1 / 16

PCR reaction to be visualized “in real time” as the reaction progresses

REAL TIME QUANTIFICATION. PCR reaction to be visualized “in real time” as the reaction progresses to quantify the amount of DNA in the sample at the start of the reaction. 5’. 3’. 5’. 3’. 3’. 3’. 3’. 3’. 3’. 3’. 5’. 5’. 5’. 5’. 5’. 5’. 5’. 5’. 5’. 5’. 5’. 5’. 3’. 3’. 3’.

jamil
Download Presentation

PCR reaction to be visualized “in real time” as the reaction progresses

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. REAL TIME QUANTIFICATION • PCR reaction to be visualized “in real time” as the reaction progresses • to quantify the amount of DNA in the sample at the start of the reaction

  2. 5’ 3’ 5’ 3’ 3’ 3’ 3’ 3’ 3’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 3’ 3’ 3’ 3’ 3’ 3’ 5’ 3’ 5’ 3’ THE REACTION d.NTPs Primers Thermal Stable DNA Polymerase Add to Reaction Tube Denaturation Annealing

  3. 5’ 5’ 5’ 3’ 3’ 3’ 5’ 5’ 3’ 3’ 3’ 3’ 5’ 5’ 3’ Taq Taq 5’ 5’ 5’ Taq Taq 5’ Extension Extension Continued Repeat

  4. 3’ 3’ 5’ 5’ 5’ 3’ 3’ 3’ 5’ 3’ 5’ 3’ 3’ 3’ 3’ 3’ 3’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 3’ 3’ 3’ 3’ 3’ 3’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 3’ 3’ 3’ 3’ 5’ 5’ 5’ Cycle 2 4 Copies Cycle 3 8 Copies

  5. 2*10=1024 4*5=1024

  6. Ct values are directly related to the starting quantity of DNA, by way of the formula: Quantity = 2^Ct

  7. Ct Values: 25 23 28

  8. As shown by Higuchi et al.2, a plot of the log of initial target copy number for a set of standards versus CT is a straight line.

  9. Effect of Limiting Reagents the rate of target amplification decreases until a plateau is reached CT is a more reliable measure of starting copy number than an endpoint measurement

  10. Detection of PCR product accumulation double stranded DNA binding dye: SYBR GREEN ® fluorogenic probes: Taqman ® Probes

  11. 5’ 5’ 5’ 3’ 3’ 3’ 5’ 5’ 3’ 3’ 3’ 3’ 5’ 5’ 3’ Taq Taq 5’ 5’ 5’ Taq Taq l l l ID ID ID ID ID ID ID ID ID ID 5’ l l SYBR Green real time Extension Apply Excitation Wavelength Repeat

  12. PROBE BASED real time fluorophore covalently attached to the 5’-end of the oligonucleotide probe and a quencher at the 3’-end. Fluorophores: 6-carboxyfluorescein, acronym: FAM, tetrachlorofluorescein, acronym: TET Quenchers: tetramethylrhodamine, acronym: TAMRA,  minor groove binder, MGB

  13. PROBE BASED real time

  14. the 5' to 3' exonuclease activity of the polymerase degrades the probe that has annealed to the template. Degradation of the probe releases the fluorophore from it and breaks the close proximity to the quencher, thus relieving the quenching effect and allowing fluorescence of the fluorophore. Hence, fluorescence detected in the quantitative PCR thermal cycler is directly proportional to the fluorophore released and the amount of DNA 

  15. INTERNAL REFERENCE DYE 5700 system: able to use an internal reference dye (ROX) normalize for non-PCR-related, well-to-well fluctuations in fluorescence fluorescence readings taken at 95 ºC in the baseline region and is essential for reproducible results.

More Related