Biomedical Engineering Reference
In-Depth Information
mRNA
5'
AAAA
3'
Oligo-dT
Reverse transcriptase
+ 4 dNTPs
First strand synthesis
mRNA
5'
AAAA
3'
3'
TTTT
5'
cDNA
Terminal transferase
+ dCTP
mRNA
5'
AAAA
CCC
3'
3'
CCCC
TTT
5'
cDNA
Alkaline sucrose gradient:
(1) hydrolyses RNA
(2) recovers full length cDNA
3'
CCCC
TTT
T
5'
cDNA
Oligo-dG, reverse transcriptase
+ 4 dNTPs
Second strand synthesis
5'
3' CCCC
GGG
3'
5'
duplex cDNA
TTTT
Fig. 6.6
Improved method for cDNA
cloning. The first strand is tailed with
oligo(dC) allowing the second strand to
be initiated using an oligo(dG) primer.
Insert into vector by
either
further homopolymer
tailing
or
linkers
are usually purified from avian myeloblastosis virus
(AMV) or produced from a cloned Moloney murine
leukaemia virus (MMLV) gene in
E. coli.
Native
enzymes have poor processivity and intrinsic RNase
activity, which leads to degradation of the RNA tem-
plate (Champoux 1995). Several companies pro-
duce engineered murine reverse transcriptases that
lack RNase H activity, and these are more efficient
in the production of full-length cDNAs (Gerard &
D'Allesio 1993). An example is the enzyme Super-
Script II, marketed by Life Technologies (Kotewicz
et al
. 1988). This enzyme can also carry out reverse
transcription at temperatures of up to 50°C. The native
enzymes function optimally at 37°C and therefore
tend to stall at sequences that are rich in secondary
structure, as often found in 5
′
and 3
′
untranslated
regions.
Selection of 5
′
mRNA ends
Despite improvements in reverse transcriptases,
the generation of full-length clones corresponding
to large mRNAs remains a problem. This has been
addressed by the development of cDNA cloning
strategies involving the selection of mRNAs with
intact 5
′
ends. Nearly all eukaryotic mRNAs have
a 5
end cap, a specialized, methylated guanidine
residue that is inverted with respect to the rest of the
strand and is recognized by the ribosome prior to the
initiation of protein synthesis. Using a combination
′
