Analysis Techniques Flashcards
(41 cards)
With each blotting technique give name and what they use?
Southern- DNA
Northern- RNA
Western- proteins
Describe process of DNA/Sanger Sequencing
Uses Fluorescently labelled dideoxy nucleotides. Once incorporated, they prevent elongation after them. After PCR you TF produce lots of DNA strands of different lengths. Can be read on gel electrophoresis
What technique would you use to test for mutations present?
Restriction endonucleases, PCR, Southern blotting
What is needed for and what would you use gel electrophoresis alongside?
Restriction endonucleases, agarose gel, salt buffer solution (to allow the charge to be carried), power supply (to generate charge difference across the gel)
Why may you use restriction endonucleases?
To investigate size of DNA (check for deletions), check presence of mutations if enzyme binds/not, clone specific chunks of DNA
What do restriction endonucleases recognise?
Specific palindrome sequences
What can restriction endonucleases produce?
Sticky and blunt ends
What is the function of the agarose gel?
Acts as a sieve, smaller fragments go further than larger ones.
What enzyme is needed for PCR? Why?
Taq polymerase, it is able to withstand high temperatures
What are the three main steps of PCR?
Heat to denature, cool with DNA primers to allow to anneal, reheat to allow the taq polymerase to work, repeat
What is taq polymerase?
A thermos table DNA polymerase that can withstand high temperatures
List some characteristics of probes
Don’t have to be a 100% match- can overlap.
What is reverse transcriptase PCR? (RT-PCR)
As PCR doesn’t work with RNA, converts the RNA back to DNA
What is microarray? And benefits
Can analyse several genes at once, give different DNA different colours showing either deletions (if less of one colour) etc
What is DNA fingerprinting?
Where you compare the short random repeats of sequences that non-coding region of genes have (called VNTR- variable number tandem repeats). Only identical twins have same
How may you asses chromosomal abnormalities?
Karotyping, FISH, chromosome painting,
What is karotyping?
Visual picture of the full set of a persons chromosomes, organised according to chromosome number
The chromosomes are halted at metaphase to be able to visualise them
What may you use karotyping for?
To spot aneuploidy, monoploidy, sex,
What is FISH? Why is it so important?
Fluorescence in situ hybridisation, allows analysis to occur whilst chromosomes are still in the cell
What different probes are used in FISH?
Telomeres, centromere, loci, whole chromosome
Why may chromosomes painting be useful?
May be able to see where translocations may have happened
What two types of prenatal testing are most commonly used?
Amniocentesis and chorionic villus sampling
Which is more dangerous for miscarriage risk- amniocentesis or chorionic villus?
Chorionic villus sampling as its done earlier- weeks 11-12
What happens in chorionic villus sampling?
Some chorionic villus cells are sampled by suction through a needle through the abdomen
