Method: Polymerase Chain Reaction

Terry C. Lairmore

Principle:

The polymerase chain reaction (PCR) is a method for oligonucleotide primer directed enzymatic amplification of a specific DNA sequence of interest. This technique is capable of amplifying a sequence 105 to 106-fold from nanogram amounts of template DNA within a large background of irrelevant sequences (e.g. from total genomic DNA). A prerequisite for amplifying a sequence using PCR is to have known, unique sequences flanking the segment of DNA to be amplified so that specific oligonucleotides can be obtained. It is not necessary to know anything about the intervening sequence between the primers. The PCR product is amplified from the DNA template using a heat-stable DNA polymerase from Thermus aquaticus (Taq DNA polymerase) and using an automated thermal cycler (Perkin-Elmer/Cetus) to put the reaction through 30 or more cycles of denaturing, annealing of primers, and polymerization. After amplification by PCR, the products are separated by polyacrylamide gel electrophoresis and are directly visualized after staining with ethidium bromide.

Time required:

1. 1-2 Days
2. PCR reaction: 3-6 hours or overnight
3. Polyacrylamide gel electrophoresis using "Mighty-small II" gel apparatus: 2.5 hours
4. Ethidium bromide staining and photography: 45 minutes

1. Synthetic oligonucleotide primer pair flanking the sequence to be amplified
2. 5X PCR Buffer (250 mM KCl, 50 mM Tris-HCl pH 8.3, 7.5 mM MgCl2)
3. Mixture of four dNTPS (dGTP, dATP, dTTP, dCTP) each at 2.5 mM (Ultrapure dNTP set, Pharmacia #27-2035-01). The dNTP mixture is made by adding equal volumes of a 10 mM solution of each of the four separate dNTPs together.
4. Taq DNA Polymerase (AmpliTaqTM, Perkin-Elmer/Cetus)
5. Light mineral oil
6. Acrylamide (electrophoresis grade)
7. N,N'-Methylenebisacrylamide (electrophoresis grade, Ultra-Pure/BRL, #5516UB)
8. Ammonium persulfate (Ultra-Pure/BRL, #5523UA)
9. TEMED (N,N,N'N' Tetramethylethylenediamine, Ultra-Pure/ BRL, #5524UB)

Special Equipment:

1. Mighty-small II SE-250 vertical gel electrophoresis unit (Hoefer)
2. Perkin-Elmer/Cetus Thermal Cycler
3. Sterile Thin-wall 0.5 ml Thermocycler microfuge tubes: (TC-5, Midwest Scientific)

Instructions for preparation of oligonucleotides, strategies for optimizing the specificity of a PCR reaction, pouring and running polyacrylamide gels using the "Mighty-small II" unit, and additional helpful information appear at the end of this protocol.

Recommendations for choosing oligonucleotide primers :

The aim is to choose oligonucleotide primers complementary to relatively unique sequences flanking the segment to be amplified. Although more rigorous calculations and considerations can be employed to choose optimal primers, a few general guidelines will be given below to supply a good starting point.

Primers for PCR are generally 20-30 bp long and are chosen to be complementary to one strand (5' to 3') upstream and complementary to the opposite strand (5' to 3') downstream from the sequence to be amplified. The 5' ends of the primers define the ends of the amplified PCR product. Primers should ideally contain relatively balanced GC vs. AT content (e.g. 45-55% GC), and no long stretches of any one base. Caution should also be taken that the two primers of the primer pair do not contain complementary structures >2 bp to avoid "primer dimer" formation resulting from annealing of the two primers (especially at their 3' ends). The target sequence to be amplified is ideally 200-400 bp in length, with an upper limit probably around 3 kb.

Procedure for polymerase chain reaction:

The PCR reaction can be performed in volumes from 5 µl to 200 µl or more. The protocol below is similar to that used by the Center for Genetics in Medicine when screening the YAC library using a specific PCR assay and is carried out in a 5 µl reaction volume. This volume is recommended when the purpose of the experiment is diagnostic (to visualize whether or not a specific product is generated). A scaled up volume can be used if the PCR product will be recovered from the gel or used for sequencing. The 5 µl reaction is performed in a 0.5 ml eppendorf tube and covered by a drop of oil before placing in the thermal cycler.

The following components will make up one reaction (5 µl total volume), but a cocktail of everything except the DNA will be made first:

		Cocktail for 10 reactions
1.0 µl	5X PCR Buffer	10 µl 5X PCR Buffer
0.4 µl	dNTP mixture (each at 2.5 mM)	4 µl dNTPs
0.2 µl*	Primer pair (each primer at 25 µM)	2 µl Primer pair
	(The primer pair solution is 1:1 mixture of the
	50 µM primer solutions)
0.1 µl	Taq polymerase	1 µl Taq polymerase
2.3 µl	ddH2O	23 µl ddH20
plus,
1.0 µl	DNA (100 ng genomic template DNA or < 50 ng cloned template)

*The range of final primer pair concentrations in a normal reaction mix is 0.25 - 2.5 µM and 0.5 µM is sometimes ideal.

Because of the small volumes involved, it is convenient to make a cocktail of the first five ingredients for each primer pair to be used. For instance, if 8 PCR reactions are to be performed from 8 different genomic or cloned DNA templates using one primer pair, then a cocktail may be made (including a slight excess) for 10 reactions by mixing together each of the volumes above multiplied by 10. A 4 µl aliquot of the cocktail will then be added to the 1.0 µl of DNA in each tube.

1. Plan your experiment before adding any reagents (#primer pairs to be used, number of DNA templates, etc.). After doing so, make the appropriate cocktail/s and ensure complete mixing by tapping the tube and quick spinning. (N.B. Caution should be used to avoidcontamination of reactions with even small amounts of DNA. In addition, care should be taken to avoid contamination of pipetmen with carryover amplification products from previous reactions)
2. Pipet 4.0 µl of the appropriate cocktail directly into the bottom of a sterile microeppendorf tube for each reaction. The tubes should be labeled by placing a round sticker on the cap to prevent smearing by oil in subsequent steps.
3. Add 1.0 µl of the DNA directly into the drop of cocktail in each tube and ensure adequate mixing. Quick spin to collect the reaction mixture in the bottom of the tube.
4. Overlay each reaction with one drop of light mineral oil using a pasteur pipet. The samples may be quick spun if necessary before placing in the Perkin Elmer/Cetus PCR machine.
5. Place a drop of mineral oil into each well in the thermal cycler temperature block to be used for the samples (this ensures rapid temperature equilibration during cycling)
6. Place the tightly capped tubes in the temperature block and make sure each is firmly seated by pressing on the tubes individually.

   The PCR machine must now be programmed for the specific reaction conditions desired (See brief operating instructions for Perkin-Elmer PCR machine). Each cycle in the polymerase chain reaction involves three steps (denaturing, primer annealing, polymerization), and the products are amplified by performing many cycles one after the other with the help of the automated thermal cycler. Refer to the literature citations at the end of this protocol for detailed explanation of the reaction. The Taq polymerase is heat stable, and remains active despite the high denaturing temperature of each cycle. A representative set of reaction conditions for 25-35 cycles is:

   I.	Denature	93-94 degrees C	1.5 minutes
   II.	Anneal	50-65 degrees C	2 minutes
   III.	Polymerize	72 degrees C	2 minutes

   Strategies for optimizing PCR reactions are at the end of the protocol.

7. After completion of the PCR reaction, remove the tubes from the temperature block and wipe the outside free of excess oil before placing in an eppendorf rack.
8. Add 2.0 µl of 5X Ficoll stop dye directly into the aqueous phase "bubble" at the bottom of each tube, and then add 100 µl of chloroform:isoamyl alcohol (24:1) to each tube, shake well, and spin briefly.
9. Carefully remove only the aqueous "bubble" with a P20 pipetman set to 7-8 µl by placing the pipet tip against the bubble and slowly drawing it in. Each sample should then be placed in a separate clean eppendorf tube before loading onto the polyacrylamide gel.
10. The reaction products are conveniently separated according to size by electrophoresis through a 10% polyacrylamide "Mighty-small II" gel at 110 V for 2-2.5 hours, and visualized after staining the gel with ethidium bromide.

ADDITIONAL INFORMATION:

Preparation of Oligonucleotides:

Oligonucleotide primers are synthesized using an automated machine (we currently order primers through the Center for Genetics in Medicine) and are received in a glass vial in an ammomium solution. It is convenient to remove about one half of the total volume for each oligonucleotide and divide this volume further into two 1.5 ml eppendorf tubes (the remainder of the ammonium stock solution is stored at 4 degrees C). The oligonucleotides must be prepared as detailed below before use in PCR reactions:

1. Incubate each sample in a heating block at 55 degrees C overnight and then dry in a rotary vacuum concentrator for 4-6 hours. (Warning: a cold trap should be used when drying the samples to absorb the ammonia)
2. Resuspend each oligonucleotide (from both eppendorfs) in a total of 500 µl of TE.
3. Make a 1:200 dilution by diluting 5 µl of each oligonucleotidewith 1.0 ml of TE and measure absorbance of UV light in a spectrophotometer at 260 and 280 nm. The concentration of the stock of resuspended oligonucleotide can then be calculated:

   A260 of 1.0 = 35 µg/µl for DNA oligonucleotides.
   If A260 = 0.203 for a oligomer of 21 nucleotides, then
   0.203 x 35 x 200 (dilution) = 1421 µg/ml (original solution), or 1.421
   x 106 µg/L 
   21 (#nucleotides) x 330 µg/µmol = 6930 µg/µmol
   
   1.421 x 106 µg/L = 205 µmol/L (µM) 
   ----------------
   6930 µg/µmol

4. Make 50 µM solutions in TE of each oligonucleotide for subsequent use in PCR reactions.

Strategies for optimizing the efficiency of PCR reactions:

The conditions required for generation of a specific, essentially unique product (single strong band) will nearly always need to be optimized empirically. In particular, the annealing temperature is important in determining the specificity of the reaction (that is to say, at lower temperatures the primers may anneal to similar irrelevant sequences elsewhere in the genome and prime these, resulting in the formation of multiple products). In general, higher annealing temperatures result in more stringent conditions for primer annealing and more specific products. A good place to start is with a low annealing temperature around 50-55 degrees C, with optimization by testing at 3-5 degrees C increments until maximum specificity is reached. Theoretically, oligonucleotide primers with a high GC content may require a very high annealing temperature to maximize specificity. While this is a good rule of thumb, the optimum temperature may not correspond well to this estimate. Occasionally, specifity will reach a maximum at a certain temperature and at higher annealing temperatures, multiple new products or no products at all will be generated.

Although annealing temperature is perhaps the easiest variable to change, specificity may also be increased by reducing the concentration of primers or Taq polymerase, minimizing the times allowed for annealing and extension, or reducing the free Mg++ concentration. An optimum of Mg++ concentration usually exists in the 1-10 mM range. Too low Mg++ concentration may result in no products and an excess may result in a variety of unwanted products.

Pouring and Running Polyacrylamide Gels using the Hoefer SE-250 "Mighty-small II" gel electrophoresis unit:
(Simplified instructions are provided below, for detailed instructions, refer to the Hoefer manual). Multiple identical polyacrylamide gels can be pre-cast in the supplied SE 275 multiple gel caster. Acrylamide is a neurotoxin and should be handled with caution. Wear gloves at all times when handling acrylamide and be careful to avoid spills.

1. Clean the multiple gel caster and place flat on the bench top in front of you. Place the rubber gasket in its groove without stretching it and lubricate with a thin layer of the Cello-seal provided by Hoefer.
2. Build the gel casting units by carefully placing and seating components in the following order from the bottom up: waxed paper, notched alumina plate, T-shaped spacers (0.75 or 1.0 mm), glass plate, waxed paper, etc. Approximately 5 complete 0.75 mm gels can be cast at one time with one or two additional glass plates needed to fill extra space.
3. Place the top cover on the multiple gel caster and apply red spring clamps to side grooves, ensuring adequate sealing. Be sure that the port at the bottom of the front plate has a small piece of rubber tubing on it and is clamped off.
4. Mix the ingredients for 50 ml of acrylamide (minus the TEMED) in a clean beaker, as detailed in the recipe below for a 10% polyacrylamide gel. Add the TEMED with thorough mixing just before pouring the gels.
5. Carefully pour the acrylamide evenly into the gel casting units in the multiple gel caster until the multiple gel caster is almost overflowing.
6. Insert the appropriate sized comb (0.75mm for 0.75 mm spacers) into each gel casting unit, and allow the acrylamide to polymerize for at least 1 hour. After complete polymerization, the gels may be wrapped in cellophane and stored at 4 degrees C.

Solutions:

• 40% Acrylamide/ 2% bis stock

  acrylamide:	38 g
  N,N'-methylene bisacrylamide	2 g
  dH20:	to 100 ml

• Mini-gel ("Mighty-small II") 10% polyacrylamide (50 ml volume)

  12.5 ml 40% Acrylamide/ 2% bis stock
  25 ml water
  5 ml 10x TBE
  7.5 ml glycerol
  + 714 µl 10% APS  + 17.2 µl TEMED

References:

Mullis, K. and F. A. Faloona. (1987). "Specific synthesis of DNA in vitro via a polymerase catalyzed chain reaction." Meth. in Enzymol. 255:335-350.

Mullis, K, Faloona, F., Scharf, S., Saiki, R., Horn, G., and H. Erlich. (1986). "Specific enzymatic amplification of DNA in vitro: The polymerase chain reaction." Cold Spring Harbor Symposia on Quantitative Biology, Volume 51, Cold Spring Harbor Laboratory. p. 263-272.

Williams, J. F. (1989). "Optimization strategies for the polymerase chain reaction." Biotechniques 7(7):762-768.

PCR Technology: Principles and Applications for DNA Amplification. (1989). Erlich, H.A. (ed.), Stockton Press, New York.

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