Gene analysis & expression
Transcription & Translation
Genes are transcribed into RNA, mRNA is translated into
protein
Most eukaryotic genes contain introns, so pre-mRNA is
processed (introns removed, capped, polyadenylated) so
give mature mRNA.
Protein has 3D structure and may also be processed
Genes contain regulatory regions that control expression:
promoters, enhancers, suppressors, tissue, developmental
& control motifs. Determine start & stop of transcription.
Cloned genes & cDNAs can be used to analyse many of
these processes involve in transcription, translation & the
regulation of gene expression – the relationship between a
gene and its transcript (mRNA) can be studied by
comparing gene and cDNA sequences: introns, poly(A)
addition sites, but the start of transcription has to be
determined experimentally.
Northern hybridisation: a method often used to confirm
the size of the transcript corresponding to a cloned
gene/cDNA.
- Total RNA (no restriction digestion) separated by gel
electrophoresis (using denaturing bugger formaldehyde)
- RNA is blotted onto nylon membrane
- Hybridised with radio-labelled gene/cDNA probe
- Autoradiography on X-ray film
- Band position gives measure of size of the corresponding
mRNA
- Northern blots: because the intensity of the band is a
measure of the abundance of that particular mRNA in the
total population of mRNA loaded on to the gel, this can be
used to get a measure of the changing pattern of activity
of the gene you are interested in. Can be used to assay
changes of gene expression over a time course, in
different tissues, during development & in response to an
environmental cue
Quantitative RT-PCR: alternative to Northern blotting,
based on the doubling of DNA at each cycle of PCT
Transcription & Translation
Genes are transcribed into RNA, mRNA is translated into
protein
Most eukaryotic genes contain introns, so pre-mRNA is
processed (introns removed, capped, polyadenylated) so
give mature mRNA.
Protein has 3D structure and may also be processed
Genes contain regulatory regions that control expression:
promoters, enhancers, suppressors, tissue, developmental
& control motifs. Determine start & stop of transcription.
Cloned genes & cDNAs can be used to analyse many of
these processes involve in transcription, translation & the
regulation of gene expression – the relationship between a
gene and its transcript (mRNA) can be studied by
comparing gene and cDNA sequences: introns, poly(A)
addition sites, but the start of transcription has to be
determined experimentally.
Northern hybridisation: a method often used to confirm
the size of the transcript corresponding to a cloned
gene/cDNA.
- Total RNA (no restriction digestion) separated by gel
electrophoresis (using denaturing bugger formaldehyde)
- RNA is blotted onto nylon membrane
- Hybridised with radio-labelled gene/cDNA probe
- Autoradiography on X-ray film
- Band position gives measure of size of the corresponding
mRNA
- Northern blots: because the intensity of the band is a
measure of the abundance of that particular mRNA in the
total population of mRNA loaded on to the gel, this can be
used to get a measure of the changing pattern of activity
of the gene you are interested in. Can be used to assay
changes of gene expression over a time course, in
different tissues, during development & in response to an
environmental cue
Quantitative RT-PCR: alternative to Northern blotting,
based on the doubling of DNA at each cycle of PCT
