Adapted from a presentation written forPrinciples of Gene Manipulation · November 6, 2000

Jennifer Cooper · America Madrigal · Laleña Vellanoweth

AMPLIFICATION:The Polymerase Chain Reaction

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AMPLIFICATION: PCR and Its Applications

I. Definition of PCR

II. Requirements for PCR

III. PCR Process

A. Denaturation

B. Annealing

C. Extension

D. Cycling (repeat A-C)

IV. PCR for HLA DQ-alpha

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PCR What is it?

The Polymerase Chain Reaction (PCR) is an

in vitro method to amplify a specific region of DNA.

PCR is extremely sensitive, with the capability

of amplifying minuscule quantities of DNA.

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PCR REQUIREMENTS In the reaction

Sample – template Primers High temperature resistant polymerase; e.g., Taq Deoxynucleotide triphosphates – dNTPs

(dATP, dGTP, dCTP, dTTP) Buffer Mg++, KCl

Thermocycler – instrument programmed to change samples rapidly from one set temperature to another

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PCR METHOD

There are three basic steps in PCR

1. Denaturation (~95oC)

2. Annealing (~55oC, but varies)

3. Extension (~72oC)

Cycling repeats Steps 1-3 up to 35 times.

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PCR METHOD – DENATURATION STAGE

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(reference: library.thinkquest.org/24355/data/light/details/media/polymeraseanim.html)

High temperature separates the two strands.

PCR METHOD – ANNEALING STAGE

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(reference: library.thinkquest.org/24355/data/light/details/media/polymeraseanim.html)

Primer length is usually ~20 nucleotides.

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PCR METHOD – EXTENSION STAGE

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(reference: library.thinkquest.org/24355/data/light/details/media/polymeraseanim.html)

Thermostable polymerase adds dNTPs one at a time at this stage.

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PCR METHOD - CYCLING

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(reference: library.thinkquest.org/24355/data/light/details/media/polymeraseanim.html)

The average number of cycles = 30 for efficiency reasons. 230 = 1.07 X 109 copies

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PCR METHOD - CYCLING

DNA

Cycle 1 2 3 4 5 6 …Products 2 4 8 16 32 …

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After the first two cycles, fragments with the correct length begin to be amplified. (dictated by the placement of the primers)

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PCR for HLA DQ-alpha

Biotinylated primers amplify a region of ~250 bp. Sequence of primers is common to all individuals. Sequence between primers is polymorphic – may differ between

chromosomes and among individuals. PCR picks out the 250 bp HLA DQ- α region from 3.3 X 109 bp of DNA

present in 23 chromosomes. Note that in the kit we are using there are actually six pairs of biotinylated

primers (12 primers total). Only 1 pair will amplify the HLA DQ- α region The other five pairs of primers amplify genes that we are not interested

in for this class. The analysis for these genes employs a different probe strip than the HLA DQ- α probe strip that we will be using.

After agarose gel electrophoresis six bands will be seen, only one of which is the band of interest for this class.

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The human male karyotype: 22 homologous chromosomes + X and Y

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Products of PCR reaction >106 double strand fragments ~ 250 bp in length.

All are biotinylated (biotinylated primers). 1/2 = DQ-α allele on one copy of your Chromosome 6;

1/2 = DQ-α allele on the other copy.

PCR for HLA DQ-alpha

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PCR REFERENCES

1. Gene Cloning: an introduction. T.A. Brown

2. The world wide web:

http://sunsite.berkeley.edu/PCR/whatisPCR.html

http://www.accessexcellence.org/AB/GG/polymerase.html

http://www.sciam.com/1998/0598issue/0598working.html

http://www.faseb.org/opar/bloodsupply/pcr.html

http://faculty.plattsburgh.edu/donald.slish/PCR.html

http://library.thinkquest.org/24355/data/light/details/media/polymeraseanim.html

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After a long process, this PowerPoint presentation has now ended. We hope it is of good help in your studies.

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PCR RequirementsThe Details

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PCR REQUIREMENTS: sample - template Amount

Needed very small; intact DNA from one cell to see on a gel, need 1011 final copies;

need 104 starting copies Concern –competition with primers for annealing by

Too much starting template Too much product from excessive cycling

Remember, the association rate of two strands increases with the square root of the length of the DNA. Longer strands anneal more quickly than shorter. So . . .Templates and products are longer than primers. In high concentration, they reanneal before primers can anneal.

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PCR REQUIREMENTS: sample - template

Even degraded DNA is OK if sample is large enough Fossils Remains Old samples from crime scenes

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Two primers of known sequence flank region you are interested in anneal to opposite strands of template prime toward the region between them non-complementary to each other lack internal complementarity of sufficient length to anneal to unique site in the genome

(~20 nt) 1/420 = 1 site of identical sequence/1 X 1012 bp

chance that any one of the 4 bases will be at a given site = 1/4 have similar annealing temperatures present in excess (0.1 – 1.0 uM each)

favor annealing of primer over reannealing of strands sufficient for amplification through 25-30 cycles

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PCR REQUIREMENTS - dNTPs

• must be present in sufficient excess to complete extension through all cycles (~200 M in each dNTP)

• must not be present in such high excess that Mg++ ions are complexed and unavailable as cofactors for polymerase activity

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PCR REQUIREMENTS - “buffer” components

• The buffer, e.g., Tris base adjusted to a specific pH with HCl– maintains pH

• pH 8.3 is optimum for Taq.

• pH optimum keeps protein folded in a conformation at which it is enzymatically active.

• Different temperature-insensitive polymerases have different pH optima

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PCR REQUIREMENTS - “buffer” components

• Monovalent salt, e.g., KCl– to contribute to correct folding of enzyme and

thereby – to contribute to optimum activity of enzyme

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PCR REQUIREMENTS - “buffer” components

• Mg++ (MgCl2, MgSO4)

– Mg++ is a cofactor for DNA polymerases– for Taq, required free [Mg++] = ~2mM– calculated [Mg++] may differ from the actual free

[Mg++]• Positively charged Mg++ is complexed by ionic bonding

with the negative charges on primers, template, and dNTPs

• Mg++ must be uncomplexed (free) to act as a cofactor for the polymerase

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PCR REQUIREMENTS - “buffer” components

• Mg++ (cont’d)– Determining the optimal concentration of Mg++

is the most important step in setting up PCR conditions

• too little - polymerase can’t work

• too much - favors annealing of primers to mismatched locations on the template

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PCR REQUIREMENTS - polymerase

• 1-2 units of enzyme/100 l reaction

• High temperature resistant– able to remain active through up to 35 cycles

with DNA denaturation at 95oC– Example: Taq polymerase

• isolated from Thermophilus aquaticus

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PCR REQUIREMENTS - polymerase (cont’d)

• Many different polymerases available– some have both

polymerase and editing (exonuclease) activities

Pfu polymerase can edit. Taq polymerase can not.

Taq polymerase is more likely to misincorporate a dNTP.

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PCR REQUIREMENTS - polymerase (cont’d)

• Many different polymerases available– leave different types of ends

• blunt

• single A 3’overhang

– each isolated from a different organism which has evolved to survive at high temperatures

• deep water vents

• hot springs

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PCR REQUIREMENTS - denaturation of dsDNA

• Denaturation temperature– high enough to overcome attractive energy of H-

bonds between bases of the complementary template and product strands

– 95oC provides sufficient energy to separate even long strands

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PCR REQUIREMENTS - annealing

• The lower the temperature, the easier it is for 2 strands of DNA to pair with each other

• So the chosen annealing temperature must be– High enough to prevent hybridization of primers

to imperfectly complementary template sequences (i.e., non-specific annealing)

– Not so high that the primers can’t anneal to template DNA at all

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PCR REQUIREMENTS - annealing

– What determines the optimum annealing temp• length of primer - the longer, the higher the optimum annealing temp will

be– longer the primer, the more H-bonds– So, the more H-bonds, more likely 2 strands are to anneal or stay annealed

• % GC - the more GC, the higher the optimum annealing temp will be– GC base pairs have 3 H-bonds; AT base pairs have only 2– So, the more GC, more likely 2 strands are to anneal or stay annealed

• the the [salt], the the optimum annealing temp will be– positive ions in salt are counterions to the negatively charged sugar-phosphate

backbone of the ds DNA– positive counterions prevent repulsive forces of negative charges from pushing the

strands apart

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PCR REQUIREMENTS - annealing

• Annealing temperature for PCR is often set at 5oC below the Tm

• Tm = temperature at which 50% of the possible correct primer/template complexes are unformed

• Estimate Tm for primers 10-23 nt long in 1M salt

Tm (oC) = 4 (G+C) + 2 (A+T)

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Hot start

• Way to minimize early non-specific annealing that causes– primer dimers– amplification of incorrect product

Why might early annealing be expected to be non-specific?

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Hot start

• Enzyme is not mixed with reaction until sample has reached denaturation temperature – manual addition of enzyme at 95C– polymerase separated from other reagents by layer

of solid wax• wax melts at denaturation temperature, polymerase mixes

with reagents, wax rises to top and prevents evaporation

– start with antibody/polymerase complex • antibody denatured and releases enzyme at 95C

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PCR REQUIREMENTS - extension

• Note: the longer the expected product, the longer the extension time required– exact time depends on rate of progression of the

specific polymerase

• Extension temperature– optimal temperature for enzyme– determined for each enzyme empirically– usually around 72oC

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PCR REQUIREMENTS

• Thermocycler - instrument programmed to change samples rapidly from one set temperature to another

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PCR REQUIREMENTS

• Way to prevent evaporation of water from reaction at high temperatures– Why?

• evaporation raises concentrations of reaction solutes inhibition of reaction

– How?• Thermocycler applies heat to the top of the reaction

tube, thereby preventing condensation

• or, overlayer the reaction with mineral oil, preventing evaporation

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PCR REQUIREMENTS - thermocycler

• Way to heat and cool the sample– Solid heating/cooling block that holds samples

• Efficient conduction of heat between heating/cooling block and sample– pressure applied from top pushes walls of tube

directly against block, eliminating air space, or– mineral oil is used to fill in air space between

heating block and sample

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PCR REQUIREMENTS - controls

• No template control– should be no product– if there is, contaminating DNA is present

• Known positive (if possible)– should be a product– tells you all reaction components are working– may tell you what your product should look like

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PCR REQUIREMENTS - controls (cont’d)

• Size markers – show

• what size your product is

• and if you know what size to expect, whether you are getting the expected product

• Results usually analyzed by gel electrophoresis

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PCR REQUIREMENTS -

Minimizing Contamination

• Contamination of pipettors– Use aerosol barrier pipet tips

• Contamination of supplies and reagents– UV irradiation – base/acid treatment of reusable supplies

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PCR REQUIREMENTS -

Minimizing Contamination (cont’d)

• Contamination of work area with sample or product– Perform steps at separated benches or rooms

• sample prep

• reaction set up

• thermocycling

• product analysis

– Prevent aerosols containing PCR products• centrifuge reagents and products before opening tube

– also prevents contamination of reagents from gloves

• uncap tubes carefully

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PCR REQUIREMENTS

Minimizing Contamination (cont’d)

• Contamination of reaction mix– Use aerosol barrier

pipettor tips– Use distilled

deionized water– Add DNA to the

reaction last

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PCR REQUIREMENTS -

Troubleshooting• No yield

• Extra or incorrect products

• Primer dimers

• Misincorporation

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PCR REQUIREMENTS -Troubleshooting

• No yield– Were all reagents included?

– Insufficient denaturation?• Higher temp• Check conditions for transfer of heat from block to tube

– Active nucleases or proteases present in rx?

– Insufficient free Mg++?

– Bad primers?• Degraded• Wrong sequence

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Troubleshooting

• Extra or incorrect products– Mispriming

• annealing temp too low

• may need Hot Start

• too much Mg++; facilitates misannealing

• primer sequence insufficiently specific

– [dNTP] too high– Too much polymerase– Annealing and/or extension time too long– Too many cycles; rare misprimed products become amplified– Template contamination

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PCR REQUIREMENTS -

Troubleshooting• Primer dimers

– What are they?

5’---------------------AT3’3’TA----------------------5’

- Visible below the 100 bp marker on gel- Can appear even when 3’ ends are not

complementary

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PCR REQUIREMENTS -

Troubleshooting• Primer dimers

– Causes• Primer excess too great

• 3’ primer complementarity

• Insufficient target template

• Too many cycles

• Annealing temperature too low– Hot start may be required to avoid initial primer/primer

annealing.

• Primers insufficiently specific (too short)

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PCR REQUIREMENTS -

Troubleshooting

• Misincorporation– Excess of dNTPs too great– dNTPs present in unequal concentrations; one is

exhausted before others– Polymerase lacking exonuclease (editing) activity– Polymerase concentration too high– Extension temperature too low– [Mg++] too high

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Lane #1: no template.2: + DNA.3-5: 100X DNA, [Mg++]6,8: hair root lysate7,9: hair shaft lysate

Effects of [template] and [Mg++]on PCR products

Rx in lanes 1-5 were performed with primers.Rx in lanes 6-9 were performed with HLA-DQ alpha primers.