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SNP analysis of the inter-alpha-trypsin inhibitor family heavy chain-related protein (IHRP) gene by a fluorescence-adapted SSCP method.

Tozaki T, Choi-Miura NH, Taniyama M, Kurosawa M, Tomita M - BMC Med. Genet. (2002)

Bottom Line: One of the SNPs was a new SNP resulting in an amino acid substitution, while the other SNPs have already been reported in the public databases.Six SNPs of the IHRP were associated with two haplotypes.The fluorescence-adapted SSCP was useful for detecting and genotyping SNPs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiological Chemistry, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555, Japan. ttozaki@nyc.odn.ne.jp

ABSTRACT

Background: Single-nucleotide polymorphisms (SNPs) are considered to be useful polymorphic markers for genetic studies of polygenic traits. Single-stranded conformational polymorphism (SSCP) analysis has been widely applied to detect SNPs, including point mutations in cancer and congenital diseases. In this study, we describe an application of the fluorescent labeling of PCR fragments using a fluorescent-adapted primer for SSCP analysis as a novel method.

Methods: Single-nucleotide polymorphisms (SNPs) of the inter-alpha-trypsin inhibitor family heavy chain-related protein (IHRP) gene were analyzed using a fluorescence-adapted SSCP method. The method was constructed from two procedures: 1) a fluorescent labeling reaction of PCR fragments using fluorescence-adapted primers in a single tube, and 2) electrophoresis on a non-denaturing polyacrylamide gel.

Results: This method was more economical and convenient than the single-stranded conformational polymorphism (SSCP) methods previously reported in the detection of the labeled fragments obtained. In this study, eight SNPs of the IHRP gene were detected by the fluorescence-adapted SSCP. One of the SNPs was a new SNP resulting in an amino acid substitution, while the other SNPs have already been reported in the public databases. Six SNPs of the IHRP were associated with two haplotypes.

Conclusions: The fluorescence-adapted SSCP was useful for detecting and genotyping SNPs.

No MeSH data available.


Related in: MedlinePlus

Electrophoretic profile on non-denaturing polyacrylamide gel of adapter-labeled 322-bp DNA fragments obtained with exon 21 primer set of the IHRP gene. A) homo-type of main allele, B) hetero-type of main and minor alleles, C) homo-type of minor allele
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Figure 2: Electrophoretic profile on non-denaturing polyacrylamide gel of adapter-labeled 322-bp DNA fragments obtained with exon 21 primer set of the IHRP gene. A) homo-type of main allele, B) hetero-type of main and minor alleles, C) homo-type of minor allele

Mentions: PCR fragments with the primer sets shown in Table 1 were well amplified for SSCP analysis using the fluorescence-adapted SSCP method. Figure 2 shows the migration pattern of the adapter-labeled 322-bp DNA fragments of exon 21 in the IHRP gene on non-denaturing polyacrylamide gel. The PCR fragments labeled with fluorescence-adapted primers were well detected on an automatic DNA sequencer. Figures 2A,2B, and 2C show profiles for the alleles in homozygotes (A, C) and heterozygotes (B). The alleles were clearly sequenced on non-denaturing polyacrylamide gel. The alleles were determined by direct counting.


SNP analysis of the inter-alpha-trypsin inhibitor family heavy chain-related protein (IHRP) gene by a fluorescence-adapted SSCP method.

Tozaki T, Choi-Miura NH, Taniyama M, Kurosawa M, Tomita M - BMC Med. Genet. (2002)

Electrophoretic profile on non-denaturing polyacrylamide gel of adapter-labeled 322-bp DNA fragments obtained with exon 21 primer set of the IHRP gene. A) homo-type of main allele, B) hetero-type of main and minor alleles, C) homo-type of minor allele
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC117798&req=5

Figure 2: Electrophoretic profile on non-denaturing polyacrylamide gel of adapter-labeled 322-bp DNA fragments obtained with exon 21 primer set of the IHRP gene. A) homo-type of main allele, B) hetero-type of main and minor alleles, C) homo-type of minor allele
Mentions: PCR fragments with the primer sets shown in Table 1 were well amplified for SSCP analysis using the fluorescence-adapted SSCP method. Figure 2 shows the migration pattern of the adapter-labeled 322-bp DNA fragments of exon 21 in the IHRP gene on non-denaturing polyacrylamide gel. The PCR fragments labeled with fluorescence-adapted primers were well detected on an automatic DNA sequencer. Figures 2A,2B, and 2C show profiles for the alleles in homozygotes (A, C) and heterozygotes (B). The alleles were clearly sequenced on non-denaturing polyacrylamide gel. The alleles were determined by direct counting.

Bottom Line: One of the SNPs was a new SNP resulting in an amino acid substitution, while the other SNPs have already been reported in the public databases.Six SNPs of the IHRP were associated with two haplotypes.The fluorescence-adapted SSCP was useful for detecting and genotyping SNPs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiological Chemistry, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555, Japan. ttozaki@nyc.odn.ne.jp

ABSTRACT

Background: Single-nucleotide polymorphisms (SNPs) are considered to be useful polymorphic markers for genetic studies of polygenic traits. Single-stranded conformational polymorphism (SSCP) analysis has been widely applied to detect SNPs, including point mutations in cancer and congenital diseases. In this study, we describe an application of the fluorescent labeling of PCR fragments using a fluorescent-adapted primer for SSCP analysis as a novel method.

Methods: Single-nucleotide polymorphisms (SNPs) of the inter-alpha-trypsin inhibitor family heavy chain-related protein (IHRP) gene were analyzed using a fluorescence-adapted SSCP method. The method was constructed from two procedures: 1) a fluorescent labeling reaction of PCR fragments using fluorescence-adapted primers in a single tube, and 2) electrophoresis on a non-denaturing polyacrylamide gel.

Results: This method was more economical and convenient than the single-stranded conformational polymorphism (SSCP) methods previously reported in the detection of the labeled fragments obtained. In this study, eight SNPs of the IHRP gene were detected by the fluorescence-adapted SSCP. One of the SNPs was a new SNP resulting in an amino acid substitution, while the other SNPs have already been reported in the public databases. Six SNPs of the IHRP were associated with two haplotypes.

Conclusions: The fluorescence-adapted SSCP was useful for detecting and genotyping SNPs.

No MeSH data available.


Related in: MedlinePlus