mRNA molecules the clones of each individual mRNA have to be synthesised.
Libraries that represent the mRNA in a particular cell or tissue are termed cDNA
libraries. mRNA cannot be used directly in cloning since it is too unstable. However it
is possible to synthesise complementary DNA molecules (cDNAs) to all the mRNAs
from the selected tissue. The cDNA may be inserted into vectors and then cloned. The
production of cDNA (complementary DNA) is carried out using an enzyme termed
reverse transcriptasewhich is isolated from RNA-containing retroviruses.
Reverse transcriptase is an RNA-dependent DNA polymerase, and will synthesise a
first-strand DNA complementary to an mRNA template, using a mixture of the four
dNTPs. There is also a requirement (as with all polymerase enzymes) for a short oligonu-
cleotide primer to be present (Fig. 6.5). With eukaryotic mRNA bearing a poly(A) tail, a
complementary oligo(dT) primer may be used. Alternatively random hexamers may
be used which randomly anneal to the mRNAs in the complex. Such primers provide a
free 3^0 hydroxyl group which is used as the starting point for the reverse transcriptase.
Regardless of the method used to prepare the first-strand cDNA one absolute requirement
is high-quality undegraded mRNA (Section 5.7.2). It is usual to check the integrity of the
RNA by gel electrophoresis (Section 5.7.4). Alternatively a fraction of the extract may be
used in a cell-free translation system, which, if intact mRNA is present, will direct the
synthesis of proteins represented by the mRNA molecules in the sample (Section 6.7).
Following the synthesis of the first DNA strand, a poly(dC) tail is added to its 3^0 end,
using terminal transferase and dCTP. This will also, incidentally, put a poly(dC) tail onMessenger RNAAAAAAARandom primersAAAAAAReverse transcriptase/buffer/dNTPs
Poly(dT) primer Specific primerReverse transcriptase/buffer/dNTPs
AAAAAA
Reverse transcriptase/buffer/dNTPsAAAAAAAAAAAAcDNA–mRNA hybridAAAAAAcDNA–mRNA hybrid cDNA–mRNA hybrid
AAAAAAFig. 6.5Strategies for producing first-strand cDNA from mRNA.201 6.2 Constructing gene libraries