PCR Amplification of DNA David L. Weller University of Vermont, Burlington, VT 05405 The uolvmerase chain reaction (PCR)is a biochemical or . " enzvmatlc method for the i n vitro amplification of a nprcific rceion of DKA (4-6'). A cvcle of amnlificntion consists of three steps; denaturation of the DNA, annealing of primers to the specific sequences in the single-stranded DNA, and elongation of the primers. In the procedure primers complementary to the 3'-ends of the target, usually 20-25 deoxynucleotides long, anneal to the singlestranded DNA that is generated by heating the sample. The primers are extended during elongation to the end of the target sequence through the action of Taq polymerase, a thermostable DNAnolvmerase. k i c a l l v " 2 0 3 5 cvcles of amplification are eml;loied witb segments up to 2060 base pairs (bp) amplified (5).The product of PCR amplification of a specific region of DNA can be seen as a band of appropriate size following agarose gel electrophoresis and ethidium bromide staining (5, 8). References 1 3 are readings on the theory, development, and applications of PCR. PCR generally is simpler and technically less difficult than clonine. which utilizes recombinant DNA technolom. ~m~lificatio byn PCR requires technical skills normally associated witb biochemical urocedures. a temuerature cycler, and about 3 h to compl&. Good results hive been obtained in the PCR student exercise described using both the relatively simple and inexpensive teacup thermo&cler of Watson (8)and a commercially available thermocycler. The result is amplification of a 500 bp region of bacteriophage lambda DNA.
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Methods and Materials The temperature cycler of Watson (8) consists of a teacup heater, a 150-mL beaker that was adapted with IN and OUT (overflow) ports for water, a functioning hot or wld water solenoid valve from an automatic washine machine that was connected in the water line between t h i pressure reducing valve and the lower (IN) port of the beaker and a programmable timer/controller (Cole Parmer, Chicago, IL, catalog no. I,-0861400). The latter was urozrammed (8)to controi the heater and the solenoid valvi (cold water flow) such that the temperature cycle that required about 6 min included stops a t 92 'C (denaturation), 54 "C (annealing) a n d 72 "C (elongation). The Crocodile I1 thermocycler (Appligene, Pleasanton, CA) was programmed for block
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Journal of Chemical Education
Agarose gel electrophoretic patterns. Amplified DNA obtained using "PCR in a Teacup" method (8)lanes 1 (10 WLloaded),2 (20 pL) and Crocodile II thermocycler (Appligene)lanes 6 (20 pL) and 7 (10 pL). Lane 4 IambdaIHind Ill digest; the and >indicate the 564 and 125 bpfragments, respectively. Lanes3 (20pL) and 5 (10 pL) unamplified DNA control. Migration was toward the (+) pole that corresponds to the bottom of the photograph. Samples were loaded in wells at o.
temperatures of 95 "C (50 s), 55 "C (2 m i d and 75 "C (2 min). About 30 s was required to reach the designated temperatures so that a cycle required about 6 min. Lambda DNA and primers (PCR 01 and PCR 02) were those provided with the GeneAmp PCR kit (Perkin Elmer, Norwalk, CT). The amplification reaction contained 10 nanomoles of each of the 4 deoxynucleoside triphosphates, 50 picomoles of each primer, 0.5 nanogram of lambda template, and 6.25 units of AmpliTaq DNA polymerase (Perkin-Elmer) in 50 pL of a solution containing 10 mM TrisHCl, pH 8.3, 50 pM KCl, 1.5 mM MgC12, and 0.001% (wlv) gelatin. The reagents and their order of addition to a 0.5mL microcentrihee tube were as described in the ~rotocol provided with the-kit. The sample was covered wih 25 pL of lieht mineral oil and subiected to 25 cvcles of amdification. A sample containing all of the reagents was keld a t room temperature as a control. The 56.1-bp fragment present in the lambda phage DNA/Hind I11 digest (Pharmscia, Piscataway, NJ) was used as a size marker. Following amplification 50 pL of CHC13 was added and the lower more dense layer was removed and discarded. Then 5 pL of 50 % glycerol-0.02 % bromphenol blue was added to the sample and, 10- and 20-pL aliquots were loaded in wells of a 10-cm long horizontal composite gel made of 3% NuSieve GTG agarose and 1% FastLane agarose (FMC, Rockville, ME) and containing TBE (10.78 Der liter). e TRIS. 5.5 e boric acid. and 0.74 e Na9EDTA ~ l e e t r o ~ h o r &in i s T B E ' W ~ in S a minisub cell i~io- ad, Richmond. CAI a t 10 Vlcm a t room temoerature. The run was stopped after about 2 h. The gel wai removed, s t l n e d w ~ t heth~diumbromide, and visualized as described (9,. Twenty microliters containing 1 pg of lambda phage DNA/Hind I11 digest was loaded in a separate well and electrophoretically analyzed along with the amplified DNA as was 10 and 20 pL of the unamplified control.
(Perkin-Elmer). This corresponds to the sequence near the end of the E gene and through and beyond the FI gene (7, 8).The E and F eenes are amone those reauired for head fkrmation (lo). Twentv-five cvcles of am~lificationincreased the amount of the target DNA so tha't i t could be detected by EtBr staining following agarose gel electrophoresis (figure; lanes 1, 2 and 6, 7). The amplified DNA migrated a little faster than the 564 bp fragment in the Hind IIJLambda DNA digest (figure; lane 4) as expected given the smaller size of the amplified DNA sequence. That the band seen was the result of amplification is shown by the negative result obtained with the unampliiied sample (lanes 3 and 5, figure). This sample was held a t room temperature during PCR amplification of the target sequence. Other controls that would appear to be appropriate but were not run include omitting any one of the following from the amplification reaction, template DNA, Taq polymerase, the primers and the deoxynucleoside triphosphates. The relative simple and inexpensive " PCR in a Teacup " method of Watson (8)offers a n alternative to commercially available thermocyclers for the student laboratory exercise. Moderately sophisticated skills are required to put the apparatus together and minor adjustments in the program may be necessary as conditions may vary. Precautions as described in reference 9 should be followed for ethidium bromide staining.
Results and Discussion The two primers used target for amplification the region of lambda phage DNA between positions 7131 and 7630
8. Watson, R. in PCR Pmtada, A C u A to Methods and Applimtions: Innis, M . A,: Gelfand, D. H.:Sninaky, J. J.; White,T J..Eda.:AeademieRess: San Diego, 1990; 00 nz 12M31. 9. Weller, D. L.; Gariepy, P A J. C h .Edue. 1981,68,8142. 10.Slybslski, E. H.; Slybalski, W Cem 1979,7,217-270.
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Literature Cited l.Amhelm,N.;levenson, C.H. Chem Engimr News 1980.68 (40),3647. 2. Erlieh, H.A,; Gelland, D.: Snulsky,J. J.Schap 1991,252,1643-1651. 3. Mullis, K B. Sei.Am. 1980,262(4). 5 6 6 5 . 4. Mullis, K B.: Falmns F A . Methods Enryrnol. 1987,155,335350. 5. Ssiki. R. K; Gelland, D. H.:StoKel,S.;Schsrf, S.J.:Higuchi, R.;Ham, G. T.; MuUis,K B. ;Erlich, H. A. Sczinap ISM.239,48491. 6.Saiki.R.K.:Seharf,S. J.:FaloonaF:Mullia,K.B:Harn,G.T.:Erlich,H.A.:Amheim, N.S&nn 1985.230.135&1354. 7. Sanger, F;Coulson, A. R.:Hong, G.E:Hill, D. E:Pefe*sn,C B. J. Mol Bkl. ISsZ, -"" -ms
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Volume 71
Number 4 April 1994
341
