E

Figure 7.9. Generation and selection of genomic DNA clones.

is repeated. The dead cells give rise to a clear area on the bacterial lawn called a plaque. Each plaque contains many copies of a recombinant bacteriophage that can be transferred to a nylon membrane (Fig. 7.9). Specific DNA clones are selected by incubating the nylon membrane with a radiolabeled cDNA probe complementary to the genomic sequence being searched for. This produces a radioactive area on the nylon membrane that is identified by autoradiography. The use of a cDNA sequence as a gene probe makes the task of isolating the corresponding genomic sequence much easier.

A radioactive cDNA probe can be synthesized using the method called random priming. The cDNA clone is heated so that the two strands will separate. Each strand will act as the template for the synthesis of a new DNA strand. A mixture of random hexamers, six nucleotide sequences, containing all possible combinations of the four bases (A, T, C, G) is added to the denatured cDNA along with DNA polymerase and the four deoxynucleotides dATP, dTTP, dCTP, and dGTP. The hexamers will hydrogen-bond to their corresponding sequences on the cDNA templates and prime the synthesis of new DNA strands. If, for example, radiolabeled [a-32P]dATP is included in the reaction, the newly synthesized DNA strands will be radioactive.

Was this article helpful?

0 0

Post a comment