Genome analysis Flashcards
What are the three key methods used to analyse the genome?
Hybridisation based techniques
Enzyme based techniques
The use of antibodies
Outline the key stages to DNA hybridisation
Heat up and denature two DNA samples (produced with PCR). Around 100 degrees.
One of the samples should be fluorescently marked, but not the other.
Cool to allow strand to re-anneal.
Isolate semi conservative DNA with one fluorescent strand and one non fluorescent.
Re heat this strand and note temperature at which it denatures. The higher the temperature the more closely related.
Outline the key stages to fluorescence in situ hybridisation
Heat DNA that is being tested to separate the strands.
Add a complimentary fluorescently labelled probe to the section of the gene you are testing for.
Cool to allow probe chance to anneal to DNA. Wash away excess probe.
Observe DNA, if there is any bound to the probe that gene is present.
How can siRNAs be used to regulate gene expression (clinical use)?
Delivery of exogenous siRNAs complimentary to the mRNA of interest reduces transcription of that gene and therefore can reduce disease symptoms
How does CRISPR-Cas9 work?
Used to edit the genome. Cas9 uses RNA guide to direct nuclease to area for cleavage.
What are the three key enzymes needed for enzyme techniques?
Restriction enzymes (endonuclease) - cuts at specific palindromic sequences.
Ligase - can reattach cleaved DNA
DNA polymerases - join nucleotides together adding from 5’ to 3’
Outline the process of recombinant DNA cloning
Use restriction endonuclease with recognition sequences at each end of the gene you want to clone. The RE will cut the gene out.
Use the same RE to cut the plasmid vector so that they will have complimentary sticky ends.
Mix together with DNA ligase to ligate the new gene into the plasmid.
Insert back into bacteria.
Outline the process behind PCR
DNA is heated to 95 degrees to denature it
Cooled to 55 degrees and primers added to anneal to the strands.
Heat to 72 degrees and add nucleotides for taq DNA polymerase to add the nucleotides and join up the strands.
Repeat many times.
What is RT-PCR?
Retroviral derived polymerase (reverse transcriptase) is first employed to transcribe DNA from RNA.
Why is RT-PCR useful for mRNA analysis, what primer is used?
Share a common 3’ end (poly A tail). Therefore a poly T primer can be used to RT all mRNA into DNA (called cDNA).
Outline the process of Sanger sequencing.
Get ddNTPs of each base and label each one with a different fluorescent colour. (ddGTP, ddATP, ddCTP, ddTTP).
DNA polymerase replicate DNA but include the ddNTPs. Therefor in the sample there will be DNA of different lengths as each stopped at different random time.
Use gel electrophoresis to examine lengths of the strands. Use distance travelled and colour to get code. (e.g if Guanine is red and a red dot is the furthest along the gel plate, the first nucleotide is G).
Why do ddNTPs terminate polymerisation?
They don’t have an OH in the 3’ position so they cannot phosphodiester bond.
What underpins immunofluorescence?
Used to detect and localise a particular protein in a tissue. Specific antibody to that protein is designed and tagged with a fluorescent marker.
Outline the process of ELISA
Prepare a well plate with immobilised antigens on the surface.
Sample containing potential antibody is added and after incubation the wells are washed removing non-captured material.
A secondary antibody (complimentary to the test antibody) is added. This is coupled to an enzyme.
Excess secondary antibody is removed and the plate is washed.
Detection solution/substrate is added. Enzyme catalyses the formation of dye. This gives a positive result.
How does western blotting locate a protein?
Expose proteins to SDS to make a uniform negative charge.
Gel fractionation of the proteins to separate them out.
Blot onto a nylon membrane.
Bath membrane in antibody solution with antibody specific to that target protein.
Wash away unbound antibodies and add a detection solution. (2nd antibody)
Expose to an X-ray this enables the detection.
