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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

Item Type:Article
Title:Refinement of single-nucleotide polymorphism genotyping methods on human genomic DNA: amplifluor allele-specific polymerase chain reaction versus ligation detection reaction-TaqMan
Creators Name:Rickert, A.M. and Borodina, T.A. and Kuhn, E.J. and Lehrach, H. and Sperling, S.
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.
Keywords:Amplifluor Allele-Specific PCR, Genotyping, LDR-TaqMan, Multiplexing, Single-Nucleotide Polymorphism, SNP
Source:Analytical Biochemistry
ISSN:0003-2697
Publisher:Academic Press (U.S.A.)
Volume:330
Number:2
Page Range:288-297
Date:15 July 2004
Official Publication:https://doi.org/10.1016/j.ab.2004.03.035
PubMed:View item in PubMed

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