Recombinant DNA & Techniques

Question 1

What are the steps of DNA fractionation by gel electrophoresis?

What’s the pattern between DNA fragment size & distance?

How can you alter the gel depending on size of the fragments?

Answer 1

1. Place DNA fragments in wells of agarose gel
2. Pass electrical current through gel
3. DNA fragments move down gel towards +ve pole & migrate different distances
4. Dye added to gel & subject to autoradiography to see bands/incubate with fluorescent dye

Smaller fragments move the furthest

Change the porosity- higher % means larger fragments

Question 2

What is Southern Blotting used for? What is necessary?

What characteristics can be found?

What are the 6 steps?

Answer 2

DNA/gene detection- need to know the probe

Size & restriction sites of a gene

1. Soak gel from electrophoresis in alkali to denature & separate/hybridise DNA strands
2. Put gel on platform dish with a buffer
3. Put nitrocellulose on top of gel
4. Through capillary action- buffer drawn up carrying the DNA from gel to the nitrocellulose (DNA is fixed)
5. Hybridise DNA with radioactive labelled probes solution & rotate (probe binds to complementary DNA fragments)
6. autoradiography detects the fragments with probes.

Question 3

What is Northern Blotting used for/characteristics?

What is required?

What are the 4 steps?

Answer 3

RNA detection- to find where a gene is expressed

Probes

1. Run RNA fragments on agarose gel - separate by size
2. Transfer RNA to nitrocellulose membrane and fix with UV or heat
3. Hybridise membrane with labelled probes
4. Visualisation labelled RNA on X-ray film after autoradiography

Question 4

What does Western Blotting do?

What is required?

What are the 5 steps?

Answer 4

Detects proteins- sees where a gene is encoded.

Antibodies

1. Run agarose gel with proteins
2. Transfer to nitrocellulose membrane
3. Wash with 1st antibody
4. Wash with 2nd antibody- colourmetric reaction is attached
5. Visualise on autoradiogram

Question 5

What is a microarray used for?

What is needed?

What are the steps to evaluate mRNA amounts in a sample?

How else can this be used?

What are microarray’s limitations?

Answer 5

Identify which genes are expressed in a cDNA sample, compare 2 sets conditions, identify which genes actively expressed, quantify relative signal & profile gene expression

Several different DNA probes

1. Microarray has spots containing fixed different single stranded DNA probes
2. Extract RNA & reverse transcribe in presence of labelled nucleotide into cDNA w/ fluorescent tag
3. Hybridise & wash the cDNA to sample wells
4. Tagged cDNA pair with any complementary probe
5. Colours indicate relative amounts of mRNA in sample

Compare gene expression e.g in cancer and normal cells

Only qualitative, can’t detect post transcriptional genes as detecting mRNA levels, can’t see regulatory relationships

Question 6

What is PCR used for?

What is necessary?

What are the steps?

What techniques can PCR be applied in?

Answer 6

Amplify pieces of target DNA

Primers for DNA polymerase

1. Heat DNA to 100C to separate strands/hybridise
2. quickly cool 30C so primers bind to comp sequences on both strands
3. Heat solution 60C so DNA polymerase makes 2 new double strand DNA
4. Repeat cycle- every cycle number of target DNA doubles (2^n)

Reverse transcription RNA to cDNA, label DNA probs in hybridisation, initiate PCR, sequencing

Question 7

What is DNA cloning for?

What are DNA libraries for?

Microarray?

PCR?

Labelling probes?

Answer 7

Identify gene of interest

Hybridise & identify colonies/clones with DNA of interest

Identify genes interest & compare expression patterns

Characterise & amplify sequences of interest

Apply different types priming for techniques (random, oligo dT, based on AA sequence, known sequence priming)