Module 9: V12 - V14

Question 1

What are some questions a researcher may ask about a gene?

Answer 1

what cell types express gene1?
under what conditions do these cells express gene1?
what can change or control the expression of gene1?

Question 2

How does reverse transcriptase PCR work?

Answer 2

taking a sample and determining whether it contains mRNA of the gene of interest

Question 3

What are the first steps of reverse transcriptase?

Answer 3

RNA is extracted and treated with DNase I to remove all DNA that may be present and isolate a pure sample of RNA

Question 4

What happens after a pure sample of RNA is isolated?

Answer 4

mRNA is converted to cDNA using reverse transcriptase and then PCR is carried out to acquire the gene of interest

Question 5

How is mRNA converted to cDNA?

Answer 5

reverse transcriptase is added -> mRNA is then degraded leaving behind sscDNA -> a second strand is synthesised by adding a primer and enzyme to the sscDNA resulting in dscDNA

Question 6

How is all of the mRNA converted into cDNA and not rRNA or tRNA?

Answer 6

an oligo dT primer is added to the mRNA and will bind to all of the poly-a tails which means it will only bind to the mRNAs and none of the rRNAs or tRNAs

Question 7

What does it mean if mRNA is being converted to cDNA?

Answer 7

there will only be representatives of the genes that are expressed

Question 8

Why is PCR used?

Answer 8

to amplify the cDNA as only a small amount is produced

Question 9

What is the amount of signal relative to?

Answer 9

the amount of starting DNA - but only in the exponential phase of the PCR

Question 10

What happens to PCR when there is less cDNA to start with? Will this produce a bright or faint band if PCR is stopped prematurely?

Answer 10

PCR is much slower (curve is shifted to the right)

a faint band

Question 11

Between two different samples, what does a different amount of starting product for a particular gene tell us about the mRNA in the original sample?

Answer 11

tells us how much mRNA was in the original sample and therefore how highly expressed this gene was in that sample

Question 12

How is a Western blot taken?

Answer 12

protein is extracted from a tissue sample and then run on a gel with antibodies

Question 13

While running a gel, how are proteins run on the basis of their size?

Answer 13

a negatively charged detergent called SDS is added which unfolds the protein and coats it

Question 14

How is a specific protein identified?

Answer 14

antibodies are used in an SDS-PAGE gel

this is called a Western blot

Question 15

Why are antibodies used?

Answer 15

because each one recognises a specific protein

Question 16

What is the first step of a Western blot?

Answer 16

the gel is run, taken out and then sat on top of a membrane onto which all of the proteins are transferred

Question 17

What happens after all of the proteins are transferred onto the membrane during a Western blot?

Answer 17

membrane is taken and then washed with protein so that all of the non-specific binding can be blocked

Question 18

What happens after all of the non-specific binding is blocked on the membrane during a Western blot?

Answer 18

antibody that is specific for the protein of interest is added and will stick to the gel only in locations where the protein is found

Question 19

What happens after the initial antibody sticks to the gel during a Western blot?

Answer 19

a second antibody is added which is specific for antibodies so that it is going to bind to the gel only in locations where the initial antibodies are found

Question 20

What are secondary antibodies conjugated with and why?

Answer 20

allows for detection e.g. enzyme which can bind to a substrate and change colour

Question 21

Is the promoter on the coding strand or the template strand?

Answer 21

neither, the promoter is a double stranded structure

Question 22

Does the promoter include the operator (or in a eukaryote, the regulatory sequences)?

Answer 22

promoter is a very loosely defined term, and is commonly used to refer to the promoter + regulatory sequences