Lecture 4: DNA and RNA Flashcards
How do we use DNA and RNA gels?
Gels allow us to separate DNA based on molecular weight.
• DNA is negative, so it moves to the positive terminal.
• We have to denature RNA secondary structures with formaldehyde and glyoxal. Otherwise the folding will impact mobility.
• Use EtBr for visualisation.
• RNA is susceptible to digestion, RNases are everywhere. We use inhibitors.
We can use many different labels.
• Intercalating dyes like ethidium bromide. Viewed fluorescently.
• We can use radiolabels such as alpha phosphate-32 ATP.
• Bromodeoxyuridine (BrdU) is a thymidine analogue which can be incorporated into DNA. It is then detected by antibodies.
How do we use polyacrylamide gels?
- Polyacrylamide gels are used for shorter, smaller molecules.
- They can even be used to detect single nucleotide differences in longer DNAs.
- Urea used as a denaturant.
How can we use agarose gels?
- Based on seaweed extract.
- For small molecules, higher concentrations are used (up to 1.5%).
- For big molecules (up to 50 kb) use lower concentrations (around 0.7%).
- Use alkaline agarose to get ssDNA.
- For very big molecules use PFGE (e.g. size sorting BAC and YAC inserts).
What are DNA and RNA probes?
Probes are used to indirectly label DNA and RNA.
• Can be short synthetic oligonucleotides (less then 15 n5) or longer RE fragments (<1kb).
• DNA sequence is complementary to target.
• Will hybridise by complementary base pairing.
Probes have many uses:
• Copy number analysis.
• Evolutionary conservation.
• Locate genes in a chromosome.
• Forensics.
• Screening libraries to clone genes.
• Positional cloning of disease genes.
• Norther blots give information on transcript abundance and size.
How do we use Southern blotting?
Southern blots are used to visualise the amount and size of DNA fragments.
1) DNA is cleaved. Electrophoresis used to separate DNA.
2) DNA fragments are blotted onto nitrocellulose. Uses capillary action.
3) Filter exposed to radioactive probe.
4) Filter exposed photographic film. Film is developed.
We can use this for:
• Zoo blots (are genes present in species).
• Chromosome locations.
• Copy number (by band intensity).
How do we use Northern blotting?
Northern blots are similar to Southern blots. They have different uses though:
• Tissue specificity of expression.
• Development.
• Finding transcript size (alternative splicing).
• Level of expression in response to changes e.g. drugs.
We can use control probes such as beta actin.
What is FISH?
FISH stands for fluorescent in-situ hybridisation.
• FISH is used to tagging nucleic acids.
• It can help with looking at spatial-temporal patterns of gene expression.
How do we use libraries?
Libraries contain sets of DNA sequences. cDNA libraries have all active genes whereas gDNA libraries have every part of the genome. By using a specific DNA probe, the full-length cDNA can be cloned. Requires probe hybridisation. We use this for probe hybridisation.
1) Grow up a library.
2) Hybridise a radiolabelled probe then autoradiograph.
3) Pick positive clones and grow them up.
Screening has largely been superseded by PCR. However, the generation itself is still key to modern genetics.
What is cDNA?
cDNA is made from mRNA. There is bias in cDNA from libraries due to the amount of RNA which is produced.
1) Anneal primer.
2) Make DNA copy with RT.
3) Treat with alkali to degrade RNA.
4) DNA pol uses hairpin loop as a primer to make a complementary DNA strand.
What is the method of subtractive hybridisation?
Subtractive hybridisation which is a method for identifying genes that are expressed only under specific conditions such as mutants or drug treatments.
1) Take all mRNA from cells.
2) Use RT to make DNA.
3) Degrade mRNA.
4) Hybridise cDNA with excess mRNA from the other cell.
5) Hydroxyapatite column removes all DNA/RNA hybrids.
6) Purified cDNA corresponds to mRNA confined to the second cell.
What are microarrays?
Microarrays make it possible to analyse expression of 10,000 genes at a time.
• cDNAs or oligonucleotides are printed onto a glass slide.
• Modified inkjet printer that uses DNA.
• Make test cell and reference cell cDNA. They are given different tags.
• These T and R cDNAs are hybridised to the chip.
• If cDNA binds it lights up.
• Red means T cDNA binds. Green means R cDNA. Yellow means T and R cDNA.
