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  Refinement of single-nucleotide polymorphism genotyping methods on human genomic DNA: amplifluor allele-specific polymerase chain reaction versus ligation detection reaction-TaqMan

Rickert, A. M., Borodina, T. A., Kuhn, E. J., Lehrach, H., & Sperling, S. (2004). Refinement of single-nucleotide polymorphism genotyping methods on human genomic DNA: amplifluor allele-specific polymerase chain reaction versus ligation detection reaction-TaqMan. Analytical Biochemistry, 330(2), 288-297. doi:10.1016/j.ab.2004.03.035.

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Genre: Journal Article
Alternative Title : Anal Biochem

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 Creators:
Rickert, Andreas M.1, Author              
Borodina, Tatiana A.2, Author              
Kuhn, Eckehard J.3, Author
Lehrach, Hans1, Author              
Sperling, Silke1, Author              
Affiliations:
1Dept. of Vertebrate Genomics (Head: Hans Lehrach), Max Planck Institute for Molecular Genetics, Max Planck Society, ou_1433550              
2Technology Development(Alexey Soldatov), Dept. of Vertebrate Genomics (Head: Hans Lehrach), Max Planck Institute for Molecular Genetics, Max Planck Society, ou_1479657              
3Max Planck Society, ou_persistent13              

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Free keywords: Single-nucleotide polymorphism; SNP; Genotyping; Amplifluor allele-specific PCR; LDR-TaqMan; Multiplexing
 Abstract: Single-nucleotide polymorphisms (SNPs) have proven to be powerful genetic markers for a variety of genetic applications, e.g., association studies leading to dissection of both monogenetic and complex diseases. However, no single SNP genotyping method has been broadly accepted. In the present study, we compared and refined two promising methods with potential for research and for diagnostic SNP genotyping: Amplifluor allele-specific polymerase chain reaction (PCR) and ligation detection reaction (LDR)-TaqMan. The methods are based on allele-specific primer extension and allele-specific ligation, respectively. Since LDR-TaqMan had previously been tested on just Arabidopsis thaliana, we adjusted the method for the more complex human genome. Amplifluor allele-specific PCR has a single-step and closed-tube format, whereas the LDR-TaqMan assay comprises two simple steps. Contrary to the primer-extension-based method, the ligation-based method can be multiplexed. Refining the LDR-TaqMan technique, we successfully replaced a previously suggested three-step multiplexing procedure with a less laborious two-step approach. Comparing refined LDR-TaqMan with Amplifluor allele-specific PCR in a family-based study, both techniques appeared similar with respect to high robustness and accuracy. As both approaches utilize primers with common tails, all SNPs can be assayed with the same couple of fluorescence reporting reagents, ensuring low establishing and running expenses.

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Language(s): eng - English
 Dates: 2004-07
 Publication Status: Published in print
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Type: -
 Identifiers: eDoc: 230763
DOI: 10.1016/j.ab.2004.03.035
 Degree: -

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Title: Analytical Biochemistry
  Alternative Title : Anal Biochem
Source Genre: Journal
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Publ. Info: -
Pages: - Volume / Issue: 330 (2) Sequence Number: - Start / End Page: 288 - 297 Identifier: ISSN: 0003-2697