16.3 – In vitro cloning Polymerase Chain Reaction

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Presentation transcript:

16.3 – In vitro cloning Polymerase 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? 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.

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

The Double Stranded DNA Molecule G T C T C A G Heat to 950C to separate the DNA strands

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

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

Advantages It 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 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.