Primer Extension Reactions

While the MALDI-TOF process is relatively forgiving for the presence of small amounts of salt, the bulk of contaminating sodinm and potassium salts must be removed from the sample before analysis. This is most conveniently carried out by a simple reverse-phase desalting step [9], which in the past has been accomplisbed in pipette tips and can now be supported with 96-well filter plates. Samples can also be desalted by ethanol precipitation or by membrane float dialysis [15], although these techniques are less convenient for processing a large number of samples. 

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Schulze et al. [135] developed fused-silica chips dynamically coated with hydroxypropylmethyl cellulose and utilized them for the separation of aromatic low molecular weight compounds such as serotonin, propranolol, a diol, and tryptophan. The authors used deep UV laser-induced fluorescence detection for these compounds. Schuchert-Shi et al. [136] identified ethanol, glucose, ethyl acetate, and ethyl butyrate, byproducts obtained in enzymatic conversions using hexokinase, glucose oxidase, alcohol dehydrogenase, and esterase. The authors reported that the quantification for ethyl acetate was possible using contactless conductivity detection. Hu et al. [137] described the separation of reaction products of (3-thalassemia in a multiplex primer-extension reaction using NCE. The method developed was used for patient samples and the results coincided with those of a detection kit. 

Figure 3 The MassEXTEND reaction. Following PCR amplification of the locus of interest, a primer-extension reaction is performed using an extend primer (ME) that is designed to anneal next to the SNP. The key feature of this scheme is the use of a terminator mixture that yields extension products that differ in length in an allele-specific manner, thus creating mass separations between alleles equal to the mass of a nucleotide. In this example, a normal dG base is used along with ddA, ddC and ddT. For the A allele (A), a ddA is incorporated, extending the primer to 20 mer. For the G allele (G), the SNP calls for incorporation of the normal dG residue prior to incorporation of a ddT, extending the 19-mer primer to 21-mer.

Because of the hairpin formation, these dyes are in such a close proximity that their fluorescence is quenched (molecular beacon Box 18) unless the structure is unfolded in the course of second-strand synthesis (Figure 4.3.4b). Thus, detection of a fluorescence signal from one of both dyes is a direct measure of the progress of the reaction. These researchers also showed that primer extension reactions can be monitored directly in cleared lysates of cells overexpressing the Klenow fragment of E. coli DNA polymerase I. Thus, the molecular beacon assay might supersede extensive purification. 

Fig. 1. Structure of nucleotides and site of modification by dimethyl sulfate in single-stranded RNA. DMS-modified structures of the four normally occurring nucleotides in RNA are shown. Indicated in bold type are the methyl groups added by DMS which inhibit the progress of reverse transcriptase during primer extension reactions.

Fig. 4. Primer extension analysis of rRNAs isolated from four plant species treated with dimethyl sulfate. Sequencing lanes are indicated as A, T, G, and C. A plus sign (+) over the lane denotes RNA prepared from DMS-treated tissues a minus (—) denotes RNA isolated from mock-treated tissues. The nucleotide numbers are the coordinates at which primer extension reactions terminate (see Figs- 3 and 5A). The position of the modified base in the sequence of the 18 S rRNA is determined by comparing novel bands in the DMS-reacted RNA with the sequencing ladder and adding one nucleotide (see Fig. 2 for details). Various regions of the RNA are shown for the different plant species. Several sites of chain termination arising from nascent structure are also evident.

Two photoreactive dATP analogues (115, 116) have been incorporated into DNA using Klenow fragment. Subsequent UV irradiation of the primer extension reaction allowed specific cross-linking of the analogues to the polymerase. ... 

Zhou G, Kamahori M, Okano K, Chuan G, Harada K, Kambara H. Quantitative detection of single nucleotide polymorphisms for a pooled sample by a bioluminometric assay coupled with modified primer extension reactions (BAMPER). Nucleic Acids Res 2001 29 e93... 

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. 

Both methods described here are based on the primer extension reaction. In both platforms, oligonucleotide primers are designed that extend up to the nucleotide adjacent to the SNP site. The following primer extension reaction will use ddNTPs... 

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