Biotechniques

Magnification DNA samples are often too small for effective study 2 methods of multiplying DNA samplePCR Cloning vectors

Examples A crime scene (body tissue samples) Fragments of DNA from a long extinct animal

Polymerase Chain Reaction (PCR) Generates large amounts of DNA from a small sample, very rapidly. Each cycle takes about 5 minutes, 30 cycles  2 30 copies = about a billion

1.Denaturation 1.Denaturation - heat  breaks hydrogen bonds  DNA double helix separated

2.Annealing 2.Annealing - starter chain of nucleotides act as primers for new nucleotides to attach to. 5’ 3’ 5’ 3’ 5’ 3’

5’ 3’ 5’ 3. Extending 3. Extending – heat stable enzyme (usually Taq polymerase) joins free nucleotides to the primer  complementary DNA strand (semi-conservative).

taq polymerase isolated from thermophilic bacteria live in high temperatures so enzymes adapted for heat not denatured in PCR machine

PCR Equipment Simple-to-use PCR machines called thermal cyclers

Polymerase Chain Reaction Cycle 1 Original DNASample Cycle 2 Cycle 3 Many cycles  Exponential growth

The Process of PCR 1 Primer annealed DNA sample = target DNA 98  C for 5 min  denaturation mRNA primers are annealed

The Process of PCR 2 Nucleotides Semi-conservative copies Thermally stable DNA polymerase binds new nucleotides at 60 ° C

Limitations of PCR Normally used for DNA sequences 5kb (kilobases) long – most genes are far longer than this Can only generate moderate amounts of DNA (a billion DNA molecules is not a lot!) taq Polymerase cannot proofread to eliminate mutations if the gene is unknown, we cannot make the appropriate primer – so PCR useless

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