PCR and gel electrophoresis

what does PCR stand for and what is it used for

polymerase chain reaction is used to copy DNA to produce millions of identical DNA molecules so that a usable sample is produced

What are primers 

short single stranded sequence of ~20 bases long

outline the steps of PCR (6)

1. DNA is denatured by heating to 95˚C to break H bonds between the strands exposing the bases

2. Primers are added. Can be made into complementary base sequences found either side of the desired DNA sequence so that only that section is copied 

3. Primers anneal when the temperature is cooled (~65˚C), as they base pair with the complementary sequence on the template strand

4. Taq polymerase and free activated DNA nucleotides are added. Taq polymerase can bind to the double stranded section provided by the primer.

5. Elongation - taq polymerase moves along the strand adding the nucleotides to create a double stranded DNA molecule (occurs at 72˚C)

6. Once copied, the mixture is heated to 95˚C again to denature DNA strands

Cycle is repeated to produce many of desired DNA

what temperature is required to denature DNA

95˚C

What does Taq polymerase do

binds to double stranded section provided primer

What temperature does elongation happen during the PCR

72˚C

What type of DNA polymerase is used for the PCR and why

Taq polymerase as it is thermostable at high temperatures, isolated from bacterium Thermus aquaticus 

How are DNA strands from PCR cut up

Using restriction endonucleases giving different sized fragments of DNA

What is gel electrophoresis and why does it work

• process used to separate DNA fragments based on their size and charge

• Phosphate groups in DNA are negatively charged

how does gel electrophoresis work 

• dna fragments are placed in wells at the end of a plate of agarose gel 

• Plate of agar gel is placed into a buffer solution 

• A potential difference is passed through the gel, and the dna fragments will be repelled away from the negative electrode towards the positive electrode. 

• Shorter fragments will move faster through the gel than longer fragments 

• Fragments of different sizes separated 

How is dna visualised

• dna is heated to separate the strands and single stranded DNA probes are added which bind to DNA

• DNA probes contain radioactive phosphorous isotope, so when placed on an X-ray film, the radiation caises a patternn of bands to be formed

What is a DNA probe

Single stranded specific DNA sequence used to find DNA 

What are some examples of other things that can be separated using gel electrophoresis

• proteins - polypeptides have net negative charge (with buffer of constant pH)

• Alloenzymes (formed from different alleles of same gene)

• Variants of haemoglobin (eg sickle cell) as mutation causes an amino acid with non-polar r group to replace one that is charged therefore sickled is less negatively charged.