Analysing Nucleic Acids Flashcards
give an example of how personalised medicine is helpful:
- over-expression of HER2 leads to more aggressive breast cancer - 20% of breast cancer is HER2 positive therefore more aggressive - drugs have been developed specifically for cancer where individuals have the HER2 gene - by sequencing the genome we can figure out if the person is HER positive or not
Describe in viva cloning
- Replicon and target DNA is cut with the same restriction endonuclease 2. Purify and Mix together 3. Join DNA fragments using DNA ligase 4. Transformation of recombinant DNA molecules into into host cell 5. Selective propagation on agar plate - use antibiotic resistance marker so only cells that take up replicon survive 6. expansion of cell culture Isolate target DNA
what is a replicon?
it is a sequence capable of independent replication
Describe type II restriction endonucleases
Enzymes that cut at a specific DNA sequence Recognition sequence is 4-8bp palindromic sequences they produce sticky or blunt end the longer the recognition site the less frequently it occurs in the DNA
Describe electrophoresis
- DNA is negatively charged due to the phosphate backbone so moves to the anode when electrical force is applied - Smaller and more negatively charged fragments move further - after resolution the DNA can be isolated from the gel or transferred to a membrane to form a replica for hybridisation
What are hybridisation assays?
- Target DNA is immobilised and denatured - Probes (single stranded) bind to target DNA this is hybridisation as the complementary probes will bind
What is southern blotting?
type of hybridisation - a method of transferring the electrophoresed fragments onto a membrane that immobilises them. - membrane can be washed with probes so that the probes can attach to complementary sequences. - Once the probes have annealed and excess probe has been washed off - membrane is exposed to a photographic film and kept in the dark - Bands will appear on the photographic film showing the positions of the fragments.
Southern blot Northern blot Colony blot Tissue in situ Chromosome in situ Reverse hybridisation
Southern blot - DNA target and DNA probe Northern blot - RNA target and DNA probe Colony blot - bacterial DNA target and DNA probe Tissue in situ - RNA target and RBA probe Chromosome in situ - chromosome target and DNA probe
Energy needed to denature DNA probe depends on…
Length - longer strand = more energy Base composition - more G-C = more energy Chemical environment - lots of 1+ charges outside can stabilise 1- charge of DNA
What dies stringency mean?
How specifically the probe binds to target (low stringency means the probe can bind to the target with some degree of mismatch) (high stringency means in order for the probe to bind it must be a perfect match)
Define melting temperature
The midpoint temperature of transition between double stranded to single stranded nucleic acids
how does hybridisation of DNA fragments after separation work?
- after resolution the DNA can be isolated from the gel or transferred to a membrane to form a replica for hybridisation - then mixed with labelled probes - can be detected by exposure to photographic film
Hybridisation stringency is… and can be increased by …
The power to distinguish between related sequences Increase temperature and decrease Na+
what temperature is hybridisation normally carried out at?
less than 25 degrees below the Tm Tm for mammal DNA is 87 degree
what are examples of hybridisation?
Southern blot Northern blot Colony blot Tissue in situ Chromosome in situ Reverse hybridisation
Describe in vivo cloning
Denature DNA (95 degrees) Anneal DNA with primer (50 degrees) Extend primer with DNA polymerase (72 degrees)
What to consider when designing a primer
Length - 20 nucleotides for specificity Base composition - no tandem repeats = hairpins (hairpins are bad) Not complementary at 3’ end as primers join to each other instead (this would cause primer dimers)
what is a DNA microarray?
A collection of microscopic DNA (or oligonucleotide) spots, commonly representing single genes, robotically arrays on a solid surface e.g. glass slide
What are DNA microarrays used for?
Used to determine expression profile or many different genes at the same time
what measurements are made from DNA microarrays?
Quantitative or Qualitative measurements can be made from microarrays which utilises the selective nature of hybridisation under high-stringency conditions
how to denature a probe DNA?
- the denaturation of a probe DNA is achieved by heating until the hydrogen bonds between the bases break
what is a polymerase chain reaction?
- this is an in vitro method that allows selective amplification of a specific target DNA within a heterogenous collection of DNA sequences
what are applications of PCR?
- detecting point mutations - cDNA cloning - gene expression - DNA sequencing - DNA microarrays
