Biotechniques. Magnification DNA samples are often too small for effective study 2 methods of...

Biotechniques

Magnification

DNA samples are often too small for effective study

2 methods of multiplying DNA samplePCRCloning 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 230 copies = about a billion

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

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

5’

3’

5’

3’

5’5’ 3’

3’

3’

5’ 3’

5’

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 EquipmentSimple-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

98C for 5 min denaturation

mRNA primers are annealed

The Process of PCR 2Nucleotides

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

PCR• http://www.biotechlearn.org.nz/themes/dna_lab

/pcr_polymerase_chain_reaction • http://www.biotechlearn.org.nz/themes/dna_lab

/video_clips/pcr_a_scientist_explains_v0089

• http://www.biotechlearn.org.nz/themes/dna_lab/video_clips/pcr_in_action_adding_genes_to_cells_v0013