MCBG Session 15 - Molecular Techniques

Question 1

How does Southern Blotting work?

Do the probes used in southern blotting need to have 100% similarity to the target sequence?

Answer 1

1) DNA fragment undergoes electrophoresis to separate by size, and transferred (blotted) onto nitrocellulose sheet
2) Fluorescent DNA probe attached and hybridises to target
3) Use autoradiogram or fluorescence image to reveal the DNA fragment present or not.

• No, however the higher the similarity the more tightly the probe will bind. Probes can also bind if there is a slight overlap with target sequence.

Question 2

What situations would using southern blot be appropriate for?

Answer 2

• To find large deletions or duplications, i.e.: changes in gene structure
• To investigate gene expansions & triplet repeats
• To investigate mutations in genetic tests, e.g.: using allele specific probes to find Sickle Cell disease
• To investigate variation

Question 3

What is a microarray and how are they done?

When are microarrays used?

Answer 3

• Microarrays allow 1000’s of genes to be analysed at once.
• Single strand oligonucleotides with sequence complimentary to coding regions of genome mixed with sample (created via reverse transcription of target mRNA to form cDNA).
• Microarrays used to monitor any changes in gene expression - i.e.: whether a gene is being expressed or not, or whether gene expression is high or low compared to normal.

Question 4

Describe the process of PCR and what is required for the cycle to occur.

Answer 4

1) DNA is heated to 95 degrees to denature (separate) the double helix by breaking hydrogen bonds.
2) Small primers are added (a forward and a reverse primer) - these primers define the sequence we want to amplify.
3) Cool to room temp to allow re-annealing of DNA and primers.
4) Raise to 72 degrees which is perfect temperature for thermostable DNA polymerase (usually Taq) to work.
5) Repeat cycle multiple times to allow for exponential increase in DNA.

• DNA, Taq enzyme, forward and reverse primers.

Question 5

Why would we use PCR?

Answer 5

1) Amplify a specific DNA sequence
2) Investigate single base mutations (combine with DNA sequencing after)
3) Investigate small deletions or insertions (combine with DNA sequencing after)
4) Investigate variation

NB: need to be able to interpret PCR results - go to slides/group work session.

Question 6

What controls are required in a PCR experiment?

Answer 6

Positive control = A known sample of DNA to show that the primers have attached to the DNA strand

Negative control = Sample without DNA, shows if contamination with any foreign DNA material has occurred.

Question 7

How does Sanger/DNA sequencing work?

Answer 7

Uses dideoxynucleotides (hydrogens at C2 and C3 on pentose sugar) which terminate chain elongation. You get a mixture of molecules where termination has occurred at different places, this is detected using fluorescence in order to determine each base.

Question 8

Describe the process of agarose gel electrophoresis.

What is this used in conjunction with?

Answer 8

• DNA is negatively charged, therefore moves towards the anode when placed in an electric field. Shorter fragments move further along the gel. DNA is then visualised via staining with ethidium bromide.
• DNA fragmentation via restriction endonucleases.

Question 9

Why is SDS used in SDS-page electrophoresis?

Answer 9

SDS modifies the proteins into a single polypeptide chain by removing tertiary and quaternary structure of a protein, thus allowing it to be separated by size across the gel.

Question 10

Outline the mechanism of F.I.S.H

When is fish used?

Answer 10

• Denature the DNA, add sequence specific probe with fluorescent dye/tag, allow for hybridisation and check for fluorescence.
• Whole chromosome probes can be used for chromosomal abnormalities such as trisomy 21.
• Locus specific probes can be used to determine whether an individual is missing certain genetic information from a particular chromosome