Biology Reference
In-Depth Information
Figure 6.9
cDNA cloning involves two steps. In the first step, an oligo(dT) anneals to the poly(A)
region of mRNA. The enzyme reverse transcriptase and dATPs, dCTPs, dGTPs, and dTTPs are pro-
vided to produce the first cDNA strand. Synthesis of the second strand of the cDNA involves addi-
tional incubation with RNAse H and DNA polymerase I. The double-stranded cDNA produced is a
complement to the mRNA and thus lacks introns or regulatory sequences.
carefully considered and the basic approach chosen after deciding how the
cDNA library will be screened to identify the gene(s) of interest (
Kimmel and
Berger 1987
). For example, if antibodies will be used to identify clones capable
of synthesizing specific peptides, the cDNA should be cloned into expression vec-
tors to produce proteins.
The cloning techniques vary in the type of primer used, the method for
second-strand synthesis, and methods for coupling the cDNA to the vector,
