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FIGURE 23.17 Rapid Amplification of cDNA Ends (RACE)

RACE can be used to isolate the 5' and/or 3' ends of a cDNA that is incomplete. The method to amplify the 3' end of the cDNA is shown on the left side of the figure. This requires an oligo(dT) primer that has an anchor sequence at the 5' end. This primer is used with reverse transcriptase to make the mRNA : DNA hybrid molecule. The mRNA portion of this is removed and a second strand of DNA is synthesized. Instead of priming the second strand from the beginning of the DNA, an internal primer closer to the 3' end of the gene is used. The same internal primer and a primer corresponding to the anchor sequence are then used in a standard PCR reaction to amplify just the 3' end of the cDNA.

The right side of the figure shows how the 5' end of a cDNA is isolated. An internal primer is designed to prime reverse transcriptase and make a hybrid molecule of mRNA : DNA. In order to add a primer binding site upstream of the end of the hybrid molecule, the enzyme, terminal transferase, is added together with dATP. This enzyme adds a run of adenines to the 3' end of the DNA half of the hybrid. The mRNA half of the hybrid is then removed and replaced with DNA by using an oligo(dT) primer carrying the anchor sequence. The oligo(dT) binds the newly synthesized poly(A) stretch on the DNA, and primes the polymerase to make a cDNA. Subsequent PCR using the internal primer and the anchor primer amplify only the 5' end of the cDNA.




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