A genomic library contains all of the DNA sequences found in an organisms genome

with short fragments of DNA consisting entirely of thymine nucleotides [oligo(dT) chains; Figure 18.14a]. As the RNA moves through the column, the poly(A) tails of mRNA molecules pair with the oligo(dT) chains and are retained in the column, whereas the rest of the RNA passes through. The mRNA can then be washed from the column by adding a buffer that breaks the hydrogen bonds between poly(A) tails and oligo(dT) chains.

The mRNA molecules are then copied into cDNA by reverse transcription. Short oligo(dT) primers are added to the mRNA. A primer pairs with the poly(A) tail at the 3' end of the mRNA, providing a 3'-OH group for the initiation of DNA synthesis (IFigure 18.14b). Reverse transcriptase, an enzyme isolated from retroviruses (see p. 000 in Chapter 8), synthesizes single-stranded complementary DNA from the RNA template by adding DNA nucleotides to the 3'-OH group of the primer.

The resulting RNA - DNA hybrid molecule is then converted into a double-stranded cDNA molecule by one of several methods. One common method is to treat the RNA - DNA hybrid with RNase to partly digest the RNA strand. Partial digestion leaves gaps in the RNA - DNA hybrid, allowing DNA polymerase to synthesize a second DNA strand by using the short undigested RNA pieces as primers and the first DNA strand as a template. DNA polymerase eventually displaces all the RNA fragments, replacing them with DNA nucleotides, and nicks in the sugar - phosphate backbone are sealed by DNA ligase.

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