Cloning from RNA
Although DNA can be extracted from cells and used in cloning steps, sometimes RNA is a better starting choice for cloning. Not only will intervening sequences be missing from mRNA, but often mRNA extracted from certain tissues is greatly enriched for specific gene se-quences.
Extraction of RNA from cells yields a preponderance of ribosomal RNA. The messenger RNA can easily be separated from this ribosomal RNA since most messenger RNA from most higher organisms contains a poly-A tail at the 3’ end. This tail can be used for isolation by passing a crude fraction of cellular RNA through a cellulose column to which poly-dT has been linked. At high salt concentrations, the poly-dT which is linked to the column and the poly-A tails of messenger molecules hybridize and bind the messenger RNAs to the column. The ribosomal RNA molecules flow through the column. The messenger RNAs are eluted by lowering the salt concentration so as to weaken the polyA-dT hybrids. Such a purification step frequently provides a several-hundred-fold enrichment for messenger RNA. The effort required to clone a specific gene is often greatly reduced by using this procedure in con-junction with choosing a particular tissue at a particular developmental time.
The single-stranded RNA obtained by the steps described above cannot be cloned directly. Either the RNA can be converted to DNA via a complementary strand, cDNA, or the RNA can be used to aid identi-fication of a clone containing the complementary DNA sequence. To generate a cDNA copy of the poly-A-containing messenger, several steps are performed (Fig. 9.12). First, a poly-dT primer is hybridized to the messenger and reverse transcriptase is used to elongate the primer to yield a DNA copy. This enzyme, which is found within the free virus particle of some animal viruses, synthesizes DNA using an RNA tem-plate. At this point the sequence exists as an RNA-DNA hybrid. It is converted to a DNA duplex by the simultaneous incubation with RNAse H which cuts the RNA strand in an RNA-DNA duplex and DNA polym-
Figure 9.12 Steps in the replication of an mRNA molecule into a double-stranded DNA copy suitable for cloning.
erase pol I which synthesizes DNA using the remaining RNA as primer. DNA pol I removes the remaining RNA by nick translation. Finally, to make the ends of the DNA duplex perfectly blunt, T4 DNA polymerase pol I is added. The resulting double-stranded DNA can then be cloned by methods already discussed.