Biomedical Engineering Reference
In-Depth Information
group. Full-length molecules can be ligated to a
specific oligonucleotide, while broken and degraded
molecules cannot. The result is an oligo-capped
population of full-length mRNAs. This selected popu-
lation is then reverse-transcribed using an oligo-dT
primer. Second-strand synthesis and cloning is then
carried out by PCR using the oligo-dT primer and
a primer annealing to the oligonucleotide cap. Only
full-length cDNAs annealing to both primers will
be amplified, thus eliminating broken or degraded
RNAs, incomplete first cDNA strands (which lack
a 5
Oligo-capping
Full-length mRNA
cap
AAAAAAA
Partial-length mRNA
P
AAAAAAA
BAP treatment
cap
AAAAAAA
OH
AAAAAAA
TAP treatment
primer annealing site) and misprimed cDNAs
(which lack a 3
′
P
AAAAAAA
′
primer annealing site).
OH
AAAAAAA
Oligo plus RNA
ligase
PCR as an alternative to cDNA cloning
Oligo capped
mRNA
AAAAAAA
Reverse transcription followed by the PCR (RT-PCR)
leads to the amplification of RNA sequences in cDNA
form. No modification to the basic PCR strategy
(p. 19) is required, except that the template for
PCR amplification is generated in the same reaction
tube in a prior reverse-transcription reaction (see
Kawasaki 1990, Dieffenbach & Dvesler 1995). Using
gene-specific primers, RT-PCR is a sensitive means
for detecting, quantifying and cloning specific cDNA
molecules. Reverse transcription is carried out using
a specific 3
OH
AAAAAAA
First strand
synthesis
AAAAAAA
TTTTTTT
OH
AAAAAAA
TTTTTTT
PCR amplification and cloning
(only molecules containing both
primers will be amplified)
primer that generates the first cDNA
strand, and then PCR amplification is initiated follow-
ing the addition of a 5
′
AAAAAAA
primer to the reaction mix.
The sensitivity is such that total RNA can be used
as the starting material, rather than the poly(A)
+
RNA which is used for conventional cDNA cloning.
Total RNA also contains ribosomal RNA (rRNA)
and transfer RNA (tRNA), which can be present in a
great excess to mRNA.
Due to the speed with which RT-PCR can be
carried out, it is an attractive approach for obtaining
a specific cDNA sequence for cloning. In contrast,
screening a cDNA library is laborious, even presum-
ing that a suitable cDNA library is already available
and does not have to be constructed for the purpose.
Quite apart from the labour involved, a cDNA library
may not yield a cDNA clone with a full-length cod-
ing region, because, as described above, generating
a full-length cDNA clone may be technically chal-
lenging, particularly with respect to long mRNAs.
Furthermore, the sought-after cDNA may be very
rare even in specialized libraries. Does this mean
′
TTTTTTT
Fig. 6.10
Oligo-capping, the addition of specific
oligonucleotide primers to full-length RNAs by
sequential treatment with alkaline phosphatase and
acid pyrophosphatase. Once the oligo cap has annealed
to the 5′ end of the mRNA, it can serve as a primer binding
site for PCR amplification.
problem by performing selection at the RNA stage
(Maruyama & Sugano 1994, Suzuki
et al
. 1997,
2000; Fig. 6.10). The basis of the method is that
RNA is sequentially treated with the enzymes
alkaline phosphatase and acid pyrophosphatase.
The first enzyme removes phosphate groups from
the 5′ ends of uncapped RNA molecules, but does
not affect full-length molecules with a 5′ cap. The
second treatment removes the cap from full-length
RNAs, leaving a 5′-terminal residue with a phosphate
