Table Of ContentIntroduction to Polymerase Chain Reaction
Mark C. Miller and Laura Cunningham
1. Introduction
The polymerase cham reaction (PCR) has become an indispensable tool of
molecular biology (I-5). Smce its discovery in 1985 the PCR process has found
its integration into all research areas mvolving the use of DNA and RNA.
Using this technique, a small starting sample of DNA or RNA can be used to
amplify a specific DNA or RNA target over a million-fold in as little as 2 h.
This allows for the detection of as little as a single copy of a gene or part of a
gene m cells, whether they be from blood, cultured cells, tissue biopsies, chro-
mosomes, or any other biological system that contains DNA or RNA, mclud-
mg archival materials (formalin-fixed, paraffin-embedded).
Thus chapter will drscuss the prmciples of PCR as it relates to the method-
ologies employed to successfully carry out the reaction on DNA targets. Proto-
cols relating to DNA extraction, PCR conditions, amplification cycles, and
post-PCR detection methods will be presented. RNA amplification (RNA PCR)
will be discussed in another chapter.
There is a wide variety of methodologies for the extraction of DNA for use in
many molecular-biology applications. When preparing DNA for use m PCR, the
DNA extracted does not have to be of the highest quality. A variety of kits are avail-
able on the market that allow for the relatively quick isolation of PCR-quality DNA.
Two methodologies will be described. One mvolves a commercrally avail-
able krt for the quick isolation of DNA from cultured cells. The second proce-
dure is for use with formalin-fixed paraffin-embedded brain tissue. Because of
the destructive nature of formalin on DNA, special PCR consideratrons need to
be taken mto account (i.e., length of PCR product). Because of the high lipid
content of brain tissue sodium dodecyl sulfate (SDS) is often used in the
extraction step. The reader is referred to the reference list for other method-
ologies mvolvmg paraffin-embedded bram tissues (6-8).
From Methods m Molecular Medrcme, vol 22 Neurodegeneratron Methods and Protocols
Edtted by J Harry and H A T&on 0 Humana Press Inc , Totowa, NJ
1
2 Miller and Cunningham
1.1. Principles of PCR
DNA replication m vivo requrres that the double-stranded DNA molecule
be made smgle-stranded (accomplished through the use of specialized proteins)
followed by the synthesis of a short RNA primer along a stretch of the smgle-
stranded DNA. A DNA polymerase then binds to this primer-DNA complex
and moves along the single-stranded DNA template polymerizing a new strand
of DNA by mcorporatmg nucleotides that are complementary to the template
(parent) strand of DNA. This highly integrated process allows for DNA to be
replicated in a relatively short penod of time and requires specialized proteins,
physiological conditions, the presence of nucleotides, and a vartety of enzymes
used to catalyze the reaction.
The PCR process, in a sense, mtmtcs this in vrvo DNA replication process.
However, since it takes place in vitro, the PCR process requires modifications to
the normal environment through the use of specialized enzymes and reaction
components, which fosters its adaptation to a tube environment. Starting with
double-stranded DNA (either genomtc or cloned), short single-stranded oligo-
nucleotrde primers complementary to known sequenceso n the template DNA, a
thermostable DNA polymerase, as well as reagents needed to synthesize new
DNA (namely deoxynucleottde trtphosphates), along with physrologrcal salts and
buffers, the PCR reaction can carry out repeated amplifications of a target stretch
of DNA until millions of copies of that target are synthesized (see Fig. 1).
To compensate for a lack of a true physrologrcal state, the PCR process
utilizes physical conditions (i.e., temperature) to accomplish the manipulations
of the DNA that would, in an in vivo environment, be accomplished by pro-
tems. Specifically, the denaturing of the double-stranded DNA (converting to
single strands) is accomplished in the PCR by raismg the temperature of the
reaction to approx 95°C for a short period of time. The dtscovery of thermo-
stable DNA polymerases (Tuq DNA polymerase) allows the PCR reactions to
be carried out at high temperatures without the destructton of the polymerase
after each hrgh-temperature step, which would occur rf a nonthermostable DNA
polymerase were used. A typical PCR cycle involves three basic steps:
1 Denaturation: The PCR reagents are heated to approx 95°C for 15-30 s Thus
causes the DNA template to denature (melt) into single strands. Cycling times
may vary depending upon the thermal cycler model used.
2 Anneahng: The temperature is rapidly lowered to approx 55°C for 15-30 s. Thts
allows the short, smgle-stranded DNA primers the opportunity to bind to then
complementary regtons on the single-stranded DNA template. As wtll be dls-
cussed later, thus part of the reactton can lead to nonspecific annealing and exten-
sion of the primers to sues on the DNA other than their true complement, resulting
m the generation of undesired, spurious products. Thus, selection of the anneal-
ing temperature 1s crltlcal to generating specific PCR products.
introduction to PCR 3
double-stranded
template DNA
Denaturatlon
t (95 o C, 15-30 set )
3’, smgle-stranded
template DNA
s13
Annealmg
(55o C, 15-30 set )
I
3’ 5’
6’ 11111111 Pnmers bind to
complementary regions
IIIIIIIIIIIIIll l Ill ll ll on template DNA
5’
30 cycles Extension
(72 oC , 30 set )
I
3’ Taq polymerase binds to
5”“““““““’ 3’ ends of primers and
-c Incorporates dNTPs as
new DNA is synthesized
in 5’4’ dlrection
New complementary
strands are synthesized
and results in doubling of
starting target DNA
Fig. 1 Diagrammatic representation of the polymerase chain reaction.
3. Extension: The temperature is then rapidly raised to approx 72°C for 15-30 s.
During this phase the AmpliTuq@ DNA Polymerase binds to the primer-DNA
complexes and begins polymerizing a new strand of DNA by mcorporating
deoxynucleottdes (dNTPs) that are complementary to the template strand. It
should be noted, however, that this is the optimal extension temperature of
AmplzTaq DNA Polymerase AmplzTaq DNA Polymerase has activity at lower
temperatures, so care needs to be taken to avoid the opportunity for misprimed
primers to be extended. These concerns will be addressedi n Note 15
A typical PCR reaction would involve approx 30 repeated cycles of the selected
temperature and time parameters. In some cases, it is possible to do a two-
temperature cycling step. A two-step PCR involves combining the anneal and
extension steps in one. The anneal/extend cycle is run between 60 and 72°C for
45 s to 1 mm. However, rf the T,,, of the primers is below 60°C a three-step
PCR cycle should be used.
Miller and Cunningham
2. Materials
2.7. Reagents
2.1.1. Extraction Reagents
1 Puregene DNA Isolation from Cultured Cells Ku (Gentra Systems) to Include*
cell lysis solution, RNase A solution, protein precipitation solutton (Mmneapo-
hs, MN), 100% tsopropanol, 70% ethanol, and DNA hydration solution.
2 DNA from formalm-fixed, paraffin-embedded tissue.
a. Phenol.chloroform.rsoamyl alcohol (25.24: 1) (AMRESCO [Solon, OH] cat.
no. 0883 [Trts-buffered]).
b. 95% ethanol (AMRESCO cat. no. E193).
c. 1X TE, pH 7.4 (Dlgene [Beltsville, MD] cat. no. 3400-1039).
d. 0.5 M EDTA (Digene cat. no 3400-1003)
e. 20% SDS (AMRESCO cat. no E195)
f. Protemase K (AMRESCO cat. no 0837)
g. Chloroform (AMRESCO cat no. 0757)
2.7.2. PC/? Reagents
1 Autoclaved ultrafiltered water
2 AmpliTaq GoZdrM DNA Polymerase (250 U) (PE Applied Btosystems [Foster
City, CA] cat. no N808-0241, (see Note 1, Table 1)
3 10X reaction buffer II, to include:
a. 100 mM Tris-HCl, pH 8.3 (at 25°C).
b. 500 mM KCl.
c. 25 mM MgCl, solution.
d. dNTPs (PE Applied Biosystems cat. no N808-0007).
e. Primers (user provided, see Note 7)
f. DNA template (user provided, see Note 22).
2.1.3. Detection Reagents
1. Ethtdmm bromide solution (AMRESCO cat. no E406, see Note 23).
2.2. Equipment and Supplies
1. Microcentrifuge tubes
2. High-speed microcentrifuge.
3 Vortex.
4 Heat block.
5. Sterile scalpels.
6 Sterile mortar and pestle
7. UV spectrophotometer.
8 Positive displacement pipe& 0 5-25 uL (PE Applied Biosystems cat no N930-
6008)
9 Positive displacement ptpet, 25-250 PL (PE Applied Blosystems cat. no N930-
6009)
Introduction to PCR 5
10. GeneAmp@ PCR System 9700 Thermal Cycler (PE Applied Blosystems cat. no.
N805-000 1; see Note 24).
11. Primer ExpressTM Software (PE Applied Biosystems cat. no. 402809).
12. Pipet tips, 0.5-25 FL (PE Apphed Biosystems cat. no. N930-6010).
13. Pipet tips, 25-250 FL (PE Applied Biosystems cat. no. N930-6011).
14 MicroAmp@ Reaction Tubes with caps (PE Applied Biosystems cat no.
N801-0540)
3. Methods
3.1. DNA Isolation from Cultured Cells
3 7.7. Cell Lysis
1. Add 0 5-l .O mllhon cells m approx 1 mL of balanced salt solution or culture
medium to 1.5-mL tube (expected yield is 2-6 pg DNA).
2. Pellet cells by placing the tube in a centrifuge and spinning at 13,000-16,OOOg for 5 s.
3. Remove supernatant, leaving behind 5-10 pL of residual liquid
4 Vortex the tube vigorously to resuspend the cells m the residual supernatant.
5. Add 150 p.L cell lysts solution to the resuspended cells and plpet up and down
approx 20 times to lyse the cells. If cell clumps are visible after mrxing, Incubate
at 37’C until the solution is homogenous. Samples m the cell lysts solution are
stable for at least 18 mo at room temperature.
3.1.2. RNase Treatment
1. Add 0 75 pL RNase A solution to the cell lysate.
2 Mix the sample by mvertmg the tube 25 times and incubate at 37°C for 15-60 mm.
3.7.3. Protein Precipitation
1. Cool sample to room temperature
2. Add 50 PL protein precipitation solution to the RNase A-treated cell lysate.
3 Vortex vigorously at high speed for 20 s to mix the protein precipitation solutton
uniformly with the cell lysate.
4. Centrifuge at 13,000-16,000g for 3 mm. The precrpitated protems will form a
tight pellet
5. If the protein pellet 1s not visible, repeat step 3 followed by incubation on ice for
5 min, then repeat step 4
3. I. 4. DNA Precipitation
1 Pour the supernatant containing the DNA (leaving behind the precipitated protein
pellet) into a 1 5-mL microfuge tube containing 150 pL isopropanol (Zpropanol).
2. Mix the sample by gently inverting 50 ttmes.
3. Centrifuge at 13,000-16,000g for 1 mm; the DNA ~111b e visible as a small white
pellet on the bottom of the tube.
4. Pour off the supernatant and drain the tube on clean absorbent paper. Add 150 l.tL
70% ethanol and invert the tube several times to wash the DNA pellet
Table 1
Feature Chart for PCR Enzymes (70)
AmpllTaq DNA
AmpliTaq Gold AmpllTaq DNA Polymerase, rTth DNA
Product name DNA Polymerase Polymerase Stoffel fragment rTth DNA Polymerase Polymerase, XL
Organism Thermus aquaticus Thermus aqua&us Thermus aquatzcus Thermus thermaphdus Thermus thermophdus;
Thermococcus lltoralls
Molecular weight 94 kDa 94 kDa 61 kDa 94 kDa N/A
Number of ammo 832 832 544 834 N/A
acids
Optimal extension 72-80°C 72-80°C 72-80°C 72-80°C 72-80°C
temperature
Extension rate 24 kb/mm 2-4 kblmin 24 kb/mm 2-4 kb/mm 2-4 kblmm
m (72°C)
Hot start Automated Manual Manual Manual Manual
Enzyme activation 10 mm at 95°C N/A N/A N/A N/A
or 10 extra cycles
RT actrvity Mimmalflow Mmrmalllow Mmtmal/low Yes, Mn2+-dependent Yes, Mn2+-dependent
Half-life 97.5WlO mm 97 5WlO mm 97 5W20 min 97 5”C/2 mm 97.5W2 mm
95W40 mm 95W40 mm 95W80 mm 95W20 mm 95W20 mm
92S”Ch130 min 92 5”C/>130 mm
Cycling half-life” 160 cycles 160 cycles 320 cycles 80 cycles 80 cycles
Magnesium ion 15-4mM 1-4mM 2-10 rrUI4 1.5-2.5 mi%Z 1.5-2 5 mM
PCR optimum
pH opttmum for 7.0-7 5 7.0-7.5 7.0-7 5 7.0-7 5 7.0-7.5
PCR amphficatron
pH optrmum for 8 3 at room N/A N/A N/A N/A
activatior+ temperature
dNTP PCR 40-2OOpM each 40-2OOpM each 40-2OOpM each 40-2OOpM each 40-2OOpM each
optimum
KC1 PCR optimum 5OlllM 5omM 10 mM 75-100 m&I 75-100 mM
Primer PCR 0.1-1.0 /.tM 0 l-l.0 FM 0 l-l.0 p.44 0.1-1.0 /.M 0.4 pM
opumum
Processivny 50-60 bases 5CL60 bases 5-10 bases 3WO bases 30-40 bases
Longest PCR -5 kb -5 kb N/A -5 kb >40 kb
product
Extra A addition Yes Yes Yes (less) Yes No
Y-3 exonuclease Yes Yes No Yes Yes
activity
3’-5’ exonuclease No No No No Yes
activity
Contammatmg No No No No No
u nucleases
Supplied 5 U/pL 5 U&L 5 U&L 5 U&L 5 U&L
concentration
Storage conditions -2OT -20°C -20°C -2OT -20°C
aAssumptions of 95”C, 15 s denaturanonlcycle
bpH optrmum measured at 72°C
8 Miller and Cunningham
5 Centrifuge at 13,000-16,000g for 1 mm. Carefully pour off the ethanol. Because
the pellet may be loose, you should pour off slowly and watch the pellet.
6 Invert and dram the tube on clean absorbent paper and allow to arr-dry for
15 min.
3.1.5. DNA Hydration
1 Add 25 ~.LLD NA hydration solution (25 pL will give a concentration of 100 pg/mL if
the total yield is 2.5 kg DNA)
2. Allow DNA to rehydrate overmght at room temperature Alternatively, heat at
65°C for 1 h. Tap the tube periodically to aid in dispersmg the DNA.
3. Store DNA at 2-8°C
3.2. DNA Extraction from Forma/in-Fixed,
Paraffin-Embedded Brain Tissue
3.2.1. Tissue Processing
1. Usmg a sterile scalpel, cut approx 100 mg from a paraffin block of brain tissue
2 Crush the tissue using a sterile mortar and pestle and mcubate with gentle agita-
tion with 500 pL xylene for 1 h at room temperature.
3. Wash m 95% ethanol and then 70% ethanol for 5 mm each.
4. Centrifuge at 13,000g for 10 mm to pellet.
5 Dry pellets m a 50°C heat block.
6 Resuspend m 1X TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.4)
7. For tissue that is formalm-fixed but not paraffin-embedded, cut a 0 1 -cm3 ptece from
the tissue using a sterile scalpel and incubate in 10 mL of 1X TE overnight at 4°C.
3.2 2. Digestion/Extraction and Purification
1 Digest sample in a 200 mL ahquot containmg
a. 0.2 M Tris, pH 8.0.
b 10 mM EDTA.
c 1% SDS.
d 4 mg/mL proteinase K.
2. Incubate for 16 h at 50°C
3. Add an equal volume of Tris-buffered, redistilled phenol.chloroform.isoamyl
alcohol and vortex briefly.
4. Centrifuge at high speed (13,000g for 15 s). Make sure to separate the phases.
Centrifuge longer if not completely separated
5. Pipet the aqueous upper phase to a fresh tube. Discard the orgamc lower phase
and interface. When pipettmg the aqueous phase, care should be taken not to
remove any of the organic phase with the aqueous phase. At this point you may
“back-extract” the organic phase and mterfaces. These contain the proteins but
some DNA may also be present. After removing the aqueous phase, add an equal
volume of 1X TE to the organic and interface phases. Mix well and centrifuge at
13,000g for 15 s Transfer the aqueous phase to the previous aqueous phase
Introduction to PCR 9
Discard the organic and interface phases in the appropriate organic waste dis-
posal contamer
6. Repeat steps l-3 until no protein is visible at the interface of the organic and
aqueous phases, This may require extractions. Add the aqueous phases to the
previously collected aqueous phases.
7. Add an equal volume of chloroform and repeat steps 4 and 5
8. Adjust the volume of the combined aqueous phases to 300 pL using 1X TE.
9. Add two volumes of me-cold 100% ethanol and mix by vortexmg. Incubate on
ice for 15-30 mm.
10. Centrifuge for 10 mm and carefully and completely remove the supernatant.
11 Half-fill the tube with 70% ethanol and recentrifuge at 12,000g for 2 mm at 4°C
in a microfuge
12 Carefully remove all of the supernatant
13 Store the open tube on the bench top until all of the hquid has evaporated.
14. Add 1X TE to the desired volume
15. Determine quantity of DNA via UV absorbance at 260 and 280 nm. See Note 20.
3.3. PCR Procedure
1. Prepare a master mix of PCR reagents (water, buffer, dNTPs, and DNA poly-
merase) for all of the samples (see Subheading 2.1.2.). The primers and template
DNA are added separately. Using a master mix is recommended because it improves
the plpettmg accuracy and mmtmlzes reagent losses on the pipet tips. To prepare
a master mix, determine the final total volume of all of the reactions (i.e., 10
tubes x 50 @/tube = 500 pL total volume) Prepare a master mix tube with the
appropriate volumes of water, buffer, dNTPs, and enzyme to accommodate the
total volume of all of your reactions (i.e., 500 FL). Be sure to take into account
the addition of primers and template DNA to your reaction tubes (see Table 2).
If AmpliTuq Gold DNA Polymerase is the enzyme bemg used, all of the com-
ponents (mcludmg primer and template DNA) can be added to the master mix.
Gently vortex the master mix and dtspense 50 mL into each tube and place tubes
in the thermal cycler.
2. Place tubes m the thermal cycler and perform cycling temperature and time
parameters( see Table 3).
3 Accurately quantitate PCR components before and after PCR (see Note 25 and
Table 4).
4. Optimize PCR reaction conditions (see Notes 26-29).
5 If necessary, utilize Hot Start PCR protocol (see Notes 30-34)
6. Low copy number templates may require further optimization (see Note 35).
7. Crosscontaminatron prevention is critical during PCR amplification (see Note 36).
3.4. Defection of PCR Products
At the completion of the PCR reaction, approx 5-10 PL of product is mixed
with a gel loading buffer and run on an agarose gel (l-2%). DNA molecular-
weight markers are also loaded mto one of the wells as fragment size refer-
10 Miller and Cunningham
Table 2
PCR Reaction Components
Components (50 pL rxn) Order Volume Final concentration
Autoclaved ultrafiltered water 1 a
[10X] Reaction buffer II 2 5.0 pL 1X
25 mM MgCI, solution 2 2-8 ~JL 1-4mMb
dATP 3 1 OpL 200 pM
dCTP 3 1OpL 200 /AM
dGTP 3 10 pL 200 FM
dTTP 3 1.0 pL 200 PM
AmpliTaq Gold DNA polymerase 4 05pL 2.5 U
User-provided primer #l 5 0.5-2.5 PL 0 2-l .O /.LM
User-provided primer #2 5 0.5-2.5 ~.LL 0 2-10 FM
User-provided DNA template 6 a cl pg/rxn”
Total mix 50 mL
aAny combmation of water and template can be used as long as the total volume of the master
mix, sample, and pnmers equals 50 pL
bThe optimal magnesmm chloride concentration may vary, depending on the primer and
template used and must be determined empmcally In most cases a final concentration of mag-
nesium chloride m the range of 1 O-4 0 nul4 m the reaction mix will work well If using the
10X PCR buffer I, which already contains 15 mM MgC12, the concentration of the M&l, will
be 1.5 mM.
CPreferably >104 copies of template but <l pg DNA/reaction.
ences. As current is passed through the gel, the fragment(s) will separate, based
on their size, as the DNA migrates through the gel. At the completion of the
electrophoretlc run the DNA fragment(s) can be detected through the use of the
fluorescent dye ethidium bromide stainmg. This intercalating dye binds in
between the stacked bases of double stranded DNA. When placed on a UV
light box (transilluminator) the dye 1s excited and emits the energy at 590 nm
in the red-orange region of the visible spectrum (II).
There are several approaches to ethidium bromide staining of gels. Ethidium
bromide 1s usually made up as a stock solution of 10 mg/mL m water. It is
stored at room temperature m a light-tight container, or one wrapped in aluml-
num foil. Staining of gels can occur in one of three ways:
1. Ethidmm bromide is added to the electrophoretic buffer at a concentration of
0 5 pg/mL prior to the run
2. Ethldium bromide can be added directly to the gel before it solldifles at a concen-
tration of 5 kg/mL.
3. Following electrophoresls the gel 1s placed in a solution of ethldium bromide and
allowed to stain for approx 30 nun. Following staining it is sometimes necessary
to “destain” by placing the stained gel m water for approx 15-20 mm.
