Nucleic acid analysis Flashcards
Describe hybridisation
Homologous single-stranded DNA/RNA combine to form double strand
labelled probe to identify target molecule
DNA on immobilised on solid support and probes are washed over it.
how do you clone DNA in a cell
identify nucleic acid sequence, remove via restriction endonucleases [sticky/blunt ends]
cut plasmid (replicon Can be Yeast Arteficial Chromosome or bacteriophage) with same endonuclease
anneal - ligase
insert recombinant DNA into bacteria and allow them to propagate
use marker eg antibiotic resistance to see which colony has taken up dna
select colony and allow to grow - have many samples of sequence.
describe the different types of hybridisation
Southern - DNA and DNA probe Northern - RNA and DNA probe Colony blot - bacterial DNA and DNA probe tissue in situ - RNA and RNA Chromosome in situ - Chromosome and DNA
What does a probe do in hybridisation
identifies which nucleic acid is present.
describe microarray
done the other way around to normal hybridisation, probe is on gel and DNA is washed over, see which genes are expressed more in cancer/normal cells and figure out which are related to cancer. Different colours show depending on where the gene is more expressed. DNA spots represent genes. Hugh stringency. flurophore-based detection. SNP detection arrays.
describe stringency in hybridisation
higher stringency more specific probe is to DNA, in PCR want really specific, sometimes less so. Higher temp and lower Na+ levels increase stringency [Na+ make DNA more stable?]
Energy breaj bond - length, CG, monovalent stabilise by neutralise charge on backbone, denaturants destabilise duplex.
describe PCR
Temperature regulated. Cell free way of cloning DNA. Use TAQ polymerase as stable and so resistant to changes in temperature that it is subject to in PCR.
DNA broken down
describe the process of PCR
Break hydrogen bond Denature - longer sequence and more CG pairs higher temperature. 25 degrees colder than Tm - temperature half double and half single strand 94 degrees
anneal primers, complementary to each strand, and DNA 50-60 degrees
nucleotides are added 5’ - 3’ to meet forming double stranded DNA 72 degrees
This is repeated until enough fragments are made for testing.
properties of primers
20 nucleotides, avoid tandem repeat CG and length equal, avoid complementary 3’ end - dimers
use of PCR
detect point mutation cDNA clone rt-PCR DNA sequencing DNA microarrray
describe the action of restriction enzymes
Host dna protected by methylation
cut at a specific point called restriction site 4-8bp and palindromic
larger the sequence, the less cuts the enzymes will make and so the fewer fragments
describe electrophoresis
Smaller fragments travel further, less resistance in porous matrix: agarose/polyacrylamide. More negative travel to anode faster. Use scalpel and remove band that desired, or transfer to membrane to form a replica for hybridisation [described above]. Use standard markers
