Polymerase Chain Reaction & It’s Modifications

•Hot Start PCR•Q-PCR•RT-PCR•Nested PCR

Kary Mullis

Polymerase Chain Reaction PCR is technology in molecular biology used to

amplify a single or a few copies of DNA across several orders generating thousands and millions copies of a particular sequence.

PCR takes a specific sequence of DNA of small amounts and amplifies it to be used for further testing.

It relies on thermal cycling consisting of cycles of repeated heating & cooling of the reaction for DNA melting and enzymatic replication of the DNA.

Processes1. Denaturation - It is the first step where DNA

stands are separated by heating At 950 C.2. Annealing - It is the process in which two

stands of DNA are allowed to form hydrogen bonds. (550 C – 650 C)

3. Extension- The process in which the nucleotides are added to primer by Taq polymerase. (720 C)

The basic protocol—what’s in the tube

Target DNA5’ 3’

3’ 5’

Primers (0.1-o.5uM)

A B FreeNucleotides (20-200uM)

Taq DNAPolymerase1-2.5units

Mg2+

Mg2+Mg2+

Mg2+

Mg2+

Mg2+

Buffercontainingmagnesium

(MgCl2) 0.5-2.5mM

The basic protocol--denaturation

Target DNA

95oC

5’ 3

3’ 5

5’ 3’

3’ 5

The basic protocol--annealing

~55oC

5 3’

3’ 5’

5’ 3

3’ 5’

5’5’

Target DNA

A

B

primersA

B

The basic protocol--extension

72oC

5’ 3

3’ 5’

5’

3’ 5

5’5’

Target DNA

Taq polymerase

3’

The basic protocol--extension

72oC

’5

3’ 5

5’ 3

3 5’

5’5’

Target DNA3’

MODIFICATIONS OF PCRHOT-START PCRQUANTITATIVE PCRREVERSE TRANSCRIPTION PCRNESTED PCR

HOT-START PCRIt is a modified form of PCR which avoids non-

specific amplification of DNA by inactivating Taq polymerase at lower temperature

In the second step in addition to primer and Taq polymerase we add specific antibodies to block Taq polymerase from annealing.

When temperature raises for amplification at 72o C, the specific antibodies detaches from Taq polymerase & amplification begins with greater specificity.

HOT-START PCR

Q-PCR (Quantitative-PCR)It is also known as Real Time PCRReal Time PCR is a laboratory technique of

molecular biology based on the PCR, which is used to amplify & simultaneously detect or quantify targeted DNA molecules.

This is the new approach compared to std. PCR where the product of the reaction is detected at it’s end.

Methods1. Non specific fluorescent dyes that

intercalate with any dsDNA.2. Sequence specific DNA probes consisting of

oligonucleotides that are labelled with a fluorescent with a fluorescent reporters which permits detection only after hybridization of probe with it’s complimentary sequence to quantify mRNA or non coding RNA in cells or tissue.

TaqMan Probe

Fluorescent dyes

Reverse Transcription PCRIn RT PCR, the RNA templates first converted

to complementary DNA (cDNA) using reverse transcriptase.

The cDNA is used as template for amplification of DNA by PCR

The amplification of RNA can be done with the help of ONE STEP PCR & TWO STEP PCR

NESTED PCRNested polymerase chain reaction (PCR) is a

modification of PCR intended to reduce the contamination in products due to the amplification of unexpected primer binding sites.

Nested PCR utilizes two different sets of primers during a two-step amplification.

The PCR reaction is run using the "outer primers“ during a first cycle of amplification which is immediately followed by a second cycle of amplification carried out with the "inner primers".

NESTED PCR• NESTED PCR is a variation of the PCR, using

two pairs of primers to amplify fragments of DNA.

• The first pair primer amplify a fragment similar to standard PCR.

• The second pair of primers called nested primers (because they are in the first PCR amplification of an internal fragment) incorporated inside the first PCR product, so that the second PCR amplified fragment is shorter than the first amplification.