Now, digest the gDNA using a desired restriction digestion endonuclease.Ĭhoose the endonuclease which can only cut the unknown flanking regions but not the known DNA regions. Restriction digestion is a process in which the DNA having the restriction site is digested using the known restriction endonuclease.įor more detail on the restriction digestion read our article: What is Restriction Digestion and how to do it? Phenol-chloroform DNA extraction method Īfter DNA extraction, quantify the DNA and check the purity of the DNA because the purity matters a lot in the amplification.Different types of DNA extraction methods.In the next step, extract DNA using any of the DNA extraction methods listed below, By identifying the known location near the unknown DNA region, design the primers complementary to the known DNA region helps to amplify the unknown region. Inverse PCR is used for identification gene rearrangements, transposons and jumping gene studies. In the very first step, we have to identify the target. Identification of known DNA region having flanking unknown DNA sequence: Amplification of ligated circular DNA moleculeġ.ligation of digested unknow DNA fragments.Identification of known DNA region having flanking unknown DNA sequence.The entire process of inverse PCR is divided into 5 steps: Ligating the flanking regions of unknown DNA generates circular DNA which is amplified using the set of primers. Restriction digestion is performed on the genomic DNA which digests only the unknown flanking regions but not the known DNA region. DNA synthesis occurs outside the known DNA region. With the help of the sequence information of known DNA region, the unknown flanking region of the DNA or the inserted DNA is amplified into the cyclic enzymatic reaction using the known DNA sequence-specific primers. No special DNA polymerase is required in the inverse PCR. See the above figure.Īlthough the Taq DNA polymerase is the same in both types of PCR. However, we design primers complementary to the known DNA region but instead of extending towards each other, the primers extend DNA away from each other. We do not have any information about what types of DNA is inserted into the genome. While in the inverse PCR, the region of the DNA is unknown. On two different single-stranded DNA, primers are bind and synthesised towards each other. Now see the figure, in the conventional PCR, our target DNA is known to us, we have the sequence information of that.ĭepending upon that the primers are designed to amplify that know complementary DNA sequence. The inverse PCR method is originally developed by Howard Ochman and coworker in the year 1988.įirstly we have to understand the basic difference between conventional PCR and the inverse PCR. Inverse PCR is just a variant of the conventional PCR. PCR is a technique in which the DNA is amplified using a set of the sequence-specific complementary primers in the enzymatic cyclic temperature dependent reaction.
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