DNA Restriction

Question 1

Restriction

Answer 1

DNA digestion using bacterial restriction enzymes.
Recognition of specific DNA sequences
Enzyme activity depends on temp, co-factors, pH and salt conditions.
Restriction sites are often palindromic sequences.
Longer the recognition site, less frequent it will be in a random genome.

Question 2

DNA fingerprints

Answer 2

Restriction fragment length polymorphism (RFLP)
>90% of DNA sequences are the same in individuals of species, enough of the DNA is different to distinguish one individual from another.
Individual genetic differences include also differences in the presence of restriction enzyme cleavage sites.
Each variant acts as an inherited genetic trait, allowing them to be used for personal or parental identification.

Question 3

RFLP procedure

Answer 3

Extraction of DNA
PCR
Restriction digest
Size separation of DNA fragments by GE
Visualisation of fragments by ethidium bromide staining

Question 4

Dideoxynucleotides

Answer 4

(ddNTP) are chain elongating inhibitors.
Also known as 2’,3’ Dideoxynucleotides as the 2’ and 3’ positions lack OH groups.
Prevents polymerisation reaction and stops extension.

Question 5

Sanger method

Answer 5

Prep of 4 polymerase mixes.
Addition of small amounts of ddNTPs to induce occasional elongation
ddNTP amounts calculated to statistically induce elongation stops in every sequence position.
Can use GE to separate fragments, new version using fluorescently labelled ddNTPs, can be PCR based.
Capillary GE uses lasers