Pcr Cloning Protocols: From Molecular Cloning to Genetic Engineering

472 Pages·1997·30.695 MB·English

by  Bruce A. White

1
PCR
Basic Principles and Routine Practice
Lori A. Kolmodin and J. Fenton Williams
1. Introduction
1.7. PC!? Definition
The polymerase chain reaction (PCR) is a primer-mediated enzymatic ampli-
fication of specifically cloned or genomtc DNA sequences (I). This PCR pro-
cess,i nvented by Kary Mullis over 10 years ago, has been automated for routine
use in laboratories worldwide. The template DNA contains the target sequence,
which may be tens or tens of thousands of nucleotides in length. A ther-
mostable DNA polymerase, Taq DNA polymerase, catalyzes the buffered
reaction in which an excess of an oligonucleotide primer pair and four deoxy-
nucleoside triphosphates (dNTPs) are used to make millions of copies of the
target sequence. Although the purpose of the PCR process is to amplify
template DNA, a reverse transcription step allows the starting point to be
RNA (2-5).
1.2. Scope of PCR Applications
PCR is widely used in molecular biology and genetic disease research to
identify new genes. Viral targets, such as HIV-l and HCV can be identified
and quantitated by PCR. Active gene products can be accurately quantitated
using RNA-PCR. In such fields as anthropology and evolution, sequences of
degraded ancient DNAs can be tracked after PCR amplification. With its
exquisite sensitivity and high selectivity, PCR has been used for wartime
human identification and validated in crime labs for mixed-sample forensic
casework. In the realm of plant and animal breeding, PCR techniques are used
to screen for traits and to evaluate living four-cell embryos. Environmental and
From Methods m Molecular Biology, Vol 67 PCR Clonmg Protocols From Molecular Cloning
to GenetIc Engmeermg Edlted by* B A White Humana Press Inc , Totowa, NJ
3
4 Kolmodin and Williams
food pathogens can be quickly identified and quantitated at high sensitivity m
complex matrices with simple sample preparation techniques,
1.3. PCR Process
The PCR process requires a repetitive series of the three fundamental steps
that defines one PCR cycle: double-stranded DNA template denaturation,
annealing of two oligonucleotide primers to the single-stranded template, and
enzymatic extension of the primers to produce copies that can serve as tem-
plates in subsequent cycles. The target copies are double-stranded and bounded
by annealing sites of the incorporated primers. The 3’ end of the primer should
complement the target exactly, but the 5’ end can actually be a noncomple-
mentary tail with restriction enzyme and promoter sites that will also be incor-
porated. As the cycles proceed, both the original template and the amplified
targets serve as substrates for the denaturation, primer annealing, and primer
extension processes, Since every cycle theoretically doubles the amount of tar-
get copies, a geometric amplification occurs. Given an efficiency factor for
each cycle, the amount of amplified target, Y, produced from an input copy
number, X; after y1c ycles is
Y = Xi 1 + efficiency) (1)
With this amplification power, 25 cycles could produce 33 million copies.
Every extra 10 cycles produces 1024 more copies. Unfortunately, the process
becomes self-limiting and amplification factors are generally between 10 5- and
log-fold. Excess primers and dNTPs help drive the reaction that commonly
occurs in 10 mMTris-HCl buffer (PH 8.3 at room temperature).I n addition, 50 mM
KC1 is present to provide proper ionic strength and magnesium ion is required
as an enzyme cofactor (6).
The denaturation step occurs rapidly at 94-96°C. Primer annealing depends
on the T,, or melting temperature, of the primer:template hybrids. Generally,
one uses a predictive software program to compute the T,,,s based on the
primer’s sequence, their matched concentrations, and the overall salt concen-
tration. The best annealing temperature is determined by optimization. Exten-
sion occurs at 72°C for most templates. PCR can also easily occur with a two
temperature cycle consisting of denaturation and annealing/extension.
1.4. Carryover Prevention
PCR has the potential sensitivity to amplify single molecules, so PCR prod-
ucts that can serve as templates for subsequent reactions must be kept isolated
after amplification. Even tiny aerosols can contain thousands of copies of car-
ried-over target molecules that can convert a true negative into a false positive.
In general, dedicated prpeters, plugged pipet tips, and separate work areas
should be designated for pre and post-PCR work. As with any high sensitivity
Basic Principles and Routine Practice 5
technique, the judicious and frequent use of positive and negative controls is
required (7-9). Through the use of dUTP instead of dTTP for all PCR samples,i t
is possible to design an internal biochemical mechanism to attack the PCR carry-
over problem. PCR products will then be dU-containing and can be cloned,
sequenced, and analyzed as usual. Pretreatment of each PCR reaction with uracil
N-glycosylase will destroy any PCR product carried over from previous reac-
tions, leaving the native T-containing sample ready for amplification (10).
1.5. Hot Start
PCR is conceptualized as a process that begins when thermal cycling ensues.
The annealing temperature sets the specificity of the reaction, assuring that the
primary primer bindmg events are the ones specific for the target in question.
In preparing a PCR reaction on ice or at room temperature, however, the reac-
tants are all present for nonspecific primer annealing to any single-stranded
DNA present. Since Taq DNA polymerase has some residual activity even at
lower temperatures, it is possible to extend these misprimed hybrids and begin
the PCR process at the wrong sites! By withholding a key reaction component,
such as Tuq DNA polymerase, until an elevated temperature can be reached,
the possibility of mispriming is avoided. This can be accomplished by a manual
addition of enzyme above 65-70°C during the first heating ramp to denatur-
ation at 94°C. Alternatively, an inactive form of the enzyme AmpliTaq Gold
can be added to all reactions to prevent misprimed extensions. Adding a pre-
PCR heat step at 92-95°C for 9-12 min synchronously reactivates the enzyme
and achieves an “invisible” hot start. In both cases, the lowest temperature
experienced by the reaction components is the stringent primer annealing tem-
perature, assuring best specificity (II).
1.6. PCR Achievements
PCR has been used to speed the gene discovery process and for early detec-
tion of viral diseases.S ingle sperm cells to measure crossover frequencies can be
analyzed and four-cell cow embryos can be typed. Trace forensic evidence of
even mixed samples can be analyzed. Single copy amplification requires some
care, but is feasible for both DNA and RNA targets.T rue needlesm haystacksc an
be found simply by ampli-fling the needles.P CR facilitates cloning of DNA sequenc-
ing and forms a natural basis for cycle sequencing by the Sanger method (12).
2. PCR Enzymes
2.1. AmpliTa@ DNA Polymerase
AmpliTaq DNA Polymerase (Perkin Elmer, Foster City, CA) is a highly
characterized recombinant enzyme for PCR. It is produced in E. coli from the
Taq DNA polymerase gene, thereby assuring high purity. It is commonly sup-
plied and used as a 5 U/pL solution in buffered 50% glycerol.
6 Kolmodin and Williams
2.2. Biophysical Properties
The enzyme is a 94-kDa protein with a 5’-3’ polymerization activity that is
most efficient in the 70-80°C range. This enzyme is very thermostable, with
a half-life at 95°C of 3540 min. In terms of thermal cycling, the half-life is
approx 100 cycles. PCR products amplified using AmphTaq DNA poly-
merase will often have single base overhangs on the 3’ ends of each polymer-
ized strand, and this artifact can be successfully exploited for use with T/A
cloning vectors.
2.3. Biochemical Reactions
AmpliTaq DNA polymerase requires magnesium ion as a cofactor and cata-
lyzes the extension reaction of a primed template at 72OC. The four dNTPs
(consisting of dATP, dCTP, dGTP, and dTTP or dUTP) are used accordmg to the
base-pairing rule to extend the primer and thereby to copy the target sequence.
Modified nucleotides (ddNTPs, biotin- 11- dNTP, dUTP, deaza-dGTP, and fluo-
rescently labeled dNTPs) can be incorporated into PCR products.
2.4. Associated Activities
AmphTaq DNA polymerase has a fork-like structure-dependent, polymer-
ization-enhanced, 5’-3’ nuclease activity. This activity allows the polymerase
to degrade downstream primers and indicates that circular targets should be
linearized before amplification. In addition, this nuclease activity has been
employed in a fluorescent signal-generating technique for PCR quantitation
(13). AmpliTaq DNA polymerase does not have an inherent 3’-5’ exonuclease
or proofreading activity, but produces amplicons of sufficiently high fidelity
for most applications.
3. PCR Primers
3. I. Design Criteria
PCR primers are short oligodeoxyribonucleotides, or oligomers, that are
designed to complement the end sequences of the PCR target amplicon. These
synthetic DNAs are usually 15-25 nucleotides long and have -50% G + C
content. Since each of the two PCR primers is complementary to a different
indrvidual strand of the target sequence duplex, the primer sequences are not
related to each other. In fact, special care must be taken to assure that the primer
sequences do not form duplex structures with each other or hairpin loops within
themselves. The 3’ end of the primer must match the target in order for poly-
merization to be efficient, and allele-specific PCR strategies take advantage of
this fact. To screen for mutants, a primer complementary to the mutant sequence
Basic Principles and Routine Practice 7
is used and results in PCR positives, whereas the same primer will be a mis-
match for the wild type and not amplify. The 5’ end of the primer may have
sequencest hat are not complementary to the target and that may contain restric-
tion sites or promotor sites that are also incorporated into the PCR product.
Primers with degenerate nucleotide positions every third base may be synthe-
sized in order to allow for amplification of targets where only the amino acid
sequence is known. In this case, early PCR cycles are performed with low,
less stringent annealing temperatures, followed by later cycles with high, more
stringent annealing temperatures.
A PCR primer can also be a homopolymer, such as oligo (dT),,, which
is often used to prime the RNA PCR process. In a technique called RAPDs
(Randomly Amplified Polymorphic DNAs), single primers as short as
decamers with random sequences are used to prime on both strands, pro-
ducing a diverse array of PCR products that form a fingerprint of a genome
(14). Often, logically designed primers are less successful in PCR than
expected, and it is usually advisable to try optimization techniques for a
practical period of time before trying new primers frequently designed near
the original sites.
3.2. T, Predlcfions
DNA duplexes, such as primer-template complexes, have a stability that
depends on the sequence of the duplex, the concentrations of the two compo-
nents, and the salt concentration of the buffer. Heat can be used to disrupt this
duplex. The temperature at which half the molecules are single-stranded and
half are double-stranded is called the T, of the complex. Because of the greater
number of intermolecular hydrogen bonds, higher G + C content DNA has a
higher T,,, than lower G + C content DNA. Often, G + C content alone is used
to predict the 7’,,o f the DNA duplex; however, DNA duplexes with the same G
+ C content may have different T, values. Computer programs are available
to perform more accurate T, predictions using sequence information (near-
est neighbor analysis) and to assure optimal primer design (see Chapter 2,
Section 3.1.4.).
Since the specificity of the PCR process depends on successful primer
binding events at each amplicon end, the annealing temperature is selected
based on the consensus of melting temperatures (within -2-4”C) of the two
primers. Usually the annealing temperature is chosen to be a few degrees
below the consensus annealing temperatures of the primers. Different strate-
gies are possible, but lower annealing temperatures should be tried first to
assess the success of amplification of the target and then higher annealing
temperatures can be investigated to find the stringency required for best
product specificity.
8 Kolmodin and Williams
4. PCR Samples
4.7. Types
The PCR sample type may be single- or double-stranded DNA of any orr-
gin-animal, bacterial, plant, or viral. RNA molecules, including total RNA,
poly (A+) RNA, viral RNA, tRNA, or rRNA, can also serve as templates for
amplification after conversion to so-called complementary DNA (cDNA) by
the enzyme reverse transcriptase (either MuLV or recombinant Thermus
thermophilus, rTth DNA polymerase).
4.2. Amount
The amount of starting material required for PCR can be as little as a single
molecule, compared to the millions of molecules needed for standard clonmg
or molecular biological analysis. As a basis, up to nanogram amounts of DNA
cloned template, up to microgram amounts of genomic DNA, or up to 1O 5D NA
target molecules are best for initial PCR testing.
4.3. Purity
Overall, the purity of the DNA sample to be subjected to PCR amplificatron
need not be high. A single cell, a crude cell lysate, or even a small sample of
degraded DNA template IS usually adequate for successful amphficatton. The
fundamental requirements of sample purity must be that the target contain at
least one intact DNA strand encompassing the amplified region and that the
impurities associated with the target be adequately dilute so as to not inhibit
enzyme activity. However, for some applications, such as long PCR, it may
be necessary to consider the quality and quantity of the DNA sample (15,I6).
For example,
1. When more template molecules are available, there is less occurrence of false
positives causedb y either crosscontaminationb etween sampleso r “carryover”
contaminationf rom previous PCR amplifications;
2. When the PCR amplification lacks specificity or efficiency, or when the target
sequencesa re limited, there IS a greater chanceo f inadequatep roduct yield; and
3. When the fraction of starting DNA available to PCR is uncertain, rt is increas-
ingly difficult to determine the target DNA content (17).
5. Thermal Cycling Considerations
5.1. PCR Vessels
PCR must be performed in vessels that are compatible with low amounts of
enzyme and nucleic acids and that have good thermal transfer characterrstics.
Typically, polypropylene is used for PCR vessels and conventional, thrck-
walled microcentrifuge tubes are chosen for many thermal cycler systems. PCR
is most often performed at a 1O -l 00 FL reaction scale and requires the preven-
Basic Principles and Routine Practice 9
tion of the evaporation/condensation processes in the closed reaction tube dur-
mg thermal cycling. A mineral oil overlay or wax layer serves this purpose.
More recently, 0.2 mL thin-walled vessels have been optimized for the PCR
process and oil-free thermal cyclers have been designed that use a heated cover
over the tubes held within the sample block.
5.2. Temperature and Time Optimization
It is essential that the reaction mixtures reach the denaturation, annealing,
and extension temperatures in each thermal cycle. If insufficient hold time is
specified at any temperature, the temperature of the sample will not be equili-
brated with that of the sample block. Some thermal cycler designs time the
hold interval based on block temperature, whereas others base the hold time on
predicted sample temperature.
In a conventional thick-walled tube used in a cycler controlled by block
temperature, a 60 s hold time is sufficient for equilibration. Extra time may be
recommended at the 72°C extension step for longer PCR products. Using a
thin-walled 0.2-mL tube in a cycler controlled by predicted sample tempera-
ture, only 15 s is required. To use existing protocols or to develop protocols for
use at multiple labs, it is very important to choose hold times according to the
cycler design and tube wall thickness.
6. Conditions for Successful PCR
6.1. Metal Ion Cofactors
Magnesium chloride is an essential cofactor for the DNA polymerase used
in PCR, and its concentration must be optimized for every primer/template
pair. Many components of the reaction bind magnesium ion, including prim-
ers, template, PCR products and dNTPs. The main 1: 1 binding agent for magne-
sium ion is the high concentration of dNTPs in the reaction. Since rt is necessary
for free magnesium ion to serve as an enzyme cofactor in PCR, the total mag-
nesium ion concentration must exceed the total dNTP concentration. Typically,
to start the optimization process, 1.5 &magnesium chloride is added to PCR
in the presence of 0.8 mM total dNTPs. This leaves about 0.7 mA4 free magne-
sium ion for the DNA polymerase. In general, magnesium ion should be varied
in a concentration series from 1 S-4 mA4 in 0.5~mM steps (I, 17).
6.2. Substrates and Substrate Analogs
Taq DNA polymerase incorporates dNTPs very efficiently, but can also
incorporate modified substrates when they are used as supplemental compo-
nents m PCR. Digoxigenin-dUTP, biotin- 1 l-dUTP, dUTP, c7deaza-dGTP,
and fluorescently labeled dNTPs all serve as substrates for Taq DNA poly-
10 Kolmodin and Williams
merase. For conventional PCR, the concentration of dNTPs remains balanced in
equrmolar ratios, but for mutagenesis, unequal concentrations should be used.
6.3. Buffers/Salts
The PCR buffer for Tuq DNA polymerase consrsts of 50 mA4 KC1 and 10
mM Tris-HCl, pH 8.3, at room temperature. This buffer provides the romc
strength and buffering capacity needed during the reaction It is important to
note that the salt concentratron affects the T,,, of the primer/template duplex,
and hence the annealing temperature. Cosolvents, such as DMSO and glycerol,
have been successfully used in PCR buffers when the targets have very high
denaturation temperatures (18).
6.4. Cycles
The number of cycles of PCR should be optimized with respect to the num-
ber of input target copies. In a typical PCR, 1O r2 copies represents the plateau
m the maximum amount of amplification possible. From a single copy, the most
efficient PCR would reach plateau m 40 cycles (1 012= 240).P CR may be 80-95%
efficient, so the amplification factors are nearer (1.9)“, where y1i s the number
of cycles. It is usually advisable to run the minimum number of cycles needed
to see the desired specific product, since unwanted nonspecific products will
interfere if the number of cycles is excessive (1,17)
6.5. Enzyme/Target
In a standard aliquot of Tag DNA polymerase used for a 100~pL reaction,
there are about 1Oro molecules. Each PCR sample should be evaluated for the
number of target copies it contams or may contain, For example 1 ng of lambda
DNA contams 1.8 x lo7 copies. For low input copy number PCR, the enzyme
is in great excess m early cycles. As the amplicon accumulates in later cycles,
the enzyme becomes limiting and it may be necessary to give the extension
process incrementally more time. Thermal cyclers can reliably perform this
automatic segment extension procedure in order to maximize PCR yield (I, J 7).
7. PCR Protocols
7.1. Reagents and Supplies
The protocol described below illustrates the basic prmciples and techniques
of PCR and can be modified to suit other particular applications. The example
chosen uses the Perkin-Elmer GeneAmplimef HIV Primer pair, SK145 and
SK43 1, in conjunction with the Perkin-Elmer GeneAmp@ PCR Reagent Kit
and PCR Carry-Over Prevention Kit (Perkin-Elmer), to amplify a 142-bp DNA
fragment from the conserved gag region of HIV- 1 using the AmpliWax@ PCR
Gem-facilitated hot start process (J I, 19).
Basic Principles and Routine Practice Ii
Reagents Stock concentrattons
10X PCR buffer II 500 mM KCI, 100 mA4 Tris-HCl, pH 8.3
MgCl, solution 25 mA4
dNTPs 10 &stocks of each of dATP, dCTP, dGTP, 20 astock
of dUTP; all neutrahzed to pH 7 0 with NaOH
Primer 1. SK145 25 r.Ln/ml 10 mM Tris-HCl, pH 8 3
5’-AGTGGGGGGACATCAAGCAGCCATGCAAAT-3’
Primer 2 SK43 1 25 cln/im 10 mMTris-HCI, pH 8.3 in 150 mA4NaC1,
30 mA4Trts-HCI, pH 7 5, 10 mM EDTA, 1 0 mA4 DTT,
0.05% Tween-20,5% (v/v) glycerol.
5’-TGCTATGTCAGTTCCCCTTGGTTCTCT-3’
AmpEraserM UNG Uractl N-glycosylase, 1 0 U/uL
AmpliTaq DNA polymerase 5 U/uL in 100 mMKC1,20 mMTris-HCl, pH 8.0,O.l mM
EDTA, 1 mMDTT, 0 5% Tween 20,0.5% Nomdet P40,
50% (v/v) glycerol
HIV- 1 posmve control DNA 1O 5 copies in 1 ug human placental DNA, 1 mM EDTA,
10 mMNaC1, 10 mMTris-HCl, pH 8.0
AmphWax PCR gems Gem 100s for 50-100 ,uL reactions or Gem 50s for
20-5 0 uL reactions
Use 0 5 mL Perkm-Elmer GeneAmp@ PCR microcentrtfuge tubes and the Perkin-Elmer
GeneAmp@ PCR instrument system
7.2. Methods
In the AmpliWax PCR gem-facilitated hot start process, a solid wax layer
is formed over a subset of PCR reactants, called the lower reagent mix, that
encompasses 30-50% of the total reaction mix volume. The remaining
reactants, called the upper reagent mix, comprise the remaining XL70% of
the total reaction mix volume and are added above the wax layer. In the
first thermal cycle, the wax layer melts during the temperature ramp to the
denaturation temperature and is displaced by the more dense upper reagent
mix. Thermal convection adequately mixes the combined lower and upper
reagent mixes, whereas the melted wax layer acts as a vapor barrier during
each PCR cycle.
1. Assemble the lower reagent mix shown m Table 1.
2. For 100 PL reactions, add 40 jtL of the lower reagent mix (which can be made up
as a batch mix) mto the bottom of each GeneAmp PCR reaction tube. Avoid
splashing liquid onto the tube. If any liquid is present on the tube walls, spin the
tube briefly in a mrcrocentrrfuge.
3. Carefully add one AmpliWax PCR Gem 100 to each tube containing the lower
reagent mix. Melt the wax gem by incubating each reaction tube at 75-80°C for
3-5 min. Solidify the wax at room temperature (25’C) for 3-5 mm
4 Assemble upper reagent mix as in Table 2.
12 Kolmodin and Williams
Table 1
Lower Reagent Mix
Volume, Final concentration,
Reagent 1X mix, uL per 100 pL volume
Sterile water 13.5 N/A
1O X PCR buffer II 4.0 1x
25 mM M&l2 10.0 25mM
10 mMdATP 2.0 200 pA4
10 mA4dCTP 2.0 200 p.A4
10 mMdGTP 2.0 200 pIi4
20 mA4 dUTP 2.0 400 pM
25 pA4primer 1 (SK145) 2.0 05W
25 PM primer 2 (SK43 1) 2.0 0.5 pM
1 U/pL AmpErase UNG 0.5 1 U/reaction
Total volume 40.0
Table 2
Upper Reagent Mix
Volume, Final concentratron,
Reagent 1X mix, uL per 100 pL volume
Sterile water 43.0-52 9 N/A
1 OX PCR buffer II 6.0 1x
5 U/pL AmpliTaq DNA polymerase 05 2.5 U/reaction
1 U/pL AmpErase UNG 0.5 1 U/reaction
lo3 Copres/pL positive control DNA 0.1-10 0 10*-l O4 copies
Total volume 60.0
5. For 100 pL reactions, carefully aliquot 60 uL of the upper reagent mix to each
GeneAmp PCR reaction tube above the wax layer. Avord splashing liqurd onto
the tube wall. If any liquid is present on the tube wall, tap the tube gently to
collect all droplets into the upper reagent layer. Do not spur the tube m a micro-
centrifuge, because this may dislodge the wax layer.
6. Amplify the PCR reactions withm a programmable thermal cycler. For the Perkin
Elmer DNA Thermal Cycler 480, program and run the following linked files:
a. Step cycle file: 95OC for 1 min, 6O“C for 2 min for 2 cycles; lmk to file (2).
b. Step cycle file. 94°C for 1 min, 60°C for 1 min for 38 cycles; lmk to file (3)
c. Time delay file: 60°C for 10 min for 1 cycle; link to file (4).
d. Soak file: 10°C for “forever” (an mfinite hold)
7. Very gently insert a prpet tip through the center of the solid wax layer to form a
small hole. To withdraw the reaction sample, use a fresh tip.