Polymerase annealing

Lower concentration of PE-PCR primers used is to avoid of self-annealing of the long adaptors, which will further block the DNA polymerase annealing on the cDNA templates. Single primer control in this PCR reaction should also be done to monitor potential faulty amplification. 

Each primer is a synthetic oligonucleotide of about 20 bases prepared so that then-sequences are complementary to the (previously determined) sequences that flank the tar get regions on opposite strands Thus one primer is annealed to one strand the other to the other strand The 3 hydroxyl end of each primer points toward the target region The stage is now set for DNA synthesis to proceed from the 3 end of each primer [Figure 28 14(c )] The solution contains a DNA polymerase and Mg " m addition to the... 

PGR amplification of a DNA sequence is faciHtated by the use of a heat-stable DNA polymerase, Taq polymerase (TM), derived from the thermostable bacterium Thermus aquaticus. The thermostable polymerase allows the repeated steps of strand separation, primer annealing, and DNA synthesis to be carried out ia a single reactioa mixture where the temperature is cycled automatically. Each cycle coasists of a high temperature step to deaature the template strands, a lower temperature annealing of the primer and template, and a higher temperature synthesis step. AH components of the reaction are present ia the same tube. 

The temperature is then raised to 72 °C, and Taq polymerase catalyzes the addition of further nucleotides to the two primed DNA strands. When replication of each strand is finished, two copies of the original DNA now exist. Repeating the denature-anneal-synthesize cycle a second time yields four DNA copies, repeating a third time yields eight copies, and so on, in an exponential series. 

FIGURE 11.10 Schematic drawing of the PCR cycle. (1) Denaturing at 95°C. (2) Annealing at 55°C. (3) Synthesizing at 72°C (P = Polymerase). (4) The first cycle is complete. The two resulting DNA strands make up the template DNA for the next cycle, thus doubling die amount of DNA duplicated for each new cycle. Source http //en.wikipedia.org/wiki/Polymerasechainreaction (see Plate 3 for color version). 

PCR is a technique for in vitro amplification of DNA sequences that involves repeated cycles of denaturation, oligonucleotide annealing, and DNA polymerase extension [29], The amplified products following PCR cycles contain double-stranded DNA fragments of discrete length. These DNAs are copies of the template DNA that are bounded at the 5 -terminus by the oligonucleotide primer for the sequence extension with a heat-resistant DNA polymerase. In quantitative assays of PCR products, therefore, nonspecific products interfere with the assay. 

Such renaturation or annealing of complementary DNA strands is an important step in probing a Southern blot and in performing the polymerase chain reaction (reviewed in Chapter 7). In these techniques, a well-characterized probe DNA is added to a mixture of target DNA molecules. The mixed sample is denatured and then renatured, When probe DNA binds to target DNA sequences of sufScient complementarity, the process is called hybridization. 

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