78 Gerard and D'Alessioand M-MLV H- RT are much less sensitive than AMV RT to inhibition
by tRNA and rRNA (16; Gerard, G., unpublished), and well suited to
copying unfractionated mRNA.
2.6. Primer
The primer for cDNA synthesis is usually oligo(dT)12_18 present in
molar excess relative to the 3' poly(A) tails of mRNA. This method of
priming usually results in a cDNA library in which the 3' ends of RNAs
are overrepresented because of the catalytic properties of RT described
in Section 2.4.2. This bias can be partially overcome by using random
hexamer primers instead ofoligo(dT) or as a supplement to an oligo(dT)-
primed reaction (10,34). When random hexamers are used, the priming
events are more evenly distributed over entire mRNA molecules. How-
ever, some sequence information corresponding to the 3'end of mRNA
is lost as the result of randomizing primer location. In addition, rRNA
in an RNA population is also copied when random hexamers are used.
The ratio of random hexamer primers to mRNA is critical. Figure
1 illustrates that there must be a balance between the desired average
length and the mass yield of cDNA product. With M-MLV H- RT,
approx 10 primers/mRNA gives a reasonable yield without sacrificing
product length. The ratio is higher with M-MLV RT, approx 40:1.
When oligo(dT) or random primers are used to prime first-strand cDNA
synthesis, the termini of the ensuing double-stranded cDNA are func-
tionally identical, precluding directional cloning of the cDNA. All
members of a directional library contain cDNA inserts cloned in a
specific orientation relative to the transcriptional polarity of the origi-
nal mRNA. Directional cloning is desirable when cDNA libraries will
be screened by expression (35,36) and is essential in some strategies
for generating subtracted, i.e., partial, cDNA libraries (37-39).
cDNA can be cloned directionally by introducing two different
restriction endonuclease recognition sites at its termini. This is most
simply achieved by using a primer-adapter to initiate first-strand syn-
thesis. A primer-adapter has the structure 5'-p(dX)n-(dT)ls-OH-3',
where (dX)n encodes sequence information for one or more restriction
endonuclease sites. The primer-adapter introduces desired restriction
endonuclease site(s) at the end of double-stranded cDNA correspond-
ing to the 3' end of mRNA. Subsequent ligation of linkers or adapters
to the DNA can be used to introduce a different restriction endonu-
clease site(s) at the opposite end of the DNA.