Genetics Polymerase Chain Reaction L7

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  • Created by: Alex
  • Created on: 12-04-13 01:19
Primer
Strand of nucleic acids that functions as the starting point for DNA synthesis
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DNA polymerase
enzyme. DNA polymerases are best-known for their role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand
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Annealing
Pairing of DNA or RNA by hydrogen bonds to a complementary sequence to form a double-stranded polynucleotide sequence
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TaqMan probes
Hydrolysis probes designed to increase the specificity of real-time PCR assays
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Helicase
Enzyme that unwind the two strands of a DNA double helix
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Replication fork
The point when two DNA strands are unwinding where the hydrogen bonds are breaking
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Polymerase Chain reaction
Method by which a targeted DNA fragment is amplified in vitro to produce millions of copies
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Requirements of PCR
double stranded(ds) template. heat resistant DNA polymerase. 4x nucluotides -dATP,dTTP,dCTP,dGTP. 2x short ssDNA molecules for primers (complementary ti strands of DNA), magnesium ions, buffer containing salt (balance acidic ph of DNA)
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How PCR works
1. heat to 94-9C to denature strands. 2.Cool to 50-65C to anneal primers to template. 3.heat to 68-72C to activate Taqpolymerase - extends primers and replicates DNA. Repeat multiple cycles.
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Heating to 94-99C
Causes strands to denature and seperate
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Cooling to 50-65C
Primers binds to template sequence and Taq polymerade binds to ds dubstrate
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Heat to 68-72C
Taq polymerase extends primer along template sequence replicating DNA
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Taq Polymerase
Heat resistant DNA polymerase enzyme- from thermus aquaticus. low replication fidelity-high error rate (16%mutations). fast
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pfu polymerase
heat-resistant hyperthermophilic - from Pyrococcus furiosu. proor reading activity -2.6%mutatios - high fidelity. slow
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Primer characteristics
>>15bp. GC content 40-60% for stability. anneal between 50-60C with higher temps being better. Primer sequences unique. No self-annealing regions. primers should not anneal each other
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Primer Dimer
when two primers attach to each other instead of attaching to a DNA sequence
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Types of PCR
DNA PCR, RNA PCR, Multiplex PCR, in situ PCR, Real time PCR
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DNA PCR
amplifies from DNA
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RNA PCR
Amplification from RNA via reverse transcriptase reaction followed by DNA PCR
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Multiplex PCR
different primer combinations simultaneously to detect and differentiate agents.
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In situ PCR
Localisation of gene/transcript
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Real Time PCR
/real-time detection using dye chemistries. high precision and sensetivity
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Other cards in this set

Card 2

Front

DNA polymerase

Back

enzyme. DNA polymerases are best-known for their role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand

Card 3

Front

Annealing

Back

Preview of the front of card 3

Card 4

Front

TaqMan probes

Back

Preview of the front of card 4

Card 5

Front

Helicase

Back

Preview of the front of card 5
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