Molecular Techniques & Tools Flashcards
How can you quantify the amount of DNA or RNA in a sample?
What ratios would you expect with:
1. a pure DNA sample
2. a pure RNA sample
3. high protein contamination
4. single stranded DNA sample?
Measure the absorbance of the samples at 260 and 280nm and calculate this to concentration
- 260:280 of 1.8
- 260:280 of 2.0
- Much smaller ratio as phenol & proteins absorb strongly at 280
- ssDNA absorbs more strongly at 260nm
What are the 4 considerations when using absorbance/spectrophotometry to quantify RNA/DNA?
How can you overcome the first two?
How can you overcome the 3rd?
What would be a positive result and how?
How can you overcome the 4th?
- More RNA in cells than DNA - falsely high quantification as absorb at same wavelength
- Protein contamination
- Hyperchromicity - melting causing single stranded structure absorbance at 260nm is greater
- DNA/RNA strands can reanneal after cooling
Ratio calculation
Use the melting temperature (midpoint of 50% double stranded 50% single) to estimate the % of GC content - higher melting temp = higher GC content
Higher Tm means more GC rich DNA - more resistant to denaturation. Normal DNA is 1:1 ratio of GC:AT
On annealing use salt in solution to minimise charge repulsion
Why does RNA run quicker in gel electrophoresis?
What can you use nucleic acid hybridisation for?
What does it require?
What is hybridisation used in?
Capable of forming local secondary structures - becomes smaller & more compact so moves quicker
Determine degree of hybridisation/how closely related two sequences are - different species
Heating & cooling
PCR (primers & probes), Southern & Northern blotting (probes), Sanger Sequencing
Why is fractionating DNA on gel electrophoresis great?
What are the drawbacks?
Why does sugar need to be added?
How else can DNA or RNA be visualised in a gel?
Migration is directly proportional to the length of DNA
- Need dye to visualise
- Resolution isn’t high enough for small DNA so require polyacrylamide instead of agarose
Prevents DNA sample from sinking to bottom of gel
Ethidium bromide
How does ethidium bromide lead to fluorescence?
Why is it better to run the gel without ethidium bromide & to add it later?
Absorbs UV at 300nm in a hydrophobic environment (lack of quenching) and re-emits fluorescence at orange wavelength
End up with ethidium bromide diffusing to the top of the gel due to the negative pole - causing a two-tone image
What does Southern Blotting analyse?
How are the DNA fragments migrated onto the nitrocellulose membrane?
To what medium is the DNA transferred to?
What are the problems with the gel?
What is a more stable membrane?
What are the basic steps?
DNA
Through capillary action through stack of paper towels
2D to 3D
Fragile, wet, 3D & can explode in the oven
Nylon
- Deanneal DNA & denature H bonds in DNA by soaking gel in NaOH
- Capillary action draws DNA from gel to membrane
- Fix DNA with heat/UV
- Add DNA probes in salt solution
- Expose membrane to X-rays or UV
What does Northern blotting analyse?
Why do you run the gel with formamide?
What is the problem with this?
What does Western blotting analyse?
How can you detect this?
RNA
Prevent formation of secondary structures
EtBr only fluoresces in double-stranded molecules - need to remove formamide
Proteins
Antibodies
What kind of enzymes are restriction enzymes?
How can they be used in DNA cloning?
How can they be used in Plasmid Mapping?
What do restriction maps in turn help with?
How do restriction enzymes detect mutations/disease in RFLP (restriction fragment length polymorphism) analysis?
Homodimers - two monomers one for each strand
Produce compatible sticky ends to form recombinant DNA
Restrict same DNA in 3 scenarios: two with one enzyme each, and 3rd with both enzymes. Proceed with gel electrophoresis & use fragment sizes to form a restriction map.
DNA cloning
Mutations can destroy restriction sites & alter DNA fragment length - running gel electrophoresis with restriction of normal & disease chromosomes identifies length & correspond with mutation
- longer DNA is, restriction site missing
What does DNA ligase do?
What does it require?
Why are blunt ligations less efficient?
What does DNA Alkaline Phosphotase do?
What does DNA polynucleotide kinase (PNK) do?
How can this help in molecular biology?
Rejoin phosphodiester bonds in backbone between 3’OH and 5’ phosphate
ATP
No overhang for hydrogen bonding - reliant on chance & spontaneous interaction
removes 5’ phosphate from DNA/RNA to prevent re-formation of phosphodiester bond
Phosphorylates 5’ of DNA with ATP
Incorporate radioactive phosphates
What does a hyperchromatic graph look like? x and y
What is the midpoint of the increasing curve?
When the y axis becomes relative % increase in absorbance at 260nm, what is a shift in the curve to the left?
Right?
What does 50% on the y axis on this graph mean?
x = temperature, y = absorbance at 260nm
Tm
Poly (AT) (lower tm)
Poly(GC)
Tm
