1. qPCR, qRTPCR
Tryton Shelp

2. Purpose of qpcr
Was originally developed to Detect and measure amplicon yield specifically during the exponential phase of PCR
To determine how much starting material of DNA was in the original sample
To compare to other PCR samples or other known standards
Can be used to quantify and compare expression of a gene of interest between two different experimental treatments
Used to determine viral load of a virus in a patient sample

3. End result-amplification curve
The Ct (comparative threshold cycle) value shows the cycle number until detection of fluorescence

4. Amplification curves for qPCR and PCR

5. Similarities/Difference between pcr and Qpcr
Uses primers, parent DNA, MgCl2, a polymerase, dNTPs
Only focuses on the product after the entire PCR process
Identifies its presence rather than how much there is
Requires a gel for gel electrophoresis to show the presence of the product
Uses a thermocycler
Uses primers, template DNA, a polymerase, dNTPs, and fluorescent dyes
Allows us to observe the amount of DNA produced during each PCR cycle
Gel electrophoresis is not the end point measurement as qPCR is not used for determining
the amount of product formed is monitored as the reaction takes place via fluorescent dyes or probes introduced into the reaction that is proportional to the amount of product formed
Uses a thermocycler along with spectrofluorometry

6. Kary Mullis, Russell Higuchi, Gavin Dollinger, P
Kary Mullis, Russell Higuchi, Gavin Dollinger, P. Walsh, and Robert Griffith
Influenced by Kari Mullis’ work with PCR
Developed the process of QPCR by considering fluorescent dye to monitor reaction progress
Developed in 1992 at Roche Molecular Systems and Chiron

7. QPCR machine

8. Process
Need to create a mix of the template DNA to amplify, DNTPs, our primers, and our heat resistant polymerase, along with a different chemical, our detector.
There are two specific types of detectors:
Non-specific detection fluorescent dye: Syber Green
Specific detection probes: TaqMan
With each cycle in the reaction, the detector binds to the DNA emitting a fluorescent signal which is detected by the spectrofluorometer

9. Non-specific detection (Sybr Green)
Dye based detector that binds to dsDNA
Binds to the dsDNA by intercalating between the bases
Fluoresces which is measured at the end of each cycle to determine the amount of dsDNA produced

10. Syber green pros and cons
Easy to produce
Cheap
Effective and easy to handle
easy to use with regular PCR protocols and primers.
Not-specific to a particular gene
Might end up getting a reading for a different gene
Fluoresces at a lower intensity than TaqMan
detects any dsDNA signal including non-specific amplicons.

11. Specific detection (TaqMan)
Probe based (essentially a third primer to add specificity)TaqMan is a single stranded DNA probe with two molecules on either end the fluorophore on the 5’ end and the quencher on the 3’ end
The quencher works as an inhibitor of the fluorophore
Attaches to the specific part of the gene via specific hybridization during the primer annealing phase
During the extension step, the polymerase moves over the probe and degrades it releasing the fluorophore

12. Taqman pros and cons Pros Cons Specific and sensitive
Different gene targets
Can be used for multiplex qRT- PCR (Can use multiple fluorescent types for different genes)
Difficult to design considering it needs to be specific to part of the gene target
Is expensive
Chance of false negatives/positives

13. Q Reverse Transcription-pcr
Used to measure and analyze the level of expression for a certain gene (transcription level analysis)
Very useful for disease and cancer research as well as gene regulation
Requires template mRNA, dNTPs, RNAse, our primers, DNA polymerase, and reverse transcriptase
Reverse transcription of mRNA generates cDNA to serve as a template for qPCR

14. Applications of qpcr
Gene Expression (mRNA) Analysis (Relative expression level=2-∆∆Ct)
Study on genes epressed by cancer cells from breast cancer patients that utilizes this equation
microRNA analysis
Genetic Variation
Mutation Detection
pathogen detection
single nucleotide polymorphism (SNP) analysis
analysis of chromosome aberrations
most recently also protein detection by real-time immuno PCR
Use of antibodies specific to a protein that can bind with TaqMan to produce a fluorescent sgnal

15. References
Higuchi R1, Dollinger G, Walsh PS, Griffith R., Simultaneous amplification and detection of specific DNA sequences., Biotechnology (N Y) Apr;10(4): Roche Molecular Systems, Inc., Emeryville, CA
reproducibility.html
Deepak, Sa et al. “Real-Time PCR: Revolutionizing Detection and Expression Analysis of Genes.” Current genomics vol. 8,4 (2007):
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