Genetics Pcr, Sanger, Protein Expression

Front 1

What type of genetic material can PCR amplification be used on

Back 1

Always on DNA

but can start with mRNA reverse transcribed into cDNA

Front 2

Outline PCR for amplifying segments of DNA. Name reagents and enzymes needed. (10 marks)

Back 2

1. Specific oligonucleotide primers to recognise dna segment of interest

2. Primers need to have a free OH-

3. Taq polymerase (thermostable enzyme) used

4. Need 4 DNPs (dATP, dCTP, dGTP, dTTP)

5. Heat DNA to denature and separate DNA strands to ssDNA (95C)

6. Lower temp to 60C to anneal primers to complementary sequences on the single stranded template DNA

7. Extension: at 70C so taq polymerase extends primers, synthesising new strands of DNA in 5-3 prime direction due to antiparallel nature

8. Repeat cycle 30x to amplify DNA exponentially

Front 3

Outline Sanger DNA sequencing , enzymes and reagents needed (10 marks)

Back 3

1) use pcr to amplify lots of DNA fragments

2) Add dideoxynucleotides so that the strands can no longer elongate (termination)

3) each dideoxynucleotide is labelled with a different colour fluorescent dye so that each fragment is labelled

4) run gel electrophoresis using polyacrylamode gel for vertical separation read from 5’ 3’ direction. Strains separated by size , fluorescent labels analyzed to create a DNA sequence

Front 4

Which technique is useful for looking at protein expression? (2 marks)

Back 4

Western Blotting as it uses antibodies to visualize proteins

Front 5

What assay is used to standardise protein levels observed between samples ?

Back 5

The Bradford Assay

Front 6

Explain how the bradford assau is used to standardise protein expression between samples (5 marks)

Back 6

1) load known about of a house protein e.g GAPDH to standardise it

2) ratio your protein expression of target protein in sample to GAPDH expression

3) Use densitometry analysis to gain values for the level of expression

This is semi quantitative.

Front 7

Describe a more quantitative way to standardise protein expression

Back 7

Use known amounts of a house keeping protein e.g. GAPDH to plot a standard curve to read protein expression against

Front 8

How would you quantify your protein sample before separating it by SDS-PAGE for analysis (5 marks)

Back 8

Use coloured Coomassie blue and known protein to create protein standards to plot a standard curve using a Bradford assay, to measure the amount of proteins in original sample

Front 9

What technique is used to look at protein-DNA interactions and why? (3marks)

Back 9

Electromobility shift assays (EMSA)

- sequence specific DNA binding proteins

- can use knowledge of specific binding sites

- investigate activation of transcription factors

- western blot and coomassie blue staining do not allow for identification of function of transcription factors

Front 10

What are the limitations of EMSA to look at protein-DNA interactions? How can it be adapted to increase specificity?

Back 10

- only tells us that transcription complex is present and active

- does not tell us what proteins are involved in the transcription complex

- Need to use supershift assays to get info on which protein subunits are active in the complex

Front 11

What do northern blots tell us?

Back 11

- info about size of mRNA

- determine whether cDNA close used as a probe is full length

Front 12

What does a southern blot tell us?

Back 12

- to identify part of genomic clone corresponds to cDNA fragment

- info about size of fragment the gene is on

- info about how many copies of gene are present in genome

- how conserved a gene is between species

Front 13

What gel does southern blotting use?

Back 13

Agarose gel