PCR
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A test tube system for amplification or enrichment of a specific nucleic acid segment
PCR (Polymerase chain reaction)
Who developed PCR?
Kary Mullis and Michael Smith
Applications of PCR
• Detects DNA sequences • Diagnoses genetic disease • Carries out DNA fingerprinting • Detects bacteria or viruses • Determines the molecular phylogenetics of organisms
Two phases of PCR
a. Screeening phase b. Amplification phase
During the first few cycles the appropriate part of the template is selected by speicfic binding and extension of the primers
Screening phase
During the sebsequent rounds of PCR the copy number of the desired sequence increases exponentially
Amplification phase
Screening phase: primer design - Requirement - Verification
• Requirements: have precise sequence matching only the desired target region • Verification- primer sequences must be checked against databases to ensure homology
Key parameters for Amplification phase of PCR that influences the reaction mix
• Buffer • dNTP conc. • primer conc. • MgCl2 conc. • Enzyme conc. • Template conc.
The three general steps of PCR
• Denaturation • Annealing • Extension or Elongation
The seperation of the strands
Denaturation
Annealing the primer to the template
Annealing
The exteion/elongation of the new strands
Extension or Elongation
Other types of PCR
• Reverse Transciption (RT-PCR) • Rapid Amplification of cDNA ends (RACE-PCR) • Real-time PCR (rt-PCR) • qRT-PCR
Goal of RT-PCR
Detect, or clone the coding sequence of a known gene
Primary goal of RACE-PCR
To map and Obtain the complete 5’ and 3’ ends of an mRNA transcript
Two main types of RACE-PCR
5’ Race 3’ Race
Used to identify the start of the mRNA (the 5’ end, including the transcription start site and the start codon).
5’ RACE
Used to identify the end of the mRNA (the 3’ end, including the stop codon and the poly-A tail).
3’ RACE
a way of quantifying the amplification of DNA as it occurs real time
real-time PCR (rt-PCR)
a type of dye that binds the minor groove
SYBR green dye
A dye that is used in fluoroghenic 5’ nuclease assay
TaqMan probe
A way to quantifying the amplication of DNA as it occurs real time • the amount of DNA is measured after each cycle via fluorescent dyes that yield increasing signal
Real-time PCR
Advanatage of real-time PCR
• ability to monitor the progress of the PCR reaction in real time • highly accurate quantification • eliminates post