Real time PCR and it's functions in diagnosis

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Template DNA that is going to be amplified.this DNA must be pure & all other contaminations like RNAs must be removed by RNase.

Primers (forward & reverse )which are attached to their complementary sequence in 3´ of each strand.

Taq or any other polymerase which must have

activity in high temprature.

dNTPs which are the main substances in DNA composition.

Buffer which makes a perfect condition to process.

Cations that are essential for polymerase activity.

more…

Advantage disadvantages

Real Time PCR

Product Detection

in medicine

Conclusion

Reference

REAL TIME PCR & IT’S FUNCTIONS IN DIAGNOSIS

Introduction

Contaminations like RNAs must be removed by RNase.

Slide 8

Advantages & disadvantages

* The most accurate & feasible technique to determine the amount & concentration of products.

* Rapid cycling (30 minutes to 2 hours).

* Specific & sensitive.

* Not much more expensive.

* * * * *

* Pollution.

* Poor precision.

* Hard to get quantitative data.

Introduction

Advantage disadvantages

Real Time PCR

Product Detection

in medicine

Conclusion

Reference

REAL TIME PCR & IT’S FUNCTIONS IN DIAGNOSIS

Slide 9

Real Time PCR

The perfect standard does not exist.

Quantitative Real-Time PCR is an important technique for quantifying messenger RNA levels (gene expression) and DNA gene levels (copy number) in biological samples.(1)

Additional benefits of Real-Time quantitative PCR include sensitivity and a wide dynamic range. As few as 10 copies of an RNA/DNA target can be detected with linearity of detection greater than six orders of magnitude.

Considered to be the most sensitive method for the detection and quantification of gene expression

Introduction

Advantage disadvantages

Real Time PCR

Product Detection

in medicine

Conclusion

Reference

REAL TIME PCR & IT’S FUNCTIONS IN DIAGNOSIS

Slide 10

The Basic of Real time PCR

Baseline – The baseline phase contains all the amplification that is below the level of detection of the real time instrument.

Threshold – where the threshold and the amplification plot intersect defines CT. Can be set manually/automatically

CT – (cycle threshold) the cycle number where the fluorescence passes the threshold

Rn – (Rn-baseline)

NTC – no template control

DRn is plotted against cycle numbers to produce the amplification curves and gives the CT value.

Introduction

Advantage disadvantages

Product Detection

in medicine

Conclusion

Reference

Real Time PCR

REAL TIME PCR & IT’S FUNCTIONS IN DIAGNOSIS