Molecular Biology Techniques Flashcards
1
Q
Polymerase Chain Reaction
A
95 – denaturation of ds DNA by the breaking of hydrogen bonds between complementary base pairs due to increased molecular vibrations due to high heat
64 – primer annealing
- each primer anneals specifically to the. 3’ end of each single stranded target DNA sequence via complementary base pairing when. the temperature is lowered
72 – Taq polymerase synthesises a complementary DNA strand from the free 3’OH end of the DNA primer by catalysing the formation of phosphodiester bonds between dNTPS
2
Q
advantages of PCR
A
- only a minute amount of DNA is required to carry out PCR with each round of PCR, the number of copies of target DNA is doubled –>. number of desired sequence increases exponentially and there will be sufficient DNA for analysis
- use of thermostable Taq polymerase allows PCR to be automated so DNA can be amplified very quickly
3
Q
limitations of PCR
A
- Taq polymerase lacks 3’ to 5’ proofreading ability, hence errors occurring early in the PCR reaction will get compounded with subsequent replication cycle
- knowledge of sequences flanking the target sequence is required in order to design appropriate primers
- Taq pol tends to fall off the DNA template before chain extension is complete if the strand is too long, there is a limit to the size of DNA fragment
- minute amounts of contaminant DNA can be exponentially amplified along with the target DNA and affect reliability of results
4
Q
Principles and procedures of gel electrophoresis
A
- separate DNA based on fragment size
- DNA sample is mixed with dense loading dye to make DNA sample denser to sink to the bottom of the well
- negatively charged DNA migrates from the negative electrode to the positive electrode
- meshwork of agarose gel fibres impede movement of longer fragments more than that of shorter fragments, hence longer fragments migrate slower and end up nearer to the well
- to visualise the bands, the gel can be treated with a staining dye (ethidium bromide) that binds DNA and fluoresces under UV light
5
Q
Principles and procedures of Southern Blotting
A
- detect specific nucleotide sequences within a sample of DNA
- gel slab is placed under a nitrocellulose membrane, placed in alkaline solution to denature double stranded DNA into ss DNA
- ss DNA is drawn upwards onto the nitrocellulose membrane and binds to the membrane.
- incubated with ss radioactive DNA probes which hybridises via complementary base pairing to part if the target sequence
- autoradiography is performed placing X ray film over membrane
6
Q
Restriction Fragment Length Polymorphism
A
- unique banding patterns among individuals when their DNA is digested by restricted enzymes and separated by gel electrophoresis.
- RFLPs arise due to polymorphic nature of DNA, there will be variations in number and location of restriction sites and number of tandemly repeated sequences
7
Q
A