DNA Techniques Flashcards
What is DNA hybridisation? Give some examples of variant hybridisation techniques.
Where you denature DNA either with heat or alkaline, which breaks the H+ bonds between two strands giving 2 x single strand DNA, then add a radiolabelled probe in before cooling and reannealing. Probe is complementary to a single-strand of the DNA so will attach and this can be seen photographically. Southern blot, northern blot, western IS NOT.
What 4 tests can southern blotting combine? What is it used for? Give disease examples
PCR to amplify a single or few copies of a piece of DNA (or clone/genomic DNA(from chromosomes rather than plasmids)), followed by Restriction enzyme digestion of DNA followed by DNA gel electrophoresis then DNA hybridisation - detect using X-Ray film. It is used to see a fragment of DNA of interest, could be a gene or stretch of DNA. E.g.
- Gene structure - large deletions of duplications
- Gene expansions triplet repeats e.g. Huntington’s
- Allele specific e.g. Sickle cell disease
- DNA variation e.g. DNA fingerprinting
Which two tests does northern blotting combine? What is it used to see?
RNA gel electrophoresis then hybridisation either with a DNA or RNA probe to see whether a gene is being over or under expressed e.g. An oncogene being over expressed may have lots of mRNA when compared to a normal control.
Why use a radiolabelled probe in Southern blot?
Allows you see small fragments of DNA that may not be visible by just staining DNA in a gel.
DNA probes in blotting - do they have 100% similarity to target DNA/RNA? Do they affect the position of the target sequence on the gel in electrophoresis?
No but it can still bind to the target- they don’t have to completely align as can still see the DNA fragment, there can be some overlap. Probes will not have an effect on the position the target sequence takes in the gel.
What is DNA sequencing and what can it be used to see? Give a disease example
Using dNTPs for ATGC and ddNTPs (which stop replication) along with a primer to initiate DNA replication, can then either use gel to separate the nucleotides on the basis of size to then read off the sequence (Sanger Chain) or nowadays use laser - Can see specific nucleotide sequences e.g. CAG repeats in Huntingtons
What is restriction enzyme digestion and when would it be used?
Enzymes that cut DNA at specific nucleotides into smaller fragments to then be selectively amplified by PCR before running tests e.g. DNA sequencing, electrophoresis. Can also digest before cloning a gene of interest
What is PCR and what is it used for? How much is DNA amplified per cycle?
PCR is used to amplify a focused segment of DNA so there is enough DNA for diagnostic tests to be used - usually 0.1 to 10Kbp (1Kbp is 1000 base pairs). DNA template in + 2 x DNA primers (bind to 3’ ends of the send and antisense strand as Taq polymerase reads 3’-5’ for the complementary DNA to be synthesised 5’-3’). Temperature cycles of denature, anneal and polymerise - heat to 95, cool to 55, heat to 75 for Taq to work. DNA is doubled every cycle.
What is a DNA primer vs a DNA probe?
DNA primer (used in PCR) is a short single strand of DNA that binds and attaches to a specific region of the DNA - enables DNA polymerase (e.g. Taq polymerase) to come and begin replication of DNA fragment (DNA polymerase can only replicate to an existing strand so needs primer). Probes are primers/DNA sequences with an addition e.g. A fluorescent/radiolabelled tag which allows you to see where the sequence is. Both can be custom made to ensure region of interest is targeted.
What is special about Taq polymerase as a DNA polymerase for PCR
It is heat resistant
What is DNA gel electrophoresis? Which probe do DNA fragments move towards? How are DNA fragments made? What is 2D gel electrophoresis?
DNA electrophoresis separates fragments of DNA on the basis of size. Move towards + electrodes, smaller fragments move further. Stain to detect DNA or use fluorescent/radiolabel if difficult to see/larger fragments (This is DNA hybridisation used in Southern Blotting). DNA fragments made by using specific Restriction Enzymes. 2D is separating DNA on size and charge.
How would you clone a gene(4 stages)? Give 3 uses of this in diabetes, and in Huntington’s/ BRCA1/2 / CF. What 4th use might gene cloning have in the future (e.g. In CF)
1) Isolate relevant gene with restriction enzymes
2) Using a plasmid you can introduce a specific gene (separated by restriction enzymes) into a vector. Ligase gene into plasmid DNA. I
3) Insert this ‘recombinant DNA’ into a host cell - Bacteria - usually Ecoli. Grow then
4) Isolate the cloned gene (Bacteria self-replicate identical copies of the DNA). Useful in forming proinsulin for diabetes and useful to clone Huntington’s HTT gene to find out its use, useful in getting enough DNA for genetic screening for diseases e.g. Huntington’s, BRCA 1/2 and CF.
1) Make useful proteins 2) Find out what genes do 3) Genetic Screening 4) Genetic therapy for e.g. CF
What is protein gel electrophoresis and what are 2 types? What are these types combined? Why would you need to combine these two processes? What 3 characteristics can proteins be separated by?
It is like DNA electrophoresis but instead of separating fragments of DNA it separates proteins. Proteins can be separated by shape, size and charge.
SDS-Page technique uses a detergent to denature proteins - so they are only then separated on size (removes tertiary/secondary structures so they are just polypeptide chains).
Isoelectric focusing - is where the gel has a pH gradient so proteins move based on their pI (pH at which they have no overall net charge).
Together they are 2D-Page - separate first by isoelectric focussing, then SDS page by side. The reason you use both is that some proteins with same isoelectric point have different sizes so this alone does not separate the proteins sufficiently.
What is Western blot? Give a disease example of when this is useful
SDS- page separation
Then add antibody specific to 1 protein - will bind on blot
Use 2ndary labelled antibody to check binding of the previous antibody
Used to see normal vs abnormal proteins e.g. In cancers
What is ELIZA?
Measuring concentration of proteins in a solution e.g. Insulin using a radiolabelled primary antibody
