Single-base extension

Steemers, E.J., Chang, W., Lee, G., Barker, D.L., Shen, R., and Gunderson, K.L. (2006) Whole-genome genotyping with the single-base extension assay. Nat. Methods. 3, 31-33. 

Single-nucleotide polymorphisms (SNPs) have been analysed on a capillary gel electrophoresis (CGE) microchip with EC detection [150]. The genetic section that contained the SNP was amplified by PCR and purified. Then, it was used in a single-base extension (SBE) reaction with a redox-labeled chain terminator, ferrocene-acycloATP. Products of the SBE, ferrocene-labeled SNP and free ferrocene-acycloATP, were separated employing CGE on microchip and detected using sinusoidal voltammetric detection at a pyrolysed photoresist film (PPF) electrode. 

In minisequencing single nucleotide primer extension (SNE) or single base extension (SBE), a DNA polymerase is used to extend a detection primer, which anneals immediately adjacent to the site of the SNP, with a labeled nucleotide analog (23,24). In the microarray format of... 

Biotin is a common reagent for labelling oligonucleotides, and a new amidite (175) has been prepared for biotinylated oligonucleotides, but the biotin group may be removed by fluoride treatment.Biotinylated ddNTPs have been used for single base extension for multiplex genotyping by mass spectros-... 

Fig. 13. Schematic of the single-base extension assay applied to Tag probe arrays. Regions containing known SNP sites (A or G in this example) are first amplified by PCR. The PCR product serves as the template for an extension reaction from a chimeric primer consisting of a 5 tag sequence and a 3 sequence that abuts the polymorphic site. The two dideoxy-NTPs that could be incorporated are labeled with different flurophors in this example, ddUTP is incorporated in the case of the A allele, and ddCTP for the G allele. Multiple SBE reactions can be done in a single tube. The tag sequence, unique for each SNP, directs the extension products to a particular address on the Tag probe array. The proportion of a fluorophor at an address reflects the abundance of the corresponding allele in the original DNA. (Reprinted with permission from [45])...

The method used for allele-specific quantification will depend on facilities available in the laboratory. A number of approaches for accurate quantification of cDNA have been published for allele-specific quantification, notably using single base extension methods in the presence of fluorescently labeled nucleotides with detection on a DNA sequence detector (5,11) or by restriction enzyme digestion and real-time PCR amplification (12). 

Key Words Denaturing high-performance liquid chromatography (DHPLC) genotyping mutation detection single-nucleotide polymorphism (SNP) single-base extension (SEE). 

Incubated the mixture at 37°C for 45 min to degrade the excess PCR primers and excess dNTP. Heat inactivate the enzymes were at 80°C for 15 min before the single-base extension reaction. 

Thomas, G., et al., Capillary and microelectrophoretic separations of ligase detection reaction products produced from low-abundant point mutations in genomic DNA, Electrophoresis, 25,1668, 2004. BraziU, S.A. and Kuhr, W.G., A single base extension technique for the analysis of known mutations utilizing capillary gel electrophoresis with electrochemical detection. Anal Chem, 74, 3421, 2002. 

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