1-2-3 Isolation of DNA fragment that is to be cloned
Select and prepare a DNA fragment that is to be incorporated into a host cell, where it will be replicated and translated into a protein product
1-2-5 Polymerase chain reaction
Polymerase chain reaction (PCR) enables the rapid synthesis of large number of copies of a specific DNA fragment from a complex mixture of DNA. This technique was developed by Kary Mullis in the year 1983 and is now a very commonly used technique in the field of medical science. Researchers can thus obtain large quantities of specific pieces of DNA for experimental and diagnostic purposes. PCR requires a series of repeated reactions called cycles. Each cycle has three steps which are executed in a machine called there Mo cycler. In the first step, the target DNA containing the sequence to be amplified is denatured by heating to make it single-stranded. Next, the temperature is lowered so that the primers and hydrogen bond or anneal to the DNA on both sides of the target sequence. Finally, DNA polymerase extend the primers and aynthesizes copies of the target sequence using deoxyribonucleoside triphosphates (dNTPs). At the end of the cycle, the targeted sequences on both strands have been copied. When the three step cycle is repeated the two strands from the first cycle are copied to produce four fragments. These are amplified in the third-cycle to yield eight doubler stranded products. Thus, each cycle increases the number of target DNA molecules exponentially. Thus continuous heating and cooling will allow DNA stands to denature and anneal to form many copies of the targeted DNA fragment. The greatest advantage of PCR over southern blot analysis is that in PCR, small portion of genome ranging from 100bp to 1000bp can be amplified by specific primers. It also requires less time and is less expensive than Southern blot analysis. PCR or polymerase chain reaction can be used to detect microsatellites. It can be used in DNA fingerprinting. Microsatellite is not a gene. PCR analysis can use different types of microsatellites. Hence PCR is more useful than southern blot analysis.