PCR in Genetic Engineering

A whole plethora of modifications has been made to the basic PCR scheme. Some of these are used to alter genes rather than to analyze them. We can divide these modifications into two broad categories: a) rearranging large stretches of DNA and b) changing one or two bases of a DNA sequence. The latter is discussed below as "directed mutagenesis".

As already illustrated, we can amplify any segment of DNA provided we have primers that match its ends. Such segments of DNA may be joined or rearranged in a variety of ways. In order to make a hybrid gene, segments of two different genes must be amplified by PCR and then joined together. There are several protocols that vary in their details. But the crucial point is to use an overlap primer that matches part of both gene segments (Fig. 23.18).

The PCR reaction is run using a primer for the front end of the first gene, a primer for the rear end of the second gene, and the overlap primer. The result is a hybrid gene. Some variants of this "molecular sewing" make the two halves separately and mix and join them later; other versions of this technique mix all three primers plus both templates in a single large reaction. By making hybrid genes using components from various sources it is sometimes possible to work out in detail which regions of a gene or protein are responsible for precisely which properties. The approach can also be used in biotechnology to construct artificial genes made up of modules from different sources.

anchor sequence Sequence added to primers or probes that may be used for binding to a support or may incorporate convenient restriction sites, primer binding sites for future manipulations, or primer bindings sites for subsequent PCR reactions molecular sewing Creation of a hybrid gene by joining segments from multiple sources using PCR

overlap primer PCR primer that matches small regions of two different gene segments and is used in joining segments of DNA from different sources

RACE See rapid amplification of cDNA ends rapid amplification of cDNA ends (RACE) RT-PCR-based technique that generates the complete 5' or 3' end of a cDNA sequence starting from a partial sequence

S" RACE-PCR

5" RACE-PCR

Iaaaaaaa S"

Rt with anchor primer

Anchor sequence

Anchor sequence

AAAAAAA S"

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