Polymerase primer extension

The elongation of the primers with the complementary order of nucleotides by the synthesis of the new DNA strand by the enzymatic activity of the taq-polymerase (primer extension)... 

Steps in the polymerase chain reaction (PCR). The DNA to be amplified is denatured and annealed with two oligonucleotides that flank the region of interest. These oligonucleotides (or primers) are extended. Extension continues to the ends of the DNA strands. The products are again denatured and annealed to primers for a second round of extension. This process of denaturation, annealing, and primer extension is repeated many times. The primary product of the reaction is duplex DNA, bounded by the sequences of the primers. (From J. L. 

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... 

Allele-specific differences between regulatory polymorphisms associated with the ability of RNA polymerase II to bind and assemble its transcription complex at the start site of transcription for several eukaryotic promoters has also been measured using MALDI-TOF MS coupled with primer extension (42). This technique provides a powerful tool for identifying important regulatory SNPs and haplotypes in vivo. 

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. 

Figure 15.5 Polymerase chain reaction step 1, separation of strands by heating (98°C) step 2, anneal primers (60°C) step 3, primer extension by polymerase.

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. 

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. ... 

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