SUBMITTED TO : DR.SABA RIAZ

Submitted by :Asma Gul -34

TYPES OF PCR

What is PCR?

• PCR is a technique that takes specific sequence of DNA and amplifies it to be

used for further testing.• In vitro technique

Principle of PCR

Purpose

• to analyze a short sequence of DNA (or RNA) even in samples containing only minute quantities of DNA or RNA. PCR is used to amplify selected sections of DNA

or RNA with same (identical) size and sequence by enzymatic method and cycling condition.

CONDITION

Denaturation

• Temperature: 92-94C• Double stranded DNA melts single stranded

DNA

92C

3’5’

3’ 5’

+

5’3’

5’ 3’

Annealing

• Temperature: ~50-70C (dependant on the melting temperature of the expected duplex)

• Primers bind to their complementary sequences

5’3’

5’ 3’

Forward primer Reverse primer

Extension

• Temperature: ~72C• Time: 0.5-3min• DNA polymerase binds to the annealed primers and

extends DNA at the 3’ end of the chain

Taq

5’3’

Taq5’

Cycling

Products of Extension3’5’

3’ 5’

3’5’

3’ 5’

Taq

Taq

Overall Principle of PCR

• DNA – 1 copy

• Sequence of interest

• PCR

Chemical Components

• Magnesium chloride: .5-2.5mM

• Buffer: pH 8.3-8.8

• dNTPs: 20-200µM

• Primers: 0.1-0.5µM

• DNA Polymerase: 1-2.5 units

• Target DNA: 1 µg

Variants of PCR

RT-PCR

• reverse transcriptase polymerase chain reaction• Template:RNA• Using reverse transcriptase enzyme RNA cDNA• One step and two step RT-PCR• AMV reverse transcriptase ( Avian myoblastosis

virus)• MMLV reverse transcriptase (Moloney murine

leukemia virus) Tissue sample RNA cDNA PCR

RT-master mix

Mechanism of RT-PCR

Real time PCR

• Beside normal amplification process performed by normal PCR, Real Time PCR can perform detection, analysis and quantification of the sample.

• Detection: Find out the presence of targeted gene sequence which is assured by the presence of the amplification curve.

• Quantification: Quantification of targeted DNA in a sample can be done by using the cycle no. needed to obtain the threshold value of detector and PCR efficiency.

• Analysis: Analysis of the variants can be done by studying the melting curve or comparing the melting temperature with the sequences of the database.

Working procedure

• The working procedure of Real Time PCR can be divided in two steps:

Amplification (Same like normal PCR)

Detection

Detection

• is based on fluorescence technology• the marker added to the sample and the signal is

amplified with the amplification of copy number of sample DNA.

• emitted signal is detected by an detector

• There are many different markers used as the marker of Real Time PCR.

• There are mainly two types of marker are used for this purpose.

1.Taqman probe. 2.SYBR Green

Process of signal generation

Nested polymerase chain reaction

• is used to increase the specificity of DNA amplification• Two sets of primers are used in two successive reactions• In the first PCR, one pair of primers is used to generate DNA

products, which may contain products amplified from non-target areas.

• products from the first PCR are then used as template in a second PCR

• using one ('hemi-nesting') or two different primers whose binding sites are located (nested) within the first set, thus increasing specificity.

Multiplex polymerase chain reaction

• refers to the use of PCR to amplify several different DNA targets (genes) simultaneously

• amplifies genomic DNA samples using multiple primers and a temperature-mediated DNA polymerase in a thermal cycler.

• primer design for all primers pairs has to be optimized • so that all primer pairs can work at the same annealing

temperature during PCR.

• Some of the applications of multiplex PCR include:1. Pathogen Identification2. High Throughput SNP Genotyping3. Mutation Analysis4. Gene Deletion Analysis5. Template Quantitation6. Linkage Analysis7. RNA Detection8. Forensic Studies9. Diet Analysis

Touchdown polymerase chain reaction

• the annealing temperature is gradually decreased in later cycles.

• annealing temperature in the early cycles is usually 3-5 °C above the standard Tm of the primers

• while in the later cycles it is a similar amount below the Tm

• initial higher annealing temperature leads to greater specificity for primer binding

• while the lower temperatures permit more efficient amplification at the end

• Primers will avoid amplifying nonspecific sequences

Assembly PCR • also known as Polymerase Cycling Assembly or PCA• is a method for the assembly of large DNA

oligonucleotides from shorter fragments. • uses the same technology as PCR, but takes advantage of

DNA hybridization and annealing• as well as DNA polymerase to amplify a complete sequence

of DNA in a precise order based on the single stranded oligonucleotides

• allows for the production of synthetic genes and even entire synthetic genomes

PCA polymerase cycling assembly

Example of PCR programme

• Initial denaturation 95C for 5 mins• Thermo-cycle file - 30 cycles of• Denaturation : 95C for 30 secs• Annealing : 55C for 30 secs• Extension : 72C for 45 secs• Final extension 72C for 5 mins• Holding ( soak ) file usually 4C

Applications of PCR

Molecular Identification Sequencing Genetic Engineering

Molecular Archaeology Bioinformatics Site-directed mutagenesis Molecular Epidemiology Genomic Cloning Gene Expression Studies Molecular Ecology Human Genome Project DNA fingerprinting Classification of organisms Genotyping Pre-natal diagnosis Mutation screening Drug discovery Genetic matching Detection of pathogens