Lecture 1: Introduction to SRP

Question 1

Central Dogma

Answer 1

The ‘Central Dogma’ is the process by which the instructions in DNA are converted into a functional product

• DNA makes RNA makes protein -

Question 2

What differentiates different cells within a person?

Answer 2

Every cell contains the exact same DNA but only express some of the 20,000 genes they contain

Question 3

Genomic DNA expression is regulated mainly by…

Answer 3

…promoters that are typically upstream of a gene but can be downstream of it or anywhere else along the DNA

Question 4

Sense & anti-sense strands are…

Answer 4

…complimentary to one another

Question 5

Genomic DNA transcription

Answer 5

1. Involves the unwinding of the DNA double helix, the attachment of RNA polymerase to the anti-sense, and using this strand as a template to form a transcript of RNA in the 5’ to 3’ direction (RNA polymerase reads in the 3’ to 5’ direction). The RNA generated is therefore in the sense orientation.
2. The RNA is then processed with this including the removal of introns as well as capping at the 5’ end and the addition of a polyA tail at the 3’ end. This mature mRNA is then exported to the cytoplasm and then used as a template for translation as part of a ribosome complex.

Question 6

Gene expression can be looked at either via two ways

Answer 6

In situ hybridisation that looks at the localisation of mRNA within tissue or via immunostaining through the use of antibodies

Question 7

In situ hybridisation

Answer 7

1. Involves the recognition of particular mRNA via a probe with this probe also being attached to digoxigenin that has been added to the probe in the lab
2. The probe is then hybridised to the particular mRNA of interest and anything else that has not bound to any mRNA is then washed away
3. The sample being analysed is then incubated with an antibody that recognises the digoxigenin molecule. This antibody has alkaline phosphatase attached to it
4. Any antibody that has not attached is then washed away
5. Following this, a substrate that turns dark purple upon exposure to alkaline phosphatase is added to the sample.
6. The result is regions that contain the mRNA of interest are highlighted by the dark purple staining.

Question 8

Gene expression can also be analysed by looking at the protein the gene of interest codes for…

Answer 8

An antibody that recognises an antigen from the protein coded by the gene of interest is added to the sample with this antibody then allowed to bind to its antigen.

To amplify the signal, a second antibody that recognises the immunoglobulin part of the first antibody is added and this second antibody will have something attached to it including a florescent molecule (fluorescein if green and rhodamine if red).

The different coloured fluorescent molecules allows for the analysis of the expression 2 or more proteins at the same time.

Question 9

The probe used in in situ hybridisation is made to recognise…

Answer 9

particular mRNA by using an mRNA transcript of the gene of interest and using reverse transcriptase to make cDNA from it

Question 10

This process of forming cDNA is done by using a strand of…

Answer 10

thymidine molecules to attach to the polyA tail of the mRNA with reverse transcriptase then completing the anti-sense transcript using the mRNA strand as the template.

Question 11

The cDNA is initially single stranded so is first made…

Answer 11

…double-stranded before it is amplified by PCR

This process is actually the part where the specific transcript of the gene of interest is differentiated from the other genes. This is due to the strand of Ts does not bind specifically to any particular gene (polyA tail is the same for all genes).

Question 12

The primers used in PCR however are specific meaning this is the point where…

Answer 12

the transcript of the gene of interest is differentially amplified over the other genes

Question 13

The primers also contain an RNA polymerase binding site known as

Answer 13

T3

This double-stranded DNA (cDNA) is then made into single strands with the sense strand used to generate anti-sense RNA and this is only possible due to T3 allowing RNA polymerase to bind to the sense strand.

The anti-sense RNA generated is then attached to DIG to form the probe to be used. The way this occurs is one of the nucleotides is replaced by a modified nucleotide that has DIG attached to it. The process of incubation within the sample of interest (e.g. body of zebrafish) followed by hybridisation then follows as described above.