10.1- PCR and Gel electrophoresis

what does PCR stand for?

Polymerase Chain Reaction

what is PCR used for?

• used to amplify (make copies of) DNA samples of interest

• necessary as DNA samples found are limited

what are the steps in PCR

1. Denaturing

2. Annealing (bonding)

3. Extension (elongation)

denaturing

• buffer containing DNA samples heated (approx 94-98 degrees)

• this breaks the hydrogen bonds between double-stranded DNA molecule (need to do this for complementary base paring)

annealing

• sample now cooled (50-68 degrees)

• primers added to solution, bind to complementary base sequence on DNA

• (primers are small single strands of DNA that allow DNA [taq] polymerase to attach to DNA)

extension

• heat stable version of DNA polymerase called Taq polymerase added to solution

• heated again (68-72 degrees)

• Taq polymerase performs complementary base pairing, building new strands of DNA

• PCR uses thermocycling (process repeats 20-30x to amplify numbers of DNA

what is gel electrophoresis used for?

• used to separate DNA based on its length

• aims to produce the DNA profile of an individual (DNA fingerprint)

what can we determine from gel electrophoresis?

• how similar 2 samples of DNA are- may be used to identify similarities between organisms

• more bands that line up with each other= more similar organisms are

what is the process for gel electrophoresis?

1. use restriction enzymes to cut DNA into small sections at recognition sites

2. DNA samples of individuals placed within different wells at end of negative electrode

3. electric current run through agar gel

4. negatively charged DNA will move through the gel towards the positive electrode (smaller pieces of DNA move faster and further than larger pieces)

what do we use the results of gel electrophoresis for?

• the DNA profile can be compared to the DNA ladder (sample that contains DNA segments with known lengths of base pairs)

• used to determine the length of DNA strands in the samples