Polymerase Chain reaction (PCR)
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Polymerase chain reaction (PCR) is greatly used in molecular genetics. It entails amplification of a single DNA strand into millions of similar DNA fragments. It involves three stages in each cycle. It is repeated to about 30 cycles. This method is vital as it is used in various processes such molecular identification, genetic engineering, and sequencing. The three stages in each cycle have varying duration and temperature. A thermal cycler is involved in the regulation of temperature in various stages. Over time, ...view middle of the document...
There are various DNA polymerases that can be utilized in the PCR process. Pfu DNA polymerase that is extracted from Pyrococcus furiosus and Taq polymerase derived from bacteria Thermis aquaticus are commonly used. These DNA polymerases are stable at high temperatures; thus, they are not likely to be denatured compared to Escherichia coli DNA polymerase that was used initially. E. coli DNA polymerase has an optimal temperature of 37oc thus it is labile at high temperatures. Its utilization forced addition of more E. coli polymerase at the beginning of each cycle. However, introduction of the latter thermal stable DNA polymerases simplified and increased the efficiency of PCR amplification due to enhanced specificity of the DNA target. This process consists of three stages namely; denaturation, annealing, and extension.
The temperature applied in each stage varies. The PCR technique has many vital applications that are worth mentioning. Polymerase chain reaction has extensively been used in DNA fingerprinting, mutation, genetic correspondence, diagnosis of genetical disorders, grouping of organisms, pre-natal screening and drug discovery and development. Furthermore, it is also used in forensic science to apprehend criminals as well as in paternity or maternity tests.
Steps carried out in polymerase chain reaction.
There are three stages taken during polymerase chain reaction in each cycle. They are redone up to about 30 cycles. They involve temperature change and different duration of each stage. To elaborate further, the temperature in these stages is varied depending on the quantity of divalent ions, DNA polymerase and divalent ions used. Some DNA polymerase used require high temperature of up to 98oC so as to be activated thus this brings the variance. In addition, primers melt at different points depending on the ratio between cytosine- guanine bonds to adenine- thymine bonds. Cytosine guanine bonds are strong since they involve three hydrogen bonds while the latter has two hydrogen bonds. Hence, primers with a higher ratio of cytosine-guanine to adenine- thymine ratio require more energy to break them up.
The following stages are involved
This stage is done to DNA polymerases that require to be activated by temperature elevation. It is essentially carried out by utilization of hot-start PCR in polymerases that need high temperature for activation. Hot start PCR is an advanced type of PCR that inactivates DNA polymerase at lower temperatures so as to avoid non-particular amplification of DNA target sequence. It is done at a temperature of between 94 to 98oC for about one to nine minutes.
The goal is to obtain single-stranded DNA template that acts as the starting material for amplification of target site fragments. It does so by introduction of heat of about 94 - 98oC in the thermal cycler for about thirty seconds. High temperatures break the hydrogen bonds formed between the various...