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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

A schematic diagram of fluorescence-adapted SSCP method. F indicates a fluorescent dye (Cy-5). Shadowed boxes are specific primer sequences to amplify the IHRP gene, and black boxes are adapted sequences. These PCR reactions are performed in a single tube
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Figure 1: A schematic diagram of fluorescence-adapted SSCP method. F indicates a fluorescent dye (Cy-5). Shadowed boxes are specific primer sequences to amplify the IHRP gene, and black boxes are adapted sequences. These PCR reactions are performed in a single tube

Mentions: An outline of the fluorescence-adapted SSCP is shown in Figure 1. In the fluorescence-adapted SSCP analysis, four primers were prepared as follows: the sequence-specific forward primer conjugated with 5'-TGA CCG GCA GCA AAA TTG-3' tail at its 5' end; the sequence-specific reverse primer conjugated with 5'-TGT AAA ACG ACG GCC AGT-3' tail at its 5' end; the Cy-5 labeled 5'-TGA CCG GCA GCA AAA TTG-3' primer (Amersham Biosciences, NJ, USA); and the Cy-5 labeled 5'-TGT AAA ACG ACG GCC AGT-3' primer (Amersham Biosciences).


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)

A schematic diagram of fluorescence-adapted SSCP method. F indicates a fluorescent dye (Cy-5). Shadowed boxes are specific primer sequences to amplify the IHRP gene, and black boxes are adapted sequences. These PCR reactions are performed in a single tube
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: A schematic diagram of fluorescence-adapted SSCP method. F indicates a fluorescent dye (Cy-5). Shadowed boxes are specific primer sequences to amplify the IHRP gene, and black boxes are adapted sequences. These PCR reactions are performed in a single tube
Mentions: An outline of the fluorescence-adapted SSCP is shown in Figure 1. In the fluorescence-adapted SSCP analysis, four primers were prepared as follows: the sequence-specific forward primer conjugated with 5'-TGA CCG GCA GCA AAA TTG-3' tail at its 5' end; the sequence-specific reverse primer conjugated with 5'-TGT AAA ACG ACG GCC AGT-3' tail at its 5' end; the Cy-5 labeled 5'-TGA CCG GCA GCA AAA TTG-3' primer (Amersham Biosciences, NJ, USA); and the Cy-5 labeled 5'-TGT AAA ACG ACG GCC AGT-3' primer (Amersham Biosciences).

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