Gene Cloning

322 Gene Cloning
mRNA
5' 3'
3' 5'
Single-stranded
radiolabeled
DNA or RNA probe
S1 nuclease (DNA probe)
or RNAse (RNA probe)
5' 3'
3' 5'
Site of DNA-DNA or RNA-DNA
duplex protected from digestion
indicates distance from 5' end of
the DNA/RNA probe to the
transcription start
G A C T *
Denaturing polyacrylamide gel
electrophoresis and autoradiography
Figure 11.3 Transcription start site mapping: Nuclease protection. RNA
is purified and a labeled RNA probe complementary to the 5′ end of the mRNA
hybridized. The probe extends from within the gene near the 5′ end to past the
likely transcription start site. A duplex forms that extends from the 5′ end of the
probe to the transcription start. The size of the duplex is determined by
digesting the single-stranded part of the probe with S1 nuclease or RNAse. The
size of the protected DNA/RNA probe (*) is then determined by denaturing
polyacrylamide gel electrophoresis. A sequencing reaction (G, A, C and T) is
used to calibrate the gel.
Box 11.2. Production of Labeled RNA Probes for Use in
the RNAse Protection Assay
It is possible to purchase a number of different plasmids which allow you
to make RNA probes from a cloned DNA template. One example is the
pGEM5Zf(+) plasmid. This plasmid contains a multiple cloning site that is
flanked by an SP6 promoter and a T7 promoter on either side. The SP6 and
T7 promoters are recognized by RNA polymerase encoded by the phages
SP6 and T7, respectively. For an RNAse protection assay a fragment covering
the transcription start of your gene is cloned into the multiple cloning
site of pGEM5Zf(+). Depending on the orientation of cloning, either SP6
RNA polymerase or T7 RNA polymerase is used to generate an RNA probe
that is complementary to the mRNA for the gene and overlaps the transcription
start. Plasmid DNA can be purified and used as template by one