dna denaturation and annealing + nucleases

what is dna denaturation?

drastic alteration in the conformation of a protein or nucleic acid due to the disruption of non-covalent interactions caused by heating/exposure to certain chemicals

how can you carry out dna denaturation?

• increase temperature of solution of dna

• thermal energy increases

• molecular motion increases

• eventually hydrogen bonds that stabilise the double helix will break

• strands will seaparte due to electrostatic repulsion (both dna backbones are negatively charged)

what is dna annealing? (hybridisation)

two complementary single strands of DNA combine via hydrogen bonding to form a double helix

reverse of denaturation

when does dna annealing occur? (conditions required)

it occurs when the temperature of a dna solution is lowered

how do dna strands associate in dna annealing?

through complementary base pairing

what are the uses of dna annealing?

find complementarity between DNA from different sources

complementary DNA strands will anneal together regardless of source

what are nucleases

types of enzymes that cleave/break phosphodiester bonds

what are the two main types of nucleases

exonucleases, endonucleases, restriction enzymes

what do exonucleases do

• 5’ → 3’ exonuclease will degrade phosphodiester bonds in a 5’ → 3’ direction

• vice versa for 3’ → 5’ exonuclease

• cuts at the end of the polynucleotide strand

• end product of exonuclease is a single nucleotide

• specific or non-specific breaking

exonuclease uses

DNA repair and proofreading

what do endonucleases do?

• cleaves phosphodiester bonds within the polynucleotide

• end product of endonuclease is a shorter polynucleotide chain

• specific breaking

endonuclease uses

DNA repair and genetic engineering

what do restriction enzymes do?

• recognise short dna sequences as targets for cleavage

• originates from bacterial defense mechanism

what happens when a restriction enzyme cleaves a dna sequence?

cleave results in sticky ends of palindromic sequences that are compatible with each other

if sticky ends are compatible they will always join together — they do not need to be from the same sample or enzyme

what are the uses of restriction enzymes?

cloning

incorporating foreign dna