V L8: DNA amplification and visualisation

Question 1

where are restriction enzymes found?

Answer 1

mainly in bacteria

Question 2

whats the full name for restriction enzyme?

Answer 2

Bacterial endonuclease enzymes

Question 3

Bacterial endonuclease enzymes are used to ___ up human ____.

Answer 3

snip

DNA

Question 4

what do restriction enzymes recognise?

Answer 4

recognise restriction site which is 4-8 base pairs

Question 5

restriction enzymes recognise sites that are _____.

Answer 5

palindromic

Question 6

palindromic is read same in every direction, true or false?

Answer 6

true

Question 7

what are the two types of cut?

Answer 7

staggered cuts

straight cut

Question 8

A staggered cuts leaves what ends?

Answer 8

sticky ends

Question 9

A straight cut leaves what end?

Answer 9

blunt ends

Question 10

How do Restriction Fragment Length Polymorphisms appear?

Answer 10

• a mutation at the particular restriction enzyme site

Question 11

Restriction Fragment Length Polymorphisms shows as?

Answer 11

only 1 fragment is labelled, the second fragment is larger.

Question 12

what causes DNA separation?

Answer 12

size

Question 13

larger fragments travel _____.

Answer 13

slower

Question 14

DNA sample is mixed with a ____ ______, this leaves cut up _____.

Answer 14

restriction enzyme

DNA

Question 15

when was the first time DNA was sued in a double murder case?

Answer 15

Colin pitchfork, 1980

Question 16

what was the technique previously step by step?

Answer 16

extraction
chopping up 
separating (electrophoreses)
southern blotting 
DNA immobilised 
xray film
DNA fingerprint

Question 17

DNA fingerprinting is an ____ technique.

Answer 17

older

Question 18

Minisatellite DNA is?

Answer 18

VNTRs

Question 19

VNTRs produce a ____ portion fo DNA

Answer 19

larger

Question 20

VNTRs are more ____.

Answer 20

variable

Question 21

whats used now VNTRs or STR?

Answer 21

STR

Question 22

STR

Answer 22

target certain portion of genome, will either have same or different number of repeats on chromosomes,

Question 23

PCR is?

Answer 23

polymerase chain reaction

Question 24

Microsatellite DNA is also known as?

Answer 24

short tandem repeats (STR)

Question 25

STRs are prone to _____ therefore are useful for ____ _______.

Answer 25

mutation | genetic profiling

Question 26

PCR was developed in?

Answer 26

1985

Question 27

PCR allows us too?

Answer 27

You can amplify DNA form a very small amount

Question 28

what are the 3 main stages of PCR?

Answer 28

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)

Question 29

with every cycle of PCR we ____ the amount of DNA.

Answer 29

double

Question 30

why are STRS used more now?

Answer 30

- Due to the amount of DNA required to perform the southern blot procedure - large PCR amplicon size of VNTR's

Question 31

what is the DNA amplification cycle?

Answer 31

1. Denaturation at high temperature (92°C-95°C) 2. Annealing at a cooler annealing temperature 3. Extension at a temperature between the annealing and denaturing temperatures (usually at 72°C)

Question 32

annealing temperature depends on?

Answer 32

primers the sequence or length of them

Question 33

Tm is..

Answer 33

melting temperature

Question 34

extension dependings on?

Answer 34

the DNA polymerase

Question 35

after PCR DNA should be form this equation?

Answer 35

DNA=2N

Question 36

what are the PCR compoennts?

Answer 36

- Template DNA (0.5-2.5 ng) | - Reagents, DNA polymerase, Oligonucleotide primers, buffer, DNTP

Question 37

DNTP are the ?

Answer 37

building blocks

Question 38

most enzymes need what to work?

Answer 38

ions

Question 39

DNA polymerase needs a co factor such as?

Answer 39

magnesium ions

Question 40

when do we produce fragments of the right size?

Answer 40

third cycle on wards

Question 41

what to look for in a PCR primer?

Answer 41

- 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