1. 16.3 – In vitro cloning Polymerase Chain Reaction

2. Objectives
You should be able to describe the biological principles underlying each of the following:
The use of the polymerase chain reaction (PCR) to make large amounts of DNA from very small samples
The use of genetic fingerprinting

3. Criminals – Watch Out!
Scientists can now identify criminals from the smallest bit of DNA.
Wherever you go, you leave a trail of DNA behind.
We consistently shed hairs and flakes of skin.
It is in these hair and skin cells that DNA is found!
You DNA is your GENETIC FINGERPRINT!

4. Polymerase Chain Reaction(PCR)
PCR is an method by which DNA can be replicated in the lab.
It can be used to create millions of copies of DNA in just a few hours.
It is essential in forensic science as very small samples of DNA are difficult to analyse.
This process amplifies DNA, so that it can be analysed.

5. What do you need?
RNA primers provide the starting sequence for DNA replication. They also stop the two DNA strands from joining together.
DNA nucleotides containing the bases adenine, guanine, cytosine and thymine.
Enzyme DNA polymerase.

6. The Stages of PCR REPEAT CYCLING Mix with Primers (RNA strands)
Strand Separation
DNA heated at 95°C for 5mins
Binding of Primers
Mixture cooled to 40°C C
Mix with
Free Nucleotides
DNA Polymerase
REPEAT CYCLING
DNA Synthesis
Mixture heated to 70°C
(optimum temp. for DNA polymerase)
With every cycle the amount
of DNA doubles

7. The Double Stranded DNA MoleculeG T C T C A G
Heat to 950C to separate the DNA strands

8. Cool to 400C to allow primers to bind (anneal) to DNA
DNA Strand A G T C C T
RNA Primers C T T C A G
DNA Strand
Cool to 400C to allow primers to bind (anneal) to DNA

9. G A G T C T C A G G C C G G A A G G T C A T C A G G
DNA Polymerase G
Original DNA strand A G T C T C A G G
Primer C C G
Nucleotides join on
Free DNA nucleotides
Nucleotides join on G A A G G T C A
Primer
Free DNA nucleotides T C A G G
Original DNA strand
Mix with DNA polymerase and free nucleotides and heat to 700C

10. Advantages It is a very rapid process Does not require living cells
Is useful when we want to introduce a gene into another organism

11. Disadvantages These are also the advantages of In vivo cloning:
There is risk of contamination by unwanted DNA
It cannot cut out specific genes
Does not produce transformed bacteria

12. Summary Questions
In the polymerase chain reaction, what are the ‘primers’?
What is the role of these primers?
Why are two different primers required?
When DNA strands are separated in the PCR, what type of bond is broken?
It is important in the PCR that the fragments of DNA used are not contaminated with any other biological material. Suggest a reason why.