Hybridisation primer

Fig. 6.2. The principle of PCR. (1) Denaturation the two DNA strands are separated at 95 °C (2) Anneahng of primers the primers are hybridised to their complementary sequences at 50-60 °C (3) Primer extension at 72 °C, the polymerase catalyses the synthesis of the complementary single stranded DNA by extending the 3 -end of the hybridised primer.

Step 3 - Primer extension The synthesis of DNA is catalysed by the polymerase. The 3 -end of the hybridised primer is extended along the original DNA strand by continuous incorporation of the complementary nucleotides into the chain. Thus, a complementary DNA strand is formed. Primer extension proceeds from the 3 -end to the 5 -end. This extension reaction stops as soon as the complementary strand has been completed. It has been empirically shown that the fidelity of the construction of the complementary strand improves at 72 °C. Hence, the temperature is usually adjusted to this value for the primer extension step. 

Fig. 8.2.2 Principle of genomic quantification by multiplex ligation-dependent probe amplification (MLPA). Synthetic oligonuleotides are designed to bind to exon 1 (oligo 1a and 1b) and 2 (2a and 2b). After specific hybridisation, the oligos are ligated. The ligation products undergo quantitative polymerase chain reaction using universal primers X and Y...

Fig. 3 Polyacrylamide gel electrophoresis result of a hybridisation mixture of an M13 reverse sequencing primer labelled with [Re(phen)(CO)3(py-3-NCS)]+ and three unmodified oligonucleotides of various lengths [40]...

Figure 5-16. Principle of the polymerase chain reaction (PCR). The polymerase chain reaction relies on primer-dependent replication of a double stranded DNA template. At the beginning of the reaction, the double stranded DNA is denatured and two primers complementary to defined regions on the two strands are hybridised to the separated strands by annealing.

High specificity, which is determined by the precise hybridisation of the two primers to the complementary sequences on the DNA template, and which can be influenced by the choice of anealing conditions, particularly temperature. 

First, a single stranded DNA template, usually PCR amplified and purified by chromatography, gel filtration or electrophoretic techniques, is immobilised onto a surface. A suitable primer is then hybridised to this single strand (Fig. 5.26). This ensemble is incubated with four enzymes (DNA polymerase, ATP sulfyrase, luciferase and apyrase) and two substrates (adenosine 5 phosphosulfate (APS) and luciferin). 

Fig. 5.26. A single stranded DNA template is immoblised onto a surface and hybridised with a suitable primer.

Primers are short oligonucleotides, which are complementary to the ends of the target sequence. Two distinctive primers are used for a PCR amplification a forward primer and a reverse primer (Fig. 6.2). Each hybridises to one of the two strands of the original dsDNA molecule (see Fig. 6.4, cycle one). DNA synthesis always proceeds in the direction from 5 to 3. The first nucleotide to be incorporated reacts with the free 3 -hydroxyl group of the primer. The 5 -end of the primer is blocked. 

Fig. 6.7. Probe based real-time PCR (a) During primer annealing the probe hybridises to a region within the target sequence and fluorescence is quenched, (h) and (c) As the primer extension proceeds, the exonuclease activity of the polymerase cleaves the reporter from the rest of the probe, (d) The reporter now fluoresces freely as it is in solution and no longer...

Deaza- and aza-analogues of guanosine have been examined primarily in hybridisation and primer extension studies. 1-Deaza-dG incorporated into ODN duplexes exhibited no preference for pairing with the natural nucleotides, and was more destabilising than a mismatch. In primer extension reactions it preferentially formed pairs with The importance of hydrogen bond... 

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