Kevin Chen

 A method of amplifying or copying DNA fragments

 DNA template  Two complementary primers  Taq polymerase or another DNA polymerase  Deoxynucleoside triphosphates (dNTPs)  Buffer solution  Magnesium or manganese ions  potassium ions.  Thermal cycler

 Initiation  Denaturation  Annealing  Elongation  Final Elongation  Final Hold

 º C  This is held for 1-9 minutes.  Only required for DNA polymerases that require heat activation by hot-start PCR.  Allows the inhibition of polymerase activity during PCR reaction preparation.  Hot Start PCR reduces non-specific amplification and increases PCR product target yield.

 º C for seconds.  This causes separation of DNA template and primers  Disrupts the hydrogen bonds between complementary bases of the DNA strands

 º C for seconds  Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers  Allows annealing of the primers to the single- stranded DNA template.  The polymerase binds to the primer-template hybrid and begins DNA synthesis.

 Temperature varies depending on DNA polymerase  Commonly used temp. for Taq Polymerase is 72 ºC  DNA Polymerase adds dNTPs in 5' to 3' direction  Polymerizes a thousand bases per minute.

 º C for 5-15 minutes after the last PCR cycle  Ensure that any remaining single-stranded DNA is fully extended

 4-15 ºC  Used for short-term storage

 Amplified DNA is quantified as it accumulates in the reaction in real time after each amplification cycle.  Capable of detecting 2-fold change  Monitors the amount of fluorescence emitted during the PCR reaction

Contains a 5’ reporter dye and a 3’ quencher dye. The probe is designed to anneal the target sequence The quencher suppresses the fluorescence of the reporter dye. During amplification, Taq DNA polymerase cleaves the probe and displaces it from the target Cleavage of the probe separates the reporter dye from the quencher dye, resulting in an increase in fluorescence.

A dye used for the quantification of double stranded DNA Fluoresces when bound to double stranded DNA

A sequence specific hybridization probe that fluoresces when it binds to a single DNA strand

TAQMAN SYBR GREEN  Reduces background and false positives.  Post-PCR processing is eliminated  A different probe has to be synthesized for each unique target sequence.  Multiple dyes can bind to a single amplified molecule, increasing sensitivity  No probes are required  It may generate false positive signals.

 en.wikipedia.org/wiki/Polymerase_chain_reaction   science.com/sis/rtpcr/lightcycler/lightcycler_docs/manuals/software_probe_desig n_2_0/probe_design_software_2_0_ch_b.pdf 