Real time PCR and it's functions in diagnosis

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REAL TIME PCR & IT’S FUNCTIONS IN DIAGNOSIS

Real Time PCR

Slide 15

Real time PCR is the most accurate method to detect(2) :

Copy number of each gene

Amount of gene expression

Efficiency of drugs

Virus infection

Different type of Pathogens

( CMV, streptococcus, mycobacterium,HIV & … )

Methylation of DNA

Different type of mutations

Adverse effect of organ transplantation

& …

Introduction

Advantage disadvantages

Product Detection

in medicine

Conclusion

Reference

REAL TIME PCR & IT’S FUNCTIONS IN DIAGNOSIS

Real Time PCR

Slide 16

Uses fluorescence as a reporter by Three general methods :

1. Hydrolysis probes

(TaqMan, Beacons, Scorpions)

2. Hybridization probes

(Light Cycler) most accurate & specific.

3. DNA-binding agents

(SYBR Green)less accuracy.

Detection in real time PCR

Introduction

Advantage disadvantages

Real Time PCR

Product Detection

in medicine

Conclusion

Reference

REAL TIME PCR & IT’S FUNCTIONS IN DIAGNOSIS

Slide 17

* DNA binding dye

* Binds to minor groove (dsDNA)

* Emits light when bound

More double stranded DNA = more binding = more fluorescence

* Forensically, can be used to calculate how much DNA was present before reaction.

* Unspecific

* Dissociation curve

SYBR® green

Introduction

Advantage disadvantages

Real Time PCR

Product Detection

in medicine

Conclusion

Reference

REAL TIME PCR & IT’S FUNCTIONS IN DIAGNOSIS

Slide 18

Dissociation curve

Fluorescence decrease as the temperature increase:

DNA strands start to separate

SYBR green looses its binding to the DNA

Fluorescence decreases

The melting temperature of the amplicon can easily be detected

Contaminating DNA, primer dimer or false priming is seen as an additional peak.

This curve lets us to detect non-specific products.

Introduction

Advantage disadvantages

Real Time PCR

Product Detection

in medicine

Conclusion

Reference

REAL TIME PCR & IT’S FUNCTIONS IN DIAGNOSIS

Slide 19

1)

2)

3)

1) Denaturation and hybridization of probe.

2) Extension of primer and strand displacement of probe.

3) Cleavage of probe and fluorescence from the reporter dye.

Fluorescence from reporter dye is directly proportional to the number of amplicons generated

REAL TIME PCR & IT’S FUNCTIONS IN DIAGNOSIS

Introduction