Molecular techniques and diagnosis Flashcards
How does the Sanger Dideoxy Chain Termination Method of DNA sequencing work?
Fluorescent ddNTPs are added to a DNA template strand (with DNA polymerase) to create a complimentary DNA strand
The new chain terminates at different places depending on which ddNTP is used.
Creates lots of DNA fragments which can be denatured and separated using gel electrophoresis.
Why does ddNTP cause the DNA chain to terminate during synthesis?
Because ddNTP is missing an OH group polymerisation cannot occur therefore the chain terminates.
What are restriction endonucleases?
Restriction enzymes that are bacterial and recognise specific DNA sequences (restriction sites) and cut the DNA here
What can restriction analysis (combined with electrophoresis) be used for?
See size of DNA fragments (Useful in seeing deletions)
Investigate mutations
Investigate DNA variation
Genetic cloning
What are plasmids?
Small sections of circular DNA that can transfer to other bacteria and sometimes contain antibiotic genes.
How can plasmids be used in Genetic cloning?
The plasmid is cut using restriction enzymes.
Gene of interest is added to create recombinant DNA molecule
Placed into the bacterium (Transformation)
Placed into an environment to multiply
Why does the plasmid often contain an antibiotic gene?
So that the bacterium which takes up the plasmid can easily be selected.
What is DNA gel electrophoresis used for?
Separation of different sized fragments
What direction do the DNA fragments move in DNA gel electeophoresis?
From the negative well to the positive anode. Heavier fragments travel slower.
Fragments of known size are used as reference.
What does DNA gel electrophoresis require?
A gel A stain A buffer A power supply Fragments of known size for reference
How does PCR amplify small DNA segments?
Repeated copying of target DNA using thermostable DNA polymerase and a pair of primers.
What are the 3 main steps to PCR amplification?
Denaturation at high temperatures
Renaturation at lower temperatures
DNA synthesis at a medium temperature
What can PCR be used for?
Amplification of a specific DNA fragment
Investigate single base mutations
Investigate small deletions or insertions
What is Southern blotting used for?
Marking unlabelled DNA from gel electrophoresis
How does Southern blotting work?
Nylon transfers the fragments from gel electrophoresis
Then this is hybridised with a labelled gene probe
Shows specific DNA fragments
Radioactive probes can be used to mark specific complimentary DNA fragments
What is Northern blotting?
The same process as Southern blotting, but it uses RNA instead of DNA
What is Western blotting?
The same principple as Southern blotting, but it analyses Proteins.
What is blotting used for?
Investigate gene structure
Investigate gene expansions
Investigate variation
How can PCR be used in Allele specific testing?
Use primers specific to the sequences either side of the allele then amplify that allele
How can Restriction analysis be used in Allele specific testing?
Use restriction enzymes that have restriction sites around and within the allele.
Analyse the size of the fragments produced
The restriction site is missing/mutated if the restriction enzyme cuts the wild type.
How can DNA hybridisation be used in allele specific testing?
Use DNA probe that is specific to either the wild type allele or mutated allele then see which binds.
What is SDS page?
A technique that allows proteins to be separated by their molecular weight only.
How does SDS page work?
SDS denatured the protein molecules
Protein has a negative charge which is proportional to molecular weight
Electrophoresis - heavier molecular weight means more negatively charged so moves further
What is isoelectric focusing?
Where proteins can be separated using their isoelectric point
