Gene analysis and expression Flashcards
What happens in post-transcriptional processing?
Introns are removed, the mRNA is capped at the 5’ end and polyadenylated at the 3’ end.
What is an example of post-translational processing?
Disulphide bond addition.
What does the regulatory region contain?
Promoters; enhancers; suppressors; tissue, developmental and environmental control motifs; areas that determine the stop and start of transcription.
How can these regulatory processes be analysed?
Using cloned genes and cDNAs. The relationship between a gene and its transcript can be studied by comparing gene and cDNA sequences, such as introns and poly(a) addition sites.
What are methods to measure gene expression?
Northern hybridisation and quantitative real time PCR.
What is Northern hybridisation?
It is similar to Southern Hybridisation but is with RNA instead of DNA.
What is the process for Northern hybridisation?
The total RNA is separated by electrophoresis on gel with a denaturing buffer to prevent 3D structures bing made in the gel by the ssRNA. It is blotted onto a nylon membrane and hybridised with radio-labelled gene probe. An autoradiography is produced on X-ray film and the band positions give a measure of size of mRNA.
Why are no restriction enzymes used in Northern hybridisation?
RE only cut DNA not RNA, RNA molecules are already small and you don’t want to make any smaller fragments.
What will be seen when the total RNA is run?
Two big bands - ribosomal RNA bands. This ensures you that your RNA has not degraded.
What can northern blots be used for?
Used to assess changes of gene expression over a time course, in different tissues, during development or in response to an environmental cue. This can be done as bend intensity gives a measure of abundance of the mRNA.
What is quantitative real time PCR based on?
The doubling of DNA at each cycle of PCR.
How do you carry out Real-Time-PCR?
mRNA is converted to cDNA using reverse transcriptase and the cDNA is amplified using gene-specific primers by PCR for a small number of cycles - around 25 to avoid saturation. The house keeping genes will also be amplified. Changes of amplification can be measured during PCR before it starts to plateau.
How is detection done in real-time PCR?
Fluorescent dyes which intercalate between the bases of the double stranded DNA. It is carried out in a thermal cycler with the ability to illuminate each sample and detect emitted fluorescence.
Where are regulatory sequences found in a gene?
In the upstream (5’) portion of the gene.
How can DNA-binding proteins be identified?
In electrophoresis, transcription factors are large and when bound to DNA slow its mobility.
