a single tube. BStag in conjunction with locus specific oligo and allele specific oligo was also useful for single nucleotide polymorphism analysis. The labeling protocol was simple and no additional operation was required. Single-tube multiplexed post-PCR labeling is useful for a wide variety of genotyping studies with maximal flexibility and minimal costs. BioMed Central 2011-05-26 /pmc/articles/PMC3126724/ /pubmed/21615927 http://dx.doi.org/10.1186/1756-0500-4-161 Text en Copyright ©2011 Shimizu et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Article Shimizu, Tokurou Yano, Kanako A post-labeling method for multiplexed and multicolored genotyping analysis of SSR, indel and SNP markers in single tube with bar-coded split tag (BStag) |
title | A post-labeling method for multiplexed and multicolored genotyping analysis of SSR, indel and SNP markers in single tube with bar-coded split tag (BStag) |
title_full | A post-labeling method for multiplexed and multicolored genotyping analysis of SSR, indel and SNP markers in single tube with bar-coded split tag (BStag) |
title_fullStr | A post-labeling method for multiplexed and multicolored genotyping analysis of SSR, indel and SNP markers in single tube with bar-coded split tag (BStag) |
title_full_unstemmed | A post-labeling method for multiplexed and multicolored genotyping analysis of SSR, indel and SNP markers in single tube with bar-coded split tag (BStag) |
title_short | A post-labeling method for multiplexed and multicolored genotyping analysis of SSR, indel and SNP markers in single tube with bar-coded split tag (BStag) |
title_sort | post-labeling method for multiplexed and multicolored genotyping analysis of ssr, indel and snp markers in single tube with bar-coded split tag (bstag) |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3126724/ https://www.ncbi.nlm.nih.gov/pubmed/21615927 http://dx.doi.org/10.1186/1756-0500-4-161 |
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