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Flashcards in Gene analysis and expression Deck (27)
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1
Q

What happens in post-transcriptional processing?

A

Introns are removed, the mRNA is capped at the 5’ end and polyadenylated at the 3’ end.

2
Q

What is an example of post-translational processing?

A

Disulphide bond addition.

3
Q

What does the regulatory region contain?

A

Promoters; enhancers; suppressors; tissue, developmental and environmental control motifs; areas that determine the stop and start of transcription.

4
Q

How can these regulatory processes be analysed?

A

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.

5
Q

What are methods to measure gene expression?

A

Northern hybridisation and quantitative real time PCR.

6
Q

What is Northern hybridisation?

A

It is similar to Southern Hybridisation but is with RNA instead of DNA.

7
Q

What is the process for Northern hybridisation?

A

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.

8
Q

Why are no restriction enzymes used in Northern hybridisation?

A

RE only cut DNA not RNA, RNA molecules are already small and you don’t want to make any smaller fragments.

9
Q

What will be seen when the total RNA is run?

A

Two big bands - ribosomal RNA bands. This ensures you that your RNA has not degraded.

10
Q

What can northern blots be used for?

A

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.

11
Q

What is quantitative real time PCR based on?

A

The doubling of DNA at each cycle of PCR.

12
Q

How do you carry out Real-Time-PCR?

A

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.

13
Q

How is detection done in real-time PCR?

A

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.

14
Q

Where are regulatory sequences found in a gene?

A

In the upstream (5’) portion of the gene.

15
Q

How can DNA-binding proteins be identified?

A

In electrophoresis, transcription factors are large and when bound to DNA slow its mobility.

16
Q

What is deletion analysis?

A

A method used to locate regulatory motifs.

17
Q

What is a consensus sequence?

A

A common pattern of bases.

18
Q

What is the basis for deletion analysis?

A

It assumes that deletion of a motif will change the regulation of expression of a cloned gene, and that this change can be analysed.

19
Q

What are some problems with deletion analysis?

A

To assess the expression the gene must be reintroduced into the organism - human gene expression cannot be analtsed in E.coli. You can also not differentiate between the endogenous gene and the modified one that has been reintroduced.

20
Q

What is a reporter gene?

A

A gene that can be used to differentiate between the endogenous gene and the modified reintroduced one.

21
Q

How does the reporter gene work?

A

It replaces the original gene. Its activity is easily measurable, has a visible phenotype and is not normally present in the organism.

22
Q

What are some examples of reporter genes?

A

BUS: Beta- glucuronidase (blue - most widely used)
LUX: luciferase (light- non-destructive)
GFP: green fluorescent protein (non-destructive)

23
Q

How can the gene expression be detected?

A

During development, in cell/tissue localisation or in response to environmental cues.

24
Q

What is promoter deletion analysis?

A

Removing promoter regions to assess how gene expression changes.

25
Q

How can deletions be made in promoter deletion analysis?

A

The removal of a restriction fragment, remove a few nucleotides with Bal31 and re-ligate and site-specific mutagenesis to change or deelete specific nucleotides.

26
Q

What is in vitro mutagenesis?

A

Mimic events that occur in mutation and see the effect on the function of the protein.

27
Q

What types of mutations can be used in in vitro mutagenesis?

A

Crude (removal of a restriction fragment) or subtle - PCR to alter a single codon and change the amino acid.