Q pcr

quantitative or real time pcr principle nad its method of quantification

1. Khuda B akhsh FA14-R02-002 Q-PCR

2. OUTLINE What is PCR and purpose of it? What is Q-PCR and purpose of it? How does Q-PCR work? Types of Q-PCR probes and comparison Advantages and Disadvantages of Q-PCR vs. PCR Questions

3. What is PCR? Stands for Polymerase Chain Reaction For the amplification of DNA fragments

4. Purpose of PCR Easy to sequence more copies For comparing DNA fragments It is used to clone specific genes

5. Material: PCR reagents: A DNA template: Primers 10x PCR buffer d NTPs Taq polymerase MgCl 2

6. Steps of PCR Denaturation : This step is the first regular cycling event and consists of heating the reaction to 94–98 °C Annealing The reaction temperature is lowered to4 5–60 °C for primer attachment Extension/elongation : At 72 ° C for taq plymerase working Final elongation:

7. What is Q-PCR? Stands for Quantitative Polymerase Chain Reaction Assay that monitors accumulation of DNA from a PCR reaction Important technique to quantify RNA(mRNA) Similar to PCR except the progress is monitored by a camera or detector

8. What Type of Instruments are used with Real-Time PCR? Real-time PCR instruments consist of TWO main components: Thermal Cycler (PCR machine) Optical Module (to detect fluorescence in the tubes during the run)

9. Amplification Plot

10. Types of Q-PCR Hydrolyzation based Assays Taqman, Beacons, DNA-binding agents SYBR Green

11. SYBR green It is used as a dye for the quantification of double stranded DNA in some methods of quantitative PCR It is also used to visualise DNA in gel electrophoresis

12. SYBR GREEN DYE

13. SYBR Green Advantages: Relative low cost of primers. No fluorescent-labeled probes required. Disadvantages: Less specific Not possible to multiplex multiple gene targets .

14. SYBR GREEN DYE

15. Double- Dye Oligonucleotides or dual labeled probes. Consists of a ssDNA probe that is complemenatry to one of the amplicon strands A fluorophore is attached to one end of the probe and a quencher to the other end. TaqMan Probes

16. Uses for TaqMan Probes DNA Quantitation Mutation Detection-Probe designed to hybridize over mutation Gene Expression Can be multiplexed Dark Quenchers- absorb emitted energy,

17. Molecular beacons Oligonucleotide hybridization probes Report the presence of specific nucleic acids in homogenous solutions . Molecular beacons are hairpin shaped whose fluorescence is restored when they bind to a target nucleic acid sequence

18. Molecular beacon

19. Applications of molecular beacons SNP detection Real-time PCR quantification Allelic discrimination and identification Multiplex PCR assays

20. Taqman vs. SYBR Green I TaqMan Probe Advantages: Increased specificity Use when the most accurate quantitation of PCR product accumulation is desired. Option of detecting multiple genes in the same well (multiplexing ). Disadvantages: Relative high cost of labeled probe. SYBR Green Advantages: Relative low cost of primers. No fluorescent-labeled probes required. Disadvantages: Less specific Not possible to multiplex multiple gene targets.

21. Application of Q-PCR Gene expression DNA quantification Pathogen detection Mutation detection SNP detection

22. Method of quantification Absolute method Relative quantification

23. Imagining Real-Time PCR Measuring Quantities We describe the position of the lines with a value that represents the cycle number where the trace crosses an arbitrary threshold. This is called the “Ct Value”. Ct values are directly related to the starting quantity of DNA, by way of the formula: Quantity = 2^ Ct 23 25 28 Ct Values:

24. Standard curve

25. SNPs detection

26. Q-PCR vs. PCR Some of the problems with End-Point Detection : Poor Precision Low sensitivity Non - Automated Size-based discrimination only Results are not expressed as numbers