cDNA Synthesis with M-MLV RT 77activity as well (7,26). This sets up a competition between the two
activities, and the extent to which the RNase H activity destroys the
hybrid prior to the initiation of polymerization determines the maxi-
mum number of priming events that can actually occur (26). This
reduces the yield of cDNA by effectively removing a portion of the
mRNA from the reaction.
When RT is synthesizing cDNA, RT RNase H activity degrades the
template that has already been copied because it is in hybrid form. If
the scissions in the mRNA occur near the point of chain growth, the
uncopied portion of the mRNA can dissociate from the transcriptional
complex, resulting in termination of cDNA synthesis for that template.
This problem is exacerbated by the tendency of RT to pause during
reverse transcription (7) and results in truncated cDNA molecules.
When M-MLV H- RT is used, RNA cleavage and its deleterious
effects are eliminated, resulting in more efficient synthesis of full-
length cDNA (7). However, M-MLV H- RT still tends to pause during
polymerization. Thus, some RNA molecules are still not copied to
their 5' ends by M-MLV H- RT, even though the RNA remains intact
during DNA synthesis. Other modifications to the RT polypeptide or
addition of accessory proteins (e.g., a nucleic acid binding protein)
will be required to reduce pausing and termination of transcription.
2.5. RNA Template
The quality of the mRNA template in a cDNA synthesis reaction
dictates the maximum amount of sequence information that can be
converted into cDNA. Thus, it is important to optimize the isolation of
mRNA from a given biological source and to prevent adventitious
introduction of RNases into a preparation that has been carefully ren-
dered RNase-free. We recommend procedures that rely on chaotropic
salts to inactivate RNases for extraction of RNA from any source
(27,28). The presence of the poly(A) tail at the 3' end of mRNA has
been exploited to fractionate mRNA from a total RNA population by
affinity chromatography on oligo(dT) cellulose (29). Most cDNA
libraries are made from poly(A)-selected mRNA. When the quantity
of the RNA source is limiting and affinity chromatography is not
feasible, it is possible to synthesize effectively cDNA from unfraction-
ated mRNA. This is done routinely in preparation for amplifying cDNA
by the polymerase chain reaction technique (30-33). Both M-MLV RT