Single-base primer extension

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

Figure 12.2. Single base primer extension. This patient is heterozygous for this Ato C substitution. The labels from both ddNTPs can be detected in this sample. In the case of a homozygous genotype, only one of the labels would be detected. Detection might be through ELISA, fluorescence, FRET, or FP.

Single Base Primer Extension. One of the most effective ways to detecting polymorphism s a technique known as singlebase primer extension (Fig. 12.2). In this... 

Primer extension reactions can be analyzed using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDl-TOF MS). Specially prepared single-base primer extension products are deposited on MALDI targets, evaporated by a laser beam, and directed into the time of flight mass spectrometer. This sensitive, label-free method can discriminate which base (A, C, G, or T) was added to the primer at the very SNP position, and affords excellent error tracking. 

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... 

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.

Preparation of Probe. Probes for genomic Southern blots have been made by a variety of methods and must be of high specific activity. A simple method that yields a probe with a very high specific activity utilizes the polymerase chain reaction (PCR).16 An advantage of PCR over methods based on nick translation or primer extension is that only one strand of DNA is synthesized, so that reassociation of denatured single-strand probe in solution is minimized. 

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