tsinghua pcr slide

8/13/2019 Tsinghua PCR Slide

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Experiment 3

The Polymerase Chain Reaction (PCR)

All rights reserved


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1. Objectives

Learn the principle and method of the

Polymerase Chain Reaction (PCR).

Comprehend the significance of PCRtechnique in DNA manipulation.


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2. Background and Concepts

The polymerase chain reaction (PCR) is a technique foramplifying DNA sequences in vitro. This method takesadvantage of thermally stable DNA polymerase and canproduce numerous copies (about 268,435,456) of DNAs from a

single template DNA molecule through tens of repeated cyclesof template denaturation, primer annealing and DNAsynthesis. It is extremely sensitive to trace contamination ofunwanted template DNA in the reaction solution.

This method is also important in biotechnology, forensicidentification, medicine and genetic research.


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PCR was invented by Kary Mullis and his colleagues in 1985.The discovery of Taq DNA polymerase, thermally stableenzyme isolated by Chien et al. in 1976, made the PCRautomation possible. In 1987, Kary Mullis et al. accomplished

the PCR automation system which made PCR practical. KaryMullis was awarded the 1993 Nobel Prize in Chemistry forinventing PCR. PCR has played a major role in the HumanGenome Project. The technique has also become invaluable inbiotechnology, medicine, disease diagnosis, forensic-scienceanalysis in convicting the guilty and freeing the falsely accused,and the study of DNA from ancient or fossil tissues.


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Applications of PCR

Gene cloning and quantification, gene mutation,DNA sequencing, and amplifying specific sequencefor 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


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Principle

Similar to DNA denaturation and renaturation at high temperature(93-95), the target double-strand DNA can be separated intosingle-strand DNA. At low temperature (37-65), two artificialoligonucleotides will anneal to the complementary sequence in

the template forming partial double strand. At 72, Taqpolymerase synthesizes new strand by extending the primersalong the direction from 5 to 3. The number of the sequences

between the primers will be doubled after this cycle. The cyclecan be repeated as the newly synthesized DNA strands can serve

as templates in the next cycle. So, the amount of target sequencedramatically increases. The amplification coefficient can beabove 109 theoretically after 25-30 cycles or 106-107practically.


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PCR Reaction Mixture

DNA template (purified or a crude extract)

Primers specific for the target DNA

Free Deoxynucleotide Triphosphates DNA polymerase

Buffer (containing Mg2+ )


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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.


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Primer Design

The number of nucleotides in a primer is usually 16-30nt. Apreferable number is 20-24nt. Sometimes restriction sitesand enhancer factors, which are not complementary totemplate, can be added to primer 5 ends in order to

accomplish gene cloning and other special tasks. Under thiscondition, additional 3-4 nucleotides as flanking sequencespacer should be added to 5 end for efficient digestion.


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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 desiredproduct. The C and G nucleotides should distribute uniformlythroughout the primer. Long stretches of any one base should

be avoided. More than three G or C nucleotides at the 3'-end ofthe primer should be avoided, as nonspecific priming mayoccur.

The melting temperature of flanking primers should not differby 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 hairpinstructures.


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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 accomplish30 amplification cycles and will decrease the PCR yield.


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Primer Annealing Temperature

Primer annealing temperature determines the PCR specificity andyield. Higher annealing temperature should result in enhanced specificitywhile 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 andincrease nonspecific products.

Optimal annealing temperature is generally 5oC lower than the meltingtemperature (Tm) of the primer-template DNA duplex. The approximateTm 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 typicalprimer concentrations (0.2 M), the annealing process will require only afew seconds.


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Primer Extension Temperature and Time

Primer extension temperature depends on the optimaltemperature of DNA polymerase. Primer extension is traditionally

performed at 70-75. Estimate for the rate of nucleotideincorporation at 72 varies from 35 to 100 nucleotides per seconddepending upon the buffer, pH, salt concentration, and the nature of

the DNA template. An extension time of one minute at 72 isconsidered sufficient for products up to 1 kb in length. So, theextension time depends on the length of target DNA fragment. Forthe fragment larger than 1kb, the extension time can be set within arange of 1-7 minutes depending on the length of the fragment.


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Deoxynucleotide Triphosphates (dNTPs)

In PCR reaction system, higher than 50 mM dNTPinhibits Taq activity. Excessive dNTP concentration mayincrease 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 therange of 50-200 M are generally appropriate. 1015 Mis minimum concentration. 4 dNTPs should be used atequivalent concentrations to minimize mis-incorporation.


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Taq DNA Polymerase

Taq DNA polymerase was isolated from Thermus aquaticus,bacteria that grows in hot springs (75). A typical PCRreaction requires 2U enzyme. The common range is 14U/100l. The enzyme requirements may vary with respect to

individual target templates or primers. Excessive polymerasemay increase the amount of non-specific PCR products.


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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.


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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 templatedirectly 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 DNAfrom a single cell may work for a PCR reaction. It isrecommended to use ng level template in order to assure the

PCR specificity.


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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 denaturationof 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.


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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 pHdecreases 1 unit, closing 7.2. The divalent cation is essentialwhich may affect PCR specificity and product amount. Mg2+

is better than Mn2+

, while Ca2+

has no effect. Reducedconcentration can prevent non-specific and undesirable PCRproducts. Increased concentration can attain more product.The concentration of Mg2+ should be optimized at first timeof association of target sequence and primers. It is within arange of 1-10 mM generally.


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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 achieveacceptable yield of PCR product and lower background of

non-specific product.


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PCR Amplification Process

Denaturation: At high temperature(90-95oC), the double-strand DNA melts and opens into single-strand DNA.

Annealing: At low temperature (35-65oC), single-strandprimer binds to the single-strand template forming partialdouble strand.

Extension: At 72oC, the DNA polymerase extends theprimers by reading the opposing strand sequence and addingnucleotides. The number of target sequences is doubled aftereach cycle.

Three major steps are involved in a PCR cycle. Thecycles are done on an automated cycler, which rapidlyheats and cools the test tubes containing the reactionmixture. Each cycle comprises the following three steps:


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Denature


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Annealing


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Extension


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Amplification


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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, gelelectrophoresis system


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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.


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In

sert

EcoR1

Xho1

1.9kb

pCMV-Myc-SIAPR


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BamH1

New P15TTC C G GAT CCC ACC ATGGCA TCA ATG CAG AAG CMyc tag

HindIII

New P2: 5TCG CAA GCT TAG TGG CAT CAG AGA CTT GCT AAT C

Tm=4GC2AT 412211700C.

Tm=4GC2AT411213700C.

Primer 1(From pCMV-Myc827-847):

Primer 2(From T10AK076127.1| 1249-1270):
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Nucleotide&list_uids=26345109&dopt=GenBankhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Nucleotide&list_uids=26345109&dopt=GenBank


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PCR Mixture:

Template DNA 1 l (10ng)

Primer 1 1 l (10M) (10 l (1M) )Primer 2 1 l (10M) (10 l (1M) )dNTPs 4 l (2.5mM)

TaqDNA polymerase 0.5 l(5U/l)10bufferMg+ 5 lddH2O 37.5l (19.5 l)

50 l


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1. Denaturation 94,5 minutes

2. 30 cycles of:

Denaturation 94,45 secondsAnnealing 65,45 seconds

Extension 72,1 minutes

3. Final extension 72,7 minutes

4. Chilling to 4

PCR Cycles


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Analysis of PCR Product

The PCR product amount and specificity are analyzed byethidium bromide-stained 1.0 agarose gel electrophoresisusing 3-5l PCR reaction mixture. The amount of PCR

product can be estimated by comparison with standard DNAladder approximately.


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PCR Product

3.0kb

0.8kb

Students PCR products (4l)

Excellent!


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PCR Purification Kit Protocol

For more detail, please see the instruction for PCR

purification Kit.


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PCR Purification Kit Protocol

TIANquick midi PCR product purification Protocal1.Column equilibration: add 500lBuffer 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 itstand 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.
http://localhost/var/www/apps/conversion/tmp/scratch_1/PCR%20purification%20Kit.pdfhttp://localhost/var/www/apps/conversion/tmp/scratch_1/PCR%20purification%20Kit.pdf


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4.To wash, add 600lBuffer 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 lbuffer 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.


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Wish your experiments

full success!

tsinghua pcr slide

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