Analysing molecular genetic variation using RFLP analysis

Question 1

Screening using sequences homology

Answer 1

-Complete mixture of DNA fragment (clones library) kept in a single strand
-probe (purified and labeled nucleic acid)
-the two are then hybirdised and wash. this lead to the detection of nucleic acid duplex

Question 2

Southern blotting

Answer 2

1/ cleave recombiniant lambda clone DNA with a restriction enzyme
2/ Seperate cleaved DNA by gel electrophoresis
3/ Denature DNA into single strand by denaturing agent
4/ transfer ss DNA to a membrane by capillary blotting

Question 3

Hybridisation and detection

Answer 3

1/ Covalently attach ss-DNA to the membrane via crosslinking
2/ Hybirdisation: incubate the DNA bound matrix with ss probe
3/ wash away the week/ unbounded probe
4/ Detect DNA duplexes with autoradiography

Question 4

Restriction mapping

Answer 4

-Digest recombiniant clone with different restriction enzy is then seperated by gel electrophoresis
-Transfer for southern blotting, hybirdisation and detection
-there must be no restriction site on the probe

Question 5

restriction fragment length polymorphism

Answer 5

-differences in restriction fragment length at a particular loci from DNA sequence polymorphism at or between restriction site

Question 6

what can cause restriction fragment polymorphism

Answer 6

-loss or gain of restriction site
indel mutaion within restriction fragment

Question 7

limits of restriction fragment length polymorphism

Answer 7

-low frequency of allesels found in a population, reduce mappable polymorphism
-low resolution- human RELP map has 400 markers that are separated by 10cM
-labour intensive and time consuming

Question 8

Use of repetative DNA as molecular markers

Answer 8

-these are simple sequence length polymorphism- microsatellite and minisatellite
-have a higher number of mutation rate, up to 10-4 per gen. high level of individual difference
-found all over the genome
-easy to detect by pcr or DNA hybirdisation