Download - Types of pcr
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