The first step in preparing a cDNA library is to isolate the total mRNA from the cell type or tissue of interest. Because of their poly(A) tails, mRNAs are easily separated from the much more prevalent rRNAs and tRNAs present in a cell extract by use of a column to which short strings of thymidyl-ate (oligo-dTs) are linked to the matrix.
The general procedure for preparing a X phage cDNA library from a mixture of cellular mRNAs is outlined in Figure 9-15. The enzyme reverse transcriptase, which is found in retroviruses, is used to synthesize a strand of DNA complementary to each mRNA molecule, starting from an oligo-dT primer (steps 1 and 2 ). The resulting cDNA-mRNA hybrid molecules are converted in several steps to double-stranded cDNA molecules corresponding to all the mRNA molecules in the original preparation (steps 3 - 5). Each double-stranded mRNA 5' ■
Hybridize mRNA with oligo-dT primer
Transcribe RNA into cDNA
Remove RNA with alkali Add poly(dG) tail
Single-stranded 3' G G G GH cDNA
Hybridize with oligo-dC primer
Synthesize complementary strand
Was this article helpful?