16.3 – In vitro cloningPolymerase Chain Reaction

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

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!

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.

What do you need?1. RNA primers provide the starting sequence

for DNA replication. They also stop the two DNA strands from joining together.

2. DNA nucleotides containing the bases adenine, guanine, cytosine and thymine.

3. Enzyme DNA polymerase.

The Stages of PCR

Strand Separation DNA heated at 95°C

for 5mins

Binding of PrimersMixture cooled to

40°C C

Mix with Primers (RNA strands)

DNA SynthesisMixture heated to

70°C (optimum temp. for DNA polymerase)

REPEAT CYCLING

Mix withFree NucleotidesDNA Polymerase

With every cycle the amount of DNA doubles

A A G G T C A C T T

T T C C A G T G A A

The Double Stranded DNA Molecule

Heat to 950C to separate the DNA strands

A A G G T C A C T T

T T C C A G T G A A

C T T

C T T

DNA Strand

DNA Strand

RNA Primers

Cool to 400C to allow primers to bind (anneal) to DNA

A A G G T C A C T T

T T C C A G T G A A

T T C C A G T

G T C A C T T

G C C

A A G

G

G

G

G

Free DNA nucleotides

Original DNA strand

Original DNA strand

DNA Polymerase

Free DNA nucleotides

Primer

Primer

Nucleotides join on

Nucleotides join on

Mix with DNA polymerase and free nucleotides and heat to 700C

AdvantagesIt is a very rapid process

Does not require living cells

Is useful when we want to introduce a gene into another organism

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

Summary QuestionsIn 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.