Single base extension assay

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. 

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])...

Single Nucleotide Extension Assay Also known as single-base primer extension or minisequencing, single nucleotide extension (SNE) assays involve the... 

Several modes of detecting a single base extension have been investigated, including measuring the incorporation of fluorescent, haptenated, or radioactive ddNTPs [6, 7] or gel electrophoresis based-detection of fluorescent primers extended by non-fluorescent nucleotides [8]. Recently, the Applied Biosystems SNapshot lYimer Extension Kit was introduced. In this assay, a primer is extended by one or more fluorescent- labelled dideoxynucleotides with subsequent detection in a fluorescent-based DNA sequencer. Several primers can be analysed within one lane of the DNA sequencer. 

MALDI-TOF mass spectrometry has recently proven an attractive means to analyse single base extensions. Several thousand samples can be analysed each day, because each analysis requires only a few seconds. Furthermore, within each sample, multiple independent loci can be simultaneously analysed. With this internal multiplexing, tens of thousands of SNP genotypes can be obtained each day. Reagent costs are comparatively low, because there are no signal molecules in the assay and the primers are unlabeled and comparatively inexpensive. Because... 

Single base extension methods such as the PinPoint assay have an advantage for multiplexed SNP marker analysis. Notably, they can combine aU assays irrespective of the alleles to be genotyped simply by using the four dideoxynucleotides... 

Chen J, Iannone MA, Li MS, Taylor JD, Rivers P, Nelsen AJ, Slentz-Kesler KA, Roses A, Weiner MP. A microsphere-based assay for multiplexed single nucleotide polymorphism analysis using single base chain extension. Genome Res 2000 10 549-557. 

Fig. 2. Primer extension assay to detect DMS-modified nucleotides in single-stranded RNA. RNAs modified with DMS are examined by primer extension. Bases at which modifications occur are unable to form complementary hydrogen bonds with the cognate nucleotide. Thus, chain termination results. Primer extension products (PE) terminate one nucleotide before the modified residue compared to a sequencing ladder.

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