Primer extension assay

The Cadet laboratory has prepared phosphoramidites of stereoisomers for 5, 8-cyclo-dA and 5, 8-cyclo-dG for incorporation into oligonucleotides. Primer extension assays using the mammalian replicative enzyme pol 8 demonstrated that 5, 8-cyclonucleosides block DNA replication in vitro and thus would be highly... 

The primer extension assay is similar to the dideoxy method commonly used in sequence analysis. In the primer extension assay, a region containing the mutation to be assayed is amplified in a first PCR reaction. A specific primer, which is designed to anneal directly upstream of the base which is the site of a known mutation, is then used in a second reaction. Radioac-tively labeled dideoxy nucleotides corresponding to either the normal or the mutant base at the potential mutation site are added in separate tubes. During the reaction, the labeled nucleotide added to the 3 end of the primer will depend on the sequence at the potential mutation site. If the normal sequence is present at the potential mutation site, only the reaction containing the normal labeled dideoxy nucleotide will produce a labeled primer. Conversely, if the mutant sequence is present at the mutation site, only... 

Primer extension assays are very sensitive for mutation detection and may be advantageous for large-scale testing with some modifications to the basic protocol. Primer extension assays have been designed to use fluorescent dye labeled nucleotides to eliminate the need for radioactivity and may also be adaptable to use on solid supports (22). For molecular diagnostics purposes, there are also commercially available kits and protocols for diagnostic applications (23). 

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.

However, quantification of the extent of editing or modifications can often be assayed by a primer extension assay. The principle of a primer extension analysis is illustrated in Fig. 5.6 that shows... 

Similar assays can be used to study modifications that inhibit the extension of the reverse transcriptase directly and also modifications which render the RNA susceptible to selective strand scission at the modified residue (e.g. m7 guanosines, Section 4.4.2.2). To quantify the extent of modification, one of the nucleotides in the extension mixture should only be present in the dideoxy form (Fig. 5.7). The primer extension assay can also be used for some modifications which do not affect the extension, provided the modification interferes with the reactivity of the nucleotide. The latter approach has been used to study the 2 -0-methylations of guanosine where the modification renders the modified guanosine resistant to RNase T1 digestion. Introduction of a complete RNase T1 digest before the primer extension allows the detection of the modification (Fig. 5.8a). However, the approach requires that there is no nucleotide as the one modified for at least 12 nucleotides 3 of the modified yresidue. If this is not the case alternative approaches can sometimes be employed (see Fig. 5.8b). 

Matyas, G., Giunta, C., Steinmann, B., Hossle, J. P., and Hellwig, R. (2002) Quantification of single nucleotide polymorphisms a novel method that combines primer extension assay and capillary electrophoresis. Hum. Mutat. 19, 58-68. 

Although a number of quantitative assays are available for allele frequency estimation, most are difficult and expensive to develop or implement. In this chapter, we describe the most versatile and least expensive assays for allele frequency estimation, namely, the template-directed dye-terminator incorporation assay with fluorescence quenching detection (the FQ-TDI assay [3]). The FQ-TDI assay is a real-time homogeneous primer extension assay based on two principles, namely, that deoxyribonucleic acid (DNA) polymerase catalyzes the allele-specific incorporation of a dye-terminator at the polymorphic site and that the fluorescence intensities of a fluorescent dye decreases significantly when it is incorporated into primers. 

Two-color primer extension assay with real time monitoring of fluorescent polarisation Mass spectrosopy - (MS)... 

PCR polishing is the process of destroying the residual dNTPs and primers, which otherwise would interfere with the primer extension assay (Figure 1). The PCR product itself need not be converted to single stranded form for the SNP assay. 

Although different DNA polymerases have distinct functions and substrate affinities, their general mechanism of action is similar. Thus, they can all be studied using the same technical principle, the primer extension assay employing radioactive tags (Lope et al. 2007). 

Lopes DO, Regis-da-SUva CG, Machado-SUva A et al (2007) Analysis of DNA polymerase activity in vitro using non-radioactive primer extension assay in an automated DNA sequencer. Genet Mol Res 6 250-255... 

The fluorescent phenoxazine derivative (45, X = O) was compared with (45, X = O) in which the ring structure was further extended to make it a spin-label, in a series of mismatched duplexes. It was shown that the parent structure exhibited greater mismatch discrimination over a series of 16 different sequence contexts.The phenoxathiazine analogue (45, X = S) has been examined with DinB (Pol IV, Y family polymerase) in primer extension assays. It was found that DinB selectively incorporated dGMP opposite the bulky nucleotide but was unable to extend the DNA chain... 

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