V L8: DNA amplification and visualisation Flashcards
where are restriction enzymes found?
mainly in bacteria
whats the full name for restriction enzyme?
Bacterial endonuclease enzymes
Bacterial endonuclease enzymes are used to ___ up human ____.
snip
DNA
what do restriction enzymes recognise?
recognise restriction site which is 4-8 base pairs
restriction enzymes recognise sites that are _____.
palindromic
palindromic is read same in every direction, true or false?
true
what are the two types of cut?
staggered cuts
straight cut
A staggered cuts leaves what ends?
sticky ends
A straight cut leaves what end?
blunt ends
How do Restriction Fragment Length Polymorphisms appear?
- a mutation at the particular restriction enzyme site
Restriction Fragment Length Polymorphisms shows as?
only 1 fragment is labelled, the second fragment is larger.
what causes DNA separation?
size
larger fragments travel _____.
slower
DNA sample is mixed with a ____ ______, this leaves cut up _____.
restriction enzyme
DNA
when was the first time DNA was sued in a double murder case?
Colin pitchfork, 1980
what was the technique previously step by step?
extraction chopping up separating (electrophoreses) southern blotting DNA immobilised xray film DNA fingerprint
DNA fingerprinting is an ____ technique.
older
Minisatellite DNA is?
VNTRs
VNTRs produce a ____ portion fo DNA
larger
VNTRs are more ____.
variable
whats used now VNTRs or STR?
STR
STR
target certain portion of genome, will either have same or different number of repeats on chromosomes,
PCR is?
polymerase chain reaction
Microsatellite DNA is also known as?
short tandem repeats (STR)
STRs are prone to _____ therefore are useful for ____ _______.
mutation
genetic profiling
PCR was developed in?
1985
PCR allows us too?
You can amplify DNA form a very small amount
what are the 3 main stages of PCR?
Denaturation (heat) - form single strand
Annealing (hybridisation of primers) - primers bind to specific area
Extension - DNA polymerase and building blocks produce new strand (extend primers)
with every cycle of PCR we ____ the amount of DNA.
double
why are STRS used more now?
- Due to the amount of DNA required to perform the southern blot procedure
- large PCR amplicon size of VNTR’s
what is the DNA amplification cycle?
- Denaturation at high temperature (92°C-95°C)
- Annealing at a cooler annealing temperature
- Extension at a temperature between the annealing and denaturing temperatures (usually at 72°C)
annealing temperature depends on?
primers the sequence or length of them
Tm is..
melting temperature
extension dependings on?
the DNA polymerase
after PCR DNA should be form this equation?
DNA=2N
what are the PCR compoennts?
- Template DNA (0.5-2.5 ng)
- Reagents, DNA polymerase, Oligonucleotide primers, buffer, DNTP
DNTP are the ?
building blocks
most enzymes need what to work?
ions
DNA polymerase needs a co factor such as?
magnesium ions
when do we produce fragments of the right size?
third cycle on wards
what to look for in a PCR primer?
- Conserved regions of DNA
- Complementary to the flanking sequence of the target region
- similar melting temperature for forward and reverse primer
- 18-30 bases long
- should NOT contain self-complementary sequences
