biotechniques. magnification dna samples are often too small for effective study 2 methods of...
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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