Module 6: Manipulating Genomes. Flashcards
What are the 3 main techniques for studying genes?
- The polymerase chain reaction (PCR)
- Cutting out DNA fragments using restriction enzymes.
- Gel electrophoresis.
What is PCR used for?
It can be used to select a fragment of DNA and amplify it to produce millions of copies in just a few hours.
What is the piece of equipment used for PCR?
Thermal cycler.
What must be added to the mixture before PCR is carried out?
DNA sample
Free DNA nucleotides.
Primers.
Taq DNA polymerase (extracted from extremophiles)
What is a Primer?
They are short pieces of DNA that are complementary to the bases at the start of the fragment/template you want.
Describe step 1 - separation of the DNA strands.
The DNA mixture is heated to 95 degree to break hydrogen bonds between the two strands of DNA
(DNA polymerases doesn’t denature at this high temperature - thermostable)
Describe step 2 - annealing of the primers.
The mixture is then cooled to 50 -65 degrees so that complementary primers can anneal (bind) to the strands.
Describe step 3 - DNA synthesis.
The temperature is increased to 72oC which is the optimum temperature for DNA polymerase.
DNA polymerase forms a new DNA strand from catalysing the formation of phosphodiester bonds between the free DNA nucleotides which align along the DNA template strand by complementary base pairing rules.
Describe step 4 - cycle repeats
2 new copies of the fragment of DNA are formed and one cycle of PCR is complete.
The cycle starts again - the mixture is heated to 95 degrees and this time all 4 strands, 2 originals and 2 new, are used as templates.
How much cycles of PCR are carried out?
Around 30-40 cycles of PCR are carried out, which generates millions of DNA fragments.
Each PCR cycle doubles the amount of DNA, so huge numbers of DNA fragments can be quickly generated.
What is the second way to get a DNA fragment from an organism’s DNA?
By using restriction enzymes.
How do restriction enzymes work?
Some sections of DNA have palindromic sequences (also known as recognition sites) of nucleotides - these sequences consist of antiparallel base pairs (base pairs that read the same in opposite directions)
Restriction enzymes recognise specific palindromic sequences
