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Experiment 3
The Polymerase Chain Reaction (PCR)
1. Objectives
Learn the principle and method of the Polymerase Chain Reaction (PCR). Comprehend the significance of PCR technique in DNA manipulation.
PCR was invented by Kary Mullis and his colleagues in 1985. The discovery of Taq DNA polymerase, thermally stable enzyme isolated by Chien et al. in 1976, made the PCR automation possible. In 1987, Kary Mullis et al. accomplished the PCR automation system which made PCR practical. Kary Mullis was awarded the 1993 Nobel Prize in Chemistry for inventing PCR. PCR has played a major role in the Human Genome Project. The technique has also become invaluable in biotechnology, medicine, disease diagnosis, forensic-science analysis in convicting the guilty and freeing the falsely accused, and the study of DNA from ancient or fossil tissues.
Applications of PCR
Gene cloning and quantification, gene mutation, DNA sequencing, and amplifying specific sequence for probing Antepartum diagnosis of genetic diseases Detection of infectious pathogens Detection and diagnosis of cancer genes Forensic medicine, DNA fingerprinting, individual identification, parentage identification Quarantine Detection of the target gene in transgenic organism
Principle
Similar to DNA denaturation and renaturation at high temperature (93-95 ), the target double-strand DNA can be separated into single-strand DNA. At low temperature (37-65 ), two artificial oligonucleotides will anneal to the complementary sequence in the template forming partial double strand. At 72 , Taq polymerase synthesizes new strand by extending the primers along the direction from 5 to 3. The number of the sequences between the primers will be doubled after this cycle. The cycle can be repeated as the newly synthesized DNA strands can serve as templates in the next cycle. So, the amount of target sequence dramatically increases. The amplification coefficient can be above 109 theoretically after 25-30 cycles or 106-107 practically.
Primers
Two primers should be designed and synthesized before amplification. They are complementary to the both ends of target DNA sequence respectively. These two primers determine the length and locus of the amplified fragment. So, the design of primer is very important.
Primer Design
The number of nucleotides in a primer is usually 16-30nt. A preferable number is 20-24nt. Sometimes restriction sites and enhancer factors, which are not complementary to template, can be added to primer 5 ends in order to accomplish gene cloning and other special tasks. Under this condition, additional 3-4 nucleotides as flanking sequence spacer should be added to 5 end for efficient digestion.
Complementation of primer pairs, especially at the 3' ends, should be avoided as this may promote the formation of primer-dimer artifacts and reduce the yield of the desired product. The C and G nucleotides should distribute uniformly throughout the primer. Long stretches of any one base should be avoided. More than three G or C nucleotides at the 3'-end of the primer should be avoided, as nonspecific priming may occur. The melting temperature of flanking primers should not differ by more than 5, so the GC content and length must be determined accordingly. The primer should not be self-complementary in order to avoid formation of internal secondary structure, especially hairpin structures.
Primer Concentrations
Primer concentration between 0.1 and 1 M is generally optimal. Higher primer concentration may easily generate primer-dimer and nonspecific product. Lower primer concentration may result in higher specificity, but too low concentration is not sufficient to accomplish 30 amplification cycles and will decrease the PCR yield.
Template
PCR can be performed using ssDNA, dsDNA or cDNA (reverse transcribed from RNA) as template. The linearized plasmid is preferred when plasmid serves as template. However, the circular plasmid can be also used as template directly in most cases. Digestion with appropriate enzyme may give better amplification result when the template is long (e.g. genomic DNA). The amount of DNA template can be so traced that the DNA from a single cell may work for a PCR reaction. It is recommended to use ng level template in order to assure the PCR specificity.
PCR Buffer
A standard buffer for PCR is 10-50 mM Tris-HCl (pH 8.3) and 1.5mM MgCl2. At 72, the reaction system pH decreases 1 unit, closing 7.2. The divalent cation is essential which may affect PCR specificity and product amount. Mg2+ is better than Mn2+, while Ca2+ has no effect. Reduced concentration can prevent non-specific and undesirable PCR products. Increased concentration can attain more product. The concentration of Mg2+ should be optimized at first time of association of target sequence and primers. It is within a range of 1-10 mM generally.
Cycle Number
The number of PCR cycles is generally 25-40. Too many cycles can increase the amount and complexity of nonspecific background products. Of course, too few cycles give low product yield. So, it is recommended that the lowest possible number of cycles should be used to achieve acceptable yield of PCR product and lower background of non-specific product.
Denature
Annealing
Extension
Amplification
This experiment amplified a piece of about 800 bp which contains a mouse gene SIPAR (previous code is T10) from the plasmid pCMV-Myc-SIPAR.
pCMV-Myc-SIAPR
EcoR1
Insert
1.9kb
Xho1
Primer 1(From pCMV-Myc827-847): New P15TTC C G GAT CCC ACC ATG GCA TCA ATG CAG AAG C BamH1 Myc tag Tm=4GC2AT 412211700C.
Primer 2(From T10AK076127.1| 1249-1270): New P2: 5TCG C AA GCT TAG TGG CAT CAG AGA CTT GCT AAT C HindIII Tm=4GC2AT411213700C.
PCR Mixture:
Template DNA
Primer 1
1 l (10ng)
1 l (10M) (10 l
(1M) ) (1M) )
Primer 2
dNTPs Taq DNA polymerase 10bufferMg+ ddH2O
1 l (10M) (10 l
4 l (2.5mM) 0.5 l(5U/l) 5 l 37.5l (19.5 l)
50 l
PCR Cycles
1. Denaturation 94 ,5 minutes 2. 30 cycles of: Denaturation 94 ,45 seconds Annealing 65 ,45 seconds Extension 72 ,1 minutes 3. Final extension 72 ,7 minutes 4. Chilling to 4
PCR Product
Students PCR products (4l)
3.0kb
0.8kb
Excellent!
For more detail, please see the instruction for PCR purification Kit.
3. Transfer the mixture to the Spin Column CB2. Let it stand for 2 min at room temperature. Centrifuge for 30s at 12,000 rpm (~13,400 X g) in a microcentrifuge. Discard the flow-through, and then place Spin Column CB2 back into the same collection tube.
4. To wash, add 600 l Buffer PW to the Spin Colum CB2 and centrifuge for 30s at 12,000 rpm (~13,400 X g). Discard the flow-through, and place Spin Column CB2 back in the same collection tube. 5. Repeat step 4 one more time, and centrifuge for an additional 2 min to remove residual wash buffer PW.
6. Place the Spin Column CB2 in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 40 l buffer EB to the center of membrane, let the column stand for 2 min, and centrifuge for 2 min at 12,000 rpm (~13,400 X g) For more detail, please see the instruction for PCR purification Kit in PDF fromat.