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The fresh gene of great interest try PCRed with oligos that contain a great area which is perfectly subservient on the layout , and one one to differs from the brand new theme by a minumum of one nucleotides . The easiest method towns the fresh mutation website to your one of many closes of the fragment where 1 of 2 oligonucleotides employed for promoting the new fragment has got the mutation. This involves just one action of PCR, yet still has got the built-in problem of requiring the right limit website near the mutation website except if a very long primer try utilized. These methods require multiple procedures away from PCR so the latest fragment getting ligated can be hold the desired mutation. The shape techniques to own generating an excellent fragment to the need mutation and associated limit internet sites will be troublesome.

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