A-level biology flashcards 6.1.3 Manipulating genomes- Common techniques

What are the three techniques for studying genes?

What are the three techniques for studying genes?

Polymerase chain reaction

Gel electrophoresis

Cutting out DNA fragments using restriction enzymes

What is PCR?

Used to select a fragment of DNA and amplify it to produce many copies

PCR part 1?

A mixture is set up containing DNA sample, Primers, Free nucleotides and DNA polymerase

The mixture is heated to 95 to break the hydrogen bonds between the DNA strands

The mixture is then cooled to 65 and primers anneal to the DNA strands

PCR part 2?

Heated to 72 so DNA polymerase work is in lining up free nucleotides on the bases alongside the template strand phosphodiester bonds form so new strand form as well

PCR part 3?

In one cycle of PCR two new copies of the fragment of DNA are made once the cycle starts again with all four strands this means the rate doubles each time the cycle happens

What is electrophoresis?

Procedure that uses an electrical current to separate out the DNA fragments (RNA or proteins)

Electrophoresis part 1?

Performed using agar gel which is left to solidify and a row of wells is a the end of the gel, the side where the wells are is closest to the negative electrode then add a buffer solution at the sides of the gel

Electrophoresis part 2?

Using a pipette add dye so the DNA can be seen clear then add the DNA samples to each well recording which is in which

Be careful not to pierce the bottom of the gel and clean the pipette each time

Electrophoresis part 3?

Connect the electrodes and the current will pass through the gel

DNA fragments are negatively charged so they move towards the positive electrode smaller move faster and further from the well then drain the wells and dye the DNA the bands will now form and can be studied

How can restriction enzymes obtain DNA fragments?

1) some sections of DNA are read the same backwards and forwards (Palindromic)

2) restriction enzymes recognise specific palindromic sequences and cut the DNA at these places

3) Different restriction enzymes cut different recognition sequences as they are only complimentary to a specific sequence (Substrate)

4) Restriction enzymes cut the DNA by a hydrolysis reaction and leave sticky ends at each end of the fragment

5) Sticky ends can be used to bind the DNA fragment to another piece of DNA that has sticky ends at complementary sequences (Genetic engineering)