Chapter 18

Sexual Reproduction, Meiosis, and Genetic Recombination

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Figure 18-31 Preparation of Complementary DNA (cDNA) for Cloning

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(1) Messenger RNA is incubated with reverse transcriptase, which uses the mRNA as a template for synthesis of a complementary DNA (cDNA) strand. Oligo(dT), a short chain of thymine deoxynucleotides, can be used as a primer, because eukaryotic mRNA always has a stretch of adenine nucleotides at its 3prime end. (2) The resulting mRNA-cDNA hybrid is treated with alkali or an enzyme to hydrolyze the RNA, leaving the single-stranded cDNA. (3) DNA polymerase can now synthesize the complementary DNA strand. The looped-around 3prime end of the first DNA strand can often be used as a primer. An enzyme called S1 nuclease is then used to cleave the loop. (4) For efficient insertion in a cloning vector, the double-stranded DNA must have single-stranded tails that are complementary to those of the vector. These can be added by incubation with terminal transferase, an enzyme that adds nucleotides one at a time to the ends of the molecule. If short stretches of cytosine (C) nucleotides, for example, are added to the cDNA and short stretches of guanine (G) nucleotides are added in the same way to a linearized cloning vector, recombinant molecules can be generated by allowing the single-stranded C tails in the cDNA to hybridize to the single-stranded G tails in the vector. (As an alternative to step 4, short synthetic "linker" molecules containing a variety of restriction sites can be ligated to the ends of both the cDNA and a blunt-ended cloning vector. The linkers are then cleaved with a restriction enzyme that generates cohesive ends.)

© 1999 by Addison Wesley Longman A division of Pearson Education