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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.

2. Background and Concepts

The polymerase chain reaction (PCR) is a technique for amplifying DNA sequences in vitro. This method takes advantage of thermally stable DNA polymerase and can produce numerous copies (about 268,435,456) of DNAs from a single template DNA molecule through tens of repeated cycles of template denaturation, primer annealing and DNA synthesis. It is extremely sensitive to trace contamination of unwanted template DNA in the reaction solution. This method is also important in biotechnology, forensic identification, medicine and genetic research.

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.

PCR Reaction Mixture

DNA template (purified or a crude extract) Primers specific for the target DNA Free Deoxynucleotide Triphosphates DNA polymerase Buffer (containing Mg2+ )

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.

Primer Annealing Temperature

Primer annealing temperature determines the PCR specificity and yield. Higher annealing temperature should result in enhanced specificity while too high annealing temperature may affect the association of primers with template and decrease the amplification efficiency. Decreasing annealing temperature can increase the amount of PCR product, but too low annealing temperature may induce mispairing and increase nonspecific products. Optimal annealing temperature is generally 5oC lower than the melting temperature (Tm) of the primer-template DNA duplex. The approximate Tm can be calculated using the following formula: Tm= 4 (G + C) + 2 (A + T) G, C, A, T - number of respective nucleotides in the primer. At typical primer concentrations (0.2 M), the annealing process will require only a few seconds.

Primer Extension Temperature and Time

Primer extension temperature depends on the optimal temperature of DNA polymerase. Primer extension is traditionally performed at 70-75. Estimate for the rate of nucleotide incorporation at 72 varies from 35 to 100 nucleotides per second depending upon the buffer, pH, salt concentration, and the nature of the DNA template. An extension time of one minute at 72 is considered sufficient for products up to 1 kb in length. So, the extension time depends on the length of target DNA fragment. For the fragment larger than 1kb, the extension time can be set within a range of 1-7 minutes depending on the length of the fragment.

Deoxynucleotide Triphosphates (dNTPs)

In PCR reaction system, higher than 50 mM dNTP inhibits Taq activity. Excessive dNTP concentration may increase the error rate. Lowering the dNTP (10-50 uM) may reduce error rate, but too low concentration will not produce enough amount of PCR products. dNTPs on the range of 50-200 M are generally appropriate. 1015 M is minimum concentration. 4 dNTPs should be used at equivalent concentrations to minimize mis-incorporation.

Taq DNA Polymerase

Taq DNA polymerase was isolated from Thermus aquaticus, bacteria that grows in hot springs (75). A typical PCR reaction requires 2U enzyme. The common range is 14U/100 l. The enzyme requirements may vary with respect to individual target templates or primers. Excessive polymerase may increase the amount of non-specific PCR products.

Activity Unit Definition of Taq (Takara)

One unit is the amount of the enzyme that will incorporate 10 nmol of dNTP into acid-insoluble products in 30 minutes at 74oC with activated salmon sperm DNA as the template-primer.

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.

Template Denaturating Temperature

At template denaturating temperature, the double-strand DNA melts and opens into single-strand DNA. Recommended denaturating temperature ranges from 90 to 95oC. The complete denaturation of the DNA template at the start of the PCR reaction is of key importance. Incomplete denaturation of DNA results in the inefficient utilization of template in the first amplification cycle and in a poor yield of PCR product. DNA denaturation requires only a few seconds. The denaturating time should be as short as possible in order to remain the polymerase activity. The denaturating temperature should not be over 95oC, as the stability of the enzyme dramatically decreases at temperature over 95C.

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.

PCR Amplification Process

Three major steps are involved in a PCR cycle. The cycles are done on an automated cycler, which rapidly heats and cools the test tubes containing the reaction mixture. Each cycle comprises the following three steps:
Denaturation: At high temperature(90-95oC), the doublestrand DNA melts and opens into single-strand DNA. Annealing: At low temperature (35-65oC), single-strand primer binds to the single-strand template forming partial double strand. Extension: At 72oC, the DNA polymerase extends the primers by reading the opposing strand sequence and adding nucleotides. The number of target sequences is doubled after each cycle.

Denature

Annealing

Extension

Amplification

Reagents and Equipments

Reagents: TaKaRa Taq (5U/ l) dNTP Mixture (each 2.5mM) Template (10ngfrom experiment I) PrimersPrimer 1 and 2, 10 M) 10x PCR Buffer (100 mM Tris-HCl, pH8.3, 500 mM KCl, 15 mM MgCl2) Equipments: PCR system, centrifuge, Eppendorf tubes, gel electrophoresis system

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

Analysis of PCR Product

The PCR product amount and specificity are analyzed by ethidium bromide-stained 1.0 agarose gel electrophoresis using 3-5l PCR reaction mixture. The amount of PCR product can be estimated by comparison with standard DNA ladder approximately.

PCR Product
Students PCR products (4l)

3.0kb

0.8kb

Excellent!

PCR Purification Kit Protocol

For more detail, please see the instruction for PCR purification Kit.

PCR Purification Kit Protocol

TIANquick midi PCR product purification Protocal 1. Column equilibration: add 500l Buffer BL to Spin Column CB2. centrifuge for 1 min at 12,000 rpm (~13,400 X g) in a table-top micro-centrifuge. Discard the flow-through, and place Spin Column CB2 into the collection tube. 2. Add 5 (250l) volumes of buffer PB to 1 (50l ) volume of the PCR reaction and mix.

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.

Wish your experiments full success!