Polymerase Chain Reaction (PCR)

History Principle Factors Identification Application Development

Part 1 History

Problem in the molecular biology

Part 1 History
In April, 1983, Kary Mullis took a drive on a moonlit California mountain road and changed the course of molecular biology. During that drive, he conceived the Polymerase Chain Reaction (PCR).

Born on December 28, 1944 Awarded the Nobel Prize Chemistry in 1993

Part 1 History

PCR(polymerase chain reaction) is an in vitro method for enzymatically synthesizing defined sequences of DNA .

One of the first thermostable DNAPolymerases was obtained from Thermus aquaticus and called Taq.

Part 2 Principle of PCR

Part 2 Principle -- PCR components

DNA template, which contains the region of the DNA fragment to be amplified One pair of primer, which determine the beginning and end of the region to be amplified DNA-Polymerase, which catalysis the region to be amplified dNTPs, from which the DNA-Polymerase builds the new DNA Buffer, which provides a suitable chemical environment for the DNA-Polymerase Mg2+ , activate the DNA-Polymerase

Part 2 Principle--Procedure
The

PCR process consists of a series of twenty to thirty cycles. steps in one circle:

Three

Melting Annealing Elongation

Principle of PCR

Principle of PCR

Part 3 Factors
Polymerase Magnesium Concentration Template Primer dNTPs Buffer Procedure

Polymerase
Activity: pH, T, ion concentration .. Types: proofreading or not Concentration: 2.5U/100ml higher: non-special copy lower: non-enough product

Magnesium Concentration
The function of Mg2+ : activate the polymerase Too higher: non-specific copies Too lower: lower polymerase activity & reduce PCR product

Template
Quality Purification : denature; enzymatical synthesis Homogeneous: reputability Quantity: efficiency; non-special

Primer
Design Primer length 15-30bpcommon use20bp Base: GC content 40%--60% Avoid secondary structure The base on the 3end Speciality Add the enzyme recognise site

Primer
Design Length:15-30bp ; common use:20bp Base components :G+C 40%--60% Avoid second structure Add enzyme cut site The base on the 3 end Concentration 0.1-1umol/L Too higher result in no-special application

dNTPs
Quantity:adjust pH 7.0-7.5 use of 1M NaOH or 1M Tris Concentration same in four dNTPs; 50-200umol/L Combine Mg2+ -----lower the dissociative Mg2+

Buffer
Give the DNA polymerase optimal condition pH ion concentration component

Procedure
Melting temperature & time 93 94 for 30s 1 min; Higher T affect polymerase activity Annealing temperature & time 30 60 for 30s 1 min; Elongation temperature & time 70 75 (common 72 ) for 1 2 min; Higher T affect primer / template complex Cycle Depend on the temple concentration; 30-40 cycles Incubation : reform the secondary structure

Annealing Temperature &Time

Depend on the primmer length ,primer base components, primer concentration & temple length Tm =4(G+C)+2(A+T) Annealing T= Tm-(5 --10 ) In the Tm permit scale , choose higher annealing T avoid non-special copy

Taq DNA polymerase activity

70 80 150n/s/p 70 60n/s/p 55 24n/s/p

n=nucleotides; s=second; p= polymerase

Part 4 Identification

Gel electrophoresis
agrose gel electrophoresis; PAGE

Enzymolysis Molecular hybridization sequencing

Part 5 Application
Genetic Fingerprinting Paternity Testing Detection of Hereditary Diseases Cloning Genes Mutagenesis Analysis of Ancient DNA

Identify suspect
Blood from a crime scene can be genetically compared to blood from a suspect. The sample may contain only a tiny amount of DNA, obtained from a source such as blood, semen, saliva, hair, etc. Then amplifies them using PCR. The amplified fragments are then separated using gel electrophoresis. The overall layout of the DNA fragments is called a DNA fingerprint.

Electrophoresis of PCR-amplified DNA fragments.

(1) Father. (2) Child. (3) Mother. The child has inherited some, but not all of the fingerprint of each of its parents, giving it a new, unique fingerprint.

Significant Lead in Treatment.

Each gene in question can easily be amplified through PCR by using the appropriate primers and then sequence to detect mutations Viral diseases, can be detected using PCR through amplification of the viral DNA

Amplify the Gene

Cloning a gene using a plasmid. (1) Chromosomal DNA of organism A. (2) PCR. (3) Multiple copies of a single gene from organism A. (4) Insertion of the gene into a plasmid. (5) Plasmid with gene from organism A. (6) Insertion of the plasmid in organism B. (7) Multiplication or expression of the gene, originally from organism A, occurring in organism B.

Site-directed mutagenesis

Analyze DNA Thousands of Years Old

Mammoth skeleton Using PCR, it becomes possible to analyze DNA that is thousands of years old

Part 6 Development
Nested primer PCR Multiplex PCR Inverse PCR or reverse PCR Asymmetric PCR Labelled primers PCR &color complement assay Race PCR Anchored PCR Slide-PCR Reverse Transcription PCR Quantitative PCR