MOLECULAR BIOLOGY LAB

what reagents do we require to replicate DNA

a template DNA strand

DNA polymerase enzyme

primer DNA
nucleotide triphosphate mix

what does DNA stand for

deoxyribonucleic acid

what is a DNA primer

a short single stranded piece of DNA or RNA which acts as a starting point for DNA polymerase to work off

it has an available 3’ OH group

what way does DNA polymerase work in

it reads in 3’ to 5’ but places in 5’ to 3’

what is the difference between dsDNA and ssDNA

double stranded DNA has 2 antiparallel strands joined by the hydrogen bonds between complementary base pairs

single stranded DNA is just one of these strands, with no hydrogen bonding to another

what occurs during denaturation

at about 95*C the hydrogen bonds between complementary base pairs in dsDNA will be broken

this allows 2 strands of ssDNA to form

what occurs during annealing

we cool the DNA slightly, allowing the primer to bind to the ssDNA

it is possible for the ssDNA to reform dsDNA, but this is not the aim

what occurs during extension

DNA polymerase begins to add nucleotides, starting at the primer

this results in dsDNA, double the amount which we originally has

at what temperature is extension done

at the optimum temperature for the specific DNA polymerase enzyme being used

what is Taq polymerase

thermus aquaticus polymerase

a commonly used DNA polymerase enzyme

how does DNA move through the electrophoresis medium

when a voltage is applied the negative DNA is repelled away from the negative electrode and attracted towards the positive electrode

how did we visualise the DNA

we added a stain which the DNA molecules bind to

the stain DNA complex will fluoresce when UV light is shone on it

the greater fluorescence observed, the greater the amount of DNA present

what is PCR

polymerase chain reaction

the process of repeatedly replicating DNA to amplify the amount present