Khuda BakhshFA14-R02-002

Q-PCR

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

What is PCR?

Stands for Polymerase

Chain Reaction

For the amplification of

DNA fragments

Purpose of PCR

Easy to sequence more copies

For comparing DNA fragments

It is used to clone specific genes

Material: PCR reagents:

A DNA template:

Primers

10x PCR buffer

dNTPs

Taq polymerase

MgCl2

Steps of PCRDenaturation:

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:

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

What Type of Instruments are

used with Real-Time PCR?

Real-time PCR instruments consist of TWO main

components:

1. Thermal Cycler (PCR machine)

2. Optical Module (to detect fluorescence in the tubes

during the run)

Amplification Plot

Types of Q-PCRHydrolyzation based Assays

Taqman,

Beacons,

DNA-binding agents

SYBR Green

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

SYBR GREEN DYE

SYBR Green

Advantages: Relative low cost of primers. No fluorescent-labeled probes

required.

Disadvantages: Less specific Not possible to multiplex multiple gene

targets.

SYBR GREEN DYE

Double- Dye Oligonucleotides or dual labeled probes.

Consists of a ssDNA probe that is complemenatry to one of the ampliconstrands

A fluorophore is attached to one end of the probe and a quencher to the other end.

TaqMan Probes

Uses for TaqMan Probes

DNA Quantitation

Mutation Detection-Probe designed to

hybridize over mutation

Gene Expression

Can be multiplexed

Dark Quenchers- absorb emitted energy,

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

Molecular beacon

Applications of molecular beacons

SNP detection

Real-time PCR quantification

Allelic discrimination and identification

Multiplex PCR assays

Taqman vs. SYBR Green ITaqMan Probe

Advantages:

Increased specificity

Use when the most accuratequantitation of PCR productaccumulation is desired.

Option of detecting multiplegenes in the same well(multiplexing).

Disadvantages:

Relative high cost of labeledprobe.

SYBR Green

Advantages:

Relative low cost ofprimers.

No fluorescent-labeledprobes required.

Disadvantages:

Less specific

Not possible tomultiplex multiple genetargets.

Application of Q-PCR

Gene expression

DNA quantification

Pathogen detection

Mutation detection

SNP detection

Method of quantification

Absolute method

Relative quantification

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

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

5000000

0 5 10 15 20 25 30 35 40

23 2528

Ct Values:

Standard curve

SNPs detection

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