Gene Cloning

324 Gene Cloning
mRNA
5' 3'
3' 5'
Gene-specific
primer
Extend primer using reverse
transcriptase in the presence
of dNTPs
5'
3' 5'
3'
cDNA
RNAse H
TdT and dATP
3'-AAAAAA 5'
RE
5'-........T TTTTTN
3'-AAAAAA 5'
3'
5'
RE
Amplify using oligo (dT)-anchor
primer and nested primer
RE
5'-........T TTTTT
3'-........AAAAAA
RE
3'
5'
Clone and sequence
Figure 11.4. Transcription start site mapping – rapid amplification of
cDNA ends (RACE). RNA is purified, a gene-specific primer is hybridised to
the RNA of the gene of interest and the primer extended by reverse
transcriptase in the presence of dNTPs. The RNA template is degraded by
RNAse H and the 3′ end of the cDNA extended using terminal transferase in
the presence of a single deoxynucleotide (dATP in this example). The cDNA is
amplified using a “nested” primer upstream to the primer used for cDNA
synthesis and a second primer complementary to the 3′ extension. Both primers
contain sites for a restriction enzyme at their 5′ end (RE). In this example an
oligo (dT)-anchor primer is used, the “primer” is in fact a mixture of four
primers which, in addition to having a run of Ts, have either G, A, C or T
(shown as N in the diagram) at their 3′ end (the anchor), this ensures that the
primer anneals to the cDNA at the junction of the polyA tract and the original
extension product. The amplification product is cloned and sequenced. The
transcription start site is adjacent to the poly T tract in the sequence.