Protocols In Human Molecular Genetics

458 Pages·1991·32.238 MB·English

by  Christopher G. Mathew

CHAPTER1
The Polymerase Chain Reaction
Getting Started
Charles R. M. Bangham
1. Introduction
The polymerase chain reaction (PCR) uses two oligonucleotide primers
to direct the synthesis of specific sequences of DNA. One primer anneals to
the coding strand of DNA and the other to the anticoding strand; the primer
binding sites are typically separated by a few hundred base pairs (loo-
1000 bp). Repeated cycles of polymerization and denaturation lead to the
exponential increase of the sequence defined by the primers. The extraordi-
nary sensitivity and specihcity of PCR have established it as a standard tech-
nique in molecular biology in the short time since it was first described (1).
The purpose of this chapter is to suggest starting conditions for a PCR
reaction and ways to overcome the main problems in PCR. It is intended as a
practical guide, so theoretical aspects will not be discussed in detail. For a
fuller account, there are excellent and comprehensive guides edited by Erlich
(2) and by Innis et al. (3‘). Protocols for special applications of PCR are de-
scribed in later chapters in this volume.
2. Choice of Primers and Target DNA Sequence
The ideal oligonucleotide primer has the following features:
Length: 18-30 bp. Shorter and longer primers may, however, work well.
l
The primers should be similar in length and composition, so that their
predicted melting temperatures (T,, the temperature at which 50% of
the strands are separated) are within 5°C.
From Methods in Molecular Biology, Vol 9 Protocols in Human Molecular GenetIcs
Edited by. C. Mathew Copyright Q 1991 The Humana Press Inc., Clifton, NJ
1
2 Bangham
. GC content should be similar to the GC content of the template and of
the other primer, ideally 5040% GC.
. Binding site on target DNA: conserved region of sequence, ending on a
nondegenerate base, e.g., first or second base of a conserved amino acid.
. No selfcomplementarity (to avoid secondary structures) or complement-
arity with the other primer. Computer programs are available to help
identify such complementarity.
. No runs of three or more Gs or Cs at the 3’ end of the primer.
. If mismatches between primer and template are known or likely to
occur, these should be minimized at the 3’ end of the primer, i.e., where
the DNA polymerase binds. Highly degenerate primers may work under
nonstringent reaction conditions, provided that at least three bases match
at the 3’ end of the primer (4).
l Restriction sites can be included in the primer to help in efficient and
directional cloning of the amplified product.
The ideal target sequence (template) to be amplified has the following
features:
. Length: 150300 bp. Lengths between 100 and 2000 bp can, however,
often be amplified efficiently.
. Unique sequence, to avoid competition from unwanted templates.
. High copy number, to minimize the number of cycles of amplification
required. PCR is, of course, highly efficient in detecting rare DNA spe-
cies, but the risk of confusion with low-abundance contaminating DNA
species increases if the target copy number is low.
. A diagnostic restriction enzyme site, to help verify amplification of the
correct product.
. An intron sequence, to distinguish genomic amplification product from
those amplified from cDNA or contaminating DNA (see Section 6).
. A sequence that can be detected specifically with a probe already in the
laboratory.
3. Reagents
Highquality reagents are necessary for efficient amplification: particu-
larly important are the DNA polymerase -usually the heat-stable enzyme from
the thermophilic bacterium Thus aquaticus (e.g., Per-kin-Elmer/Cetus
AmpliTaq@)-and the deoxynucleoside triphosphates (dNTPs). Stocks can
be prepared as follows:
1. lMKC1, 100 mL.
2. lMTris-HCl, pH 8.3 at 25”C, 100 mL.
Getting Started in PCR 3
3. O.lMMgCl,, 100 mL.
4. 0.2% Gelatin (Difco), 100 mL.
Solutions l-4 should be autoclaved and stored in 20-mL aliquots at room
temperature.
5. Oligonucleotide primers: 50 @Iupstream primer (300 pg/mL of a
20-mer) ; 50 PA4 downstream primer (300 pg/mL of a 20-mer) .
6. 100 mMdNTPs at neutral pH (e.g., Pharmacia, Central Milton Keynes,
Buckinghamshire, UK), stored at -80°C in aliquots of 5 or 10 uL.
To minimize the risk of cross-contamination with DNA templates from
plasmids or previous amplification reactions, the stock solutions may be irra-
diated with UV light at 254 nm, e.g., 10 min in a Stratalinker 1800TM
(Stratagene, Cambridge, UK). A 1-mL stock of 2x amplification solution con-
taining all components except Tuq polymerase and DNA template can be
made, and stored at -20°C in 50-p.L aliquots in siliconized 0.5-mL polypro
pylene tubes. The reaction mixture is then completed by adding the DNA
template, Taq polymerase (e.g., l-2.5 Cetus U of AmpliTaq@), and sufbcient
sterile water to bring the vol to 100 p.L
4. Design of Reaction Mixture
For many purposes, the reaction mixture given in Table 1 will give effi-
cient and specific amplification. However, there are a few variables that criti-
cally affect the efficiency and specificity of the reaction; the most important
of these are the magnesium ion concentration and the oligonucleotide primer
concentration (see below and Section 6).
The optimal number of DNA molecules in the template is between 105
and lo6 (3). For single-copy genes, this corresponds to approx 1 p.g of human
genomic DNA and 1 pg of a 6kbp plasmid.
Optimization of the reaction mixture for a particular pair of oligo
nucleotide primers frequently involves two further steps:
1. Optimize Mg2+ concentration. Amplify the template with the following
concentrations of Mg 2+: 1.5 (asabove); 3.0; 4.5; 6.0; and 7.5 mM. Certain
primer pairs may require further, finer adjustment of Mg2+ concentra-
tion, to within 0.5 mM.
2. Optimize primer concentrations. Amplify the template with the best Mg2+
concentration (as determined above), with the following concentrations
of each primer: 0.05; 0.1; 0.25; 0.5; and 1.0 PM.
Certain GGrich templates do not amplify with the above protocol, prob
ably because they rapidly adopt stable secondary structures on cooling from
94’C. The addition of dimethyl sulfoxide (DMSO) to the reaction mixture
Bangham
Table 1
Basic PCR Reaction Mixture
Final
concentration, Volume, PL, for
Reagent in lx 2x buffer, 50 p.L 2x buffer, 1 mL
1MKCl 50 mM 5 100
lMTris-HCl 10 mM 1 20
O.lMMgCI, 1.5 mM 1.5 30
0.2% gelatin 0.01% 5 100
100 mMdGTP 200 nM 0.2 4
100 mM dATP 200 nM 0.2 4
100 mM d’lTP 200 nM 0.2 4
100 mMdCTP 200 nM 0.2 4
50pM5’primer 1ClM 2 40
50 pM 3’ primer lClM 2 40
Sterile, deronized water - 33 654
(final concentrauon, 10%) may allow successful amplification, but this is not
recommended in other cases, since it decreases the efficiency of the poly-
merase enzyme by about 50% (5).
Addition of an overlay of inert mineral oil (about 50 pL) (e.g., paraffin
oil BP, British Pharmacoepia) to the reaction mixture minimizes evapora-
tion during amplification, and so increases the efficiency and reproducibility
of the reaction (6). However, it is not essential: if siliconized 0.5mL tubes are
used, the droplets that condense on the walls of the tube rapidly return
to the solution. To reduce the number of components in the mixture, and so
reduce the risk of DNA contamination, the mineral oil and gelatin, and in
some instances the KCl, may be omitted.
5. Choice of Reaction Conditions
As with the reaction mixture design (Section 4), the following condi-
tions serve to amplify efficiently and specifically in many cases. However, there
are frequent instances in which the conditions need to be changed for a
particular pair of primers. The most important variable to be optimized for a
given primer pair is the annealing temperature. This adjustment is a highly
empirical process; for example, the annealing temperature may need to be
set at, or even above, the predicted T, of a primer (note that the formula
given below for estimating the T, takes no account of the magnesium ion
concentration).
Getting Started in PCR 5
5.1. Denuturation (94°C)
Incomplete denaturation is a frequent cause of failure of PCR. In the
initial denaturation step, we use 5 min for a genomic DNA template and 2
min for a plasmid template. In subsequent cycles, 20-30 s at 94°C is adequate.
If much longer times are required for successful amplification, the tempera-
ture in the reaction mixture itself should be measured with a thermocouple
of low specific heat capacity to verify that the solution actually reaches the
temperature required for denaturation.
5.2. Annealing (30-60 s)
First calculate the approximate T, of the oligonucleotide primers,
using a simple formula (7), such as:
T,=2x(AtT)t4x(GtC)
in ‘C.
Then set the annealing temperature at 5°C below the lower of the two
predicted T,s.
If nonspecific amplification products are a particular problem, anneal-
ing and extension can be performed in a single step at between 60 and i’2”C.
5.3. Extension (72°C)
Allow 1 min/l kbp of desired product. If the required product is short
(<ZOO bp), then the extension step can be omitted, since Tuq polymerase
is sufficiently active at lower temperatures to complete the reaction during
the transition between the annealing temperature and the denaturauon
temperature.
6. Troubleshooting in PCR
It is now widely realized that the remarkable sensitivity of PCR is also its
main limitation, because a single contaminating molecule of DNA contain-
ing the target sequence may be amplified, leading to potentially serious mis-
interpretation of the results (8-11) The standards of cleanliness required in
making up the solutions are therefore higher than for almost any other
laboratory procedure, albeit for different reasons.
The main precautions to be taken to avoid false positive results in PCR
are listed in Table 2, in approximate order of importance. It is essential to
include in each experiment a tube containing all the components except the
DNA template, and to examine the products on a gel stained with ethldium
bromide, to look for contamination of the reaction mixture.
Bangham
Table 2
Precautions to Avoid DNA Contamination of PCR Reactions
1. Make up reaction mixtures in a laboratory in which plasmids containing the
target sequence are never handled. Neuertake amphfied product mto this labo-
ratory. Many workers make up therr PCR solutions in lammar-flow hoods.
2. Wear gloves when making up solutions; avoid touching the inside of the
tube cap.
3. To make up reactron mixtures, use pipets that are never used to handle plas-
mid or amplificatton productswtth the appropriate sequence. We recommend
“positive displacement” pipets (e.g., G&on “Mrcroman”), wrth ups contaunng
disposable plungers that prevent aerosol contact or direct contact between
the prpet barrel and the solution. For handling small volumes (0.5-10 uL),
calibrated drsposable glass microcapillaries are very useful (e.g , Drummond
PCR microprpets) .
4. Ahquot reagents and reaction buffers, and use each ahquot only once. See also
Sections 2 and 3.
5. Irradiate solutions used in PCR wrth UV. This was shown to abohsh the amph-
fication of plasmid that was dehberately added to PCR mixtures (8) Solutions
contaming all components except the DNA template can safely be irradiated
for 10 min on a 30.5nm-wavelength laboratory UV transrllummator, without
denaturing the primers or the enzyme.
6. Avoid reamplificauon of primary amphfied products, rf possible. If amphfica-
tion of gel-punfied DNA fragments is necessary, irradiate the agarose gel and
its running buffer in the gel apparatus with 254nm W before running: 10 min
in a Stratalinker 1800TM (Stratagene) is sufficient.
7. Some workers find that contammation is abolished only when the person mak-
ing up the solutions wears a surgical face mask and someumes a harr net (9, J.
Todd, personal communication).
In some cases (for example, in RNAviruses), it may be possible to arnpli-
fy between conserved nucleotide sequences, across a highly variable sequence.
If the frequency of nucleotide differences between two amplified products
greatly exceeds the error rate of Tuq polymerase, then DNA crosscontamin-
ation can be excluded beyond reasonable doubt (II, 12).
The dose of W radiation required to prevent amplification depends on
the size and the base composition of the potential contaminating species
(13). Ideally, the dose should be titrated with a given template and a known
contaminant with the W source used in the laboratory.
The other common problems in PCR relate to the specificity and effr-
ciency of amplification of the required product, avoiding amplification from
partial matches between the primers and template. Some of these have been
addressed above (seeSections 3 and.5); asummaryof the most frequent causes
and their remedies is given in Table 3.
Getting Started in PCR 7
Table 3
Troubleshooting in PCR
Problem Causes Remedies
No detectable product Inadequate melting of Increase ume in
after repeated at- DNA template denature step
tempts Target sequence too rare Increase number of
Annealing temperature cycles (up to 60)
too high Lower temperature by
CC content of target 5°C
sequence too high Try 10% DMSO in
Primers anneal to each reaction
other (primer dimer) Seen ote a
or to themselves
“Overamplificauon”
Multiple bands on Reduce number of
agarose gel of ampli- cycles; reduce exten-
fied product Primers too short or sron time
degenerate Seen o& a
Concentration of dNTPs
or of enzyme too high Reduce either by 2-10x
Annealing temperature
too low for CC
Continuous “smear” of content of primers Raise annealing tempera-
amplified product on ture by 5°C
agarose gel “Overamplification”
Predominance of very Reamplification of Reduce number of cycles
high mol wt amplified primary amplified Gel-purify primary
product product product before
reamplifcauon
Complementanty
between 3’ ends Seen ote a
“Primer dimer’% of pnmers
p In each case, the remedy 1st o increaset he stringency of the reacuon by increasing
the annealing temperature orreducing the primer concentratton orboth
b “Overamplification” denotes the use of too many cycles of PCR, which favors the
amphficauon of mismatched or nonspectfic DNA products. For amplification of a smgle-
copy gene from genomic DNA, 35 cycles should be enough, but more cycles may be
needed for a rare species, such as a low-copy-number mfectious agent
c The “primer dimer” results from annealing and polymerization of the 5’ pnmer on
the z)’ pnmer, and appears as a fuzzy low-molwt band on an ethtdmm bromtde stamed
agarose gel
8 Bangham
If the problem is one of persistent failure to amplify any band, it may be
necessary to choose a different sequence for one or both primers: certain
sequences are very inefficient as PCR primers, for unknown reasons. If this is
suspected, each primer should be tested in a PCR reaction with another PCR
primer of demonstrated efficacy, from the same template sequence (if avail-
able). In this way it is frequently possible to show which of the two primers is
at fault.
References
1. Saiki, R., Scharf, S., Faloona, F., Mullis, K. B., Horn, G T , Erhch, H A , and Amhelm,
N (1985) Enzymatic amplification of betaglobin genomrc sequences and restriction
analysis for diagnosis of sickle cell anemia. Snence 230, 1350-1354
Erhch, H. A., ed. (1989) PCR Technology: f+wu+!es and Aj$dwatzon f&r DNA Amplzfica-
hon. Stockton, New York.
Innis, M. A., Gelfand, D. H., Snmsky, J. J., and White, T. J , eds (1990) PCR Protocols.
A Gurde to Methods and App1rcat:on.s. Academic, New York.
Sommer, R. and Tautz, D. (1989) Muumal homology requirements for PCR primers
Nuckic Ands Res. 17,6’749.
Gelfand, D. H. and Whtte, T. J. (1990) Thermostable DNA polymerases, m PCR Protc-
cols: A Guade to Methods and Apphcatzonr Inms, M A , Gelfand, D H , Snmsky, J J , and
White, T. J., eds. Academic, New York, p. 129
6. Mezei, L. M. (1990) Effect of oil overlay on PCR amphficauon, m Amp2ajicatron.s Perkm-
Elmer, Norwalk, CT, vol. 4, p. 11.
7. Them, S. L. and Wallace, R. B. (1986) The use of synthetic ohgonucleoudes as spe-
cific hybndtzation probes m the diagnosis of genetic disorders, m Human Gen&c Dzs-
eases:A fiactrcal Ap@ach. K. E. Davies, ed. IRL, Oxford, UK, pp. 33-50
8. Sarkar G. and Sommer, S. S. (1990) Shedding light on PCR contammauon. Nature
343,27.
9. Kitchin, P. A., Szotyori, Z., Fromholc, C , and Almond, N (1990) Avoidance of false
positives. Natun 344,201.
10. Kwok, S. and Higuchi, R. (1989) Avordmg false positives wnh PCR Nature339,237,238
11. Bangham, C. R. M , Nightingale, S., Cruickshank, J K., and Daenke, S. (1989) PCR
analysis of DNA from muluple sclerosis patients for the presence of HTLV-I Sczence
246,821.
12. Daenke, S., Nightingale, S., Crurckshank, J. K, and Bangham, C R M (1990) Se-
quence vanants of human T-cell lymphotropic virus type I from patients with tropical
spasuc paraparesrs and adult T-cell leukemia do not distmgursh neurological from
leukemic isolates. j. Viral. 64,12%-l 282
13. Crmmo, G. D., Metchette, K., Isaacs, S. T., and Zhu, Y. S. (1990) More false positive
problems. Nature 345, ‘7’73,174.
CHAFTER2
Direct DNA Sequencing
of Complementary DNAAmplified
by the Polymerase Chain Reaction
Richard A. Gibbs, Phi-Nga Nmyen,
and C. Thomas Caskey
1. Introduction
Protocols for the sequence analysis of conventional single-stranded or
double-stranded DNA templates are often unsuitable for the direct sequenc-
ing of DNA fragments generated by the polymerase chain reaction (PCR)
(1,2). The features that can distinguish PCR products as templates for se-
quencing include (a) contamination of the reactions by nonspecific PCR
amplification products that are complementary to the sequencing primer,
(b) the persistence of “leftover” PCR primers from the amplification reac-
tions, and (c) the potential for competition between one strand of the ampli-
fied fragment and the oligonucleotide used for the sequencing. The various
approaches that have been used to overcome these problems include
1. The use of 5’-end-labeled DNA-sequencing primers that are comple-
mentary to regions between the PCR primers (3);
2. Gel purification of amplified DNA to remove unwanted fragments and
primer (4);
3. Spin columns for the separation of leftover primers from high mol wt
material (5, 6);
4. “Asymmetric” or knbalanced” PCR priming to generate an excess of
single strands during the initial amplification (7);
From* Methods in Molecular Bology, Vol. 9. Protocols in Human Molecular GenetIcs
Edited by C. Mathew Copyright 0 1991 The I-hJmana Press Inc., Cl&on, NJ
9
10 Gibbs, Nguyen, and Caskey
5. Addition of dimethylsulfoxide (DMSO) to sequencing reactions with
short annealing times (8); and
6. The use of several short, high-temperature, sequencing cycles (9).
In developing the protocol that is described here (summarized in Fig.
l), we have endeavored to avoid the tedious steps of gel purification or col-
umn chromatography. Instead, we have developed a twostep reaction proce-
dure for template preparation that first allows amplification of a specific
fragment and then the production of an excess of one strand. This method is
essentially a modification of the asymmetric priming protocol of Gyllensten
and Erlich (‘7). The current method can be performed comfortably in two
days and enables the reliable generation of DNA sequence ladders that can
be resolved as far as the gel system that is used will allow. The technique has
been applied for the analysis of transcribed human sequences, for which it is
preceded by a reverse transcription reaction. Equal success has been obtained
in the analysis of human gene sequences using lo-100 ng of genomic DNA as
starting material and there is no reason that virtually any DNA fragment that
can be successfully amplified by PCR would not be amenable to this analysis.
Features that are modifications of other protocols or that we regard as par-
ticularly important are further discussed below.
2. Materials (see Note 1)
1. Ribonuclease inhibitor (RNasin; Pharmacia, catalog no. 27-0815-01;
27,000 U/mL).
2. Random hexamer primers (pd(N)s, Pharmacia, catalog no. 27-2166-01;
at 5 mg/mL) .
3. 5x POL buffer (250 mMTrisHC1, pH 8.3 at 3’7”C, 40 mM MgCl,, 150
mMKC1; 50 mMdithiothreito1 [D’IT]).
4. Deoxyribonucleotide triphosphate mixture (dNTPs; mixture of 2.5 mM
each of dATP, dTTP, dCTP, and dGTP) .
5. Reverse Transcriptase (M-MuLV, Pharmacia catalog no. 27-0925
02;12,000 U/mL).
6. Modified T7 DNA polymerase (SequenaseTM; usually supplied at 12.5
U&L from United States Biochemicals [USB] catalog no. 70722).
7. Dideoxynucleotide terminator mixtures; these are 80~8 pMdeoxy:dideoxy
mixtures. The solutions supplied by USB, catalog nos. 70714 (“A” mix),
70716 (“C” mix), 70718 (“G” mix), and 70720 (,T” mix) are appropri-
ate. The solutions are thawed and stored in 20-PL aliquots. SequenaseTM
(1 .O l.tL) is added to each just before use.
a. Sequencing stop solution (STOP; 95% formamide, 20mMEDTA, 0.05%
bromophenol blue, 0.05% xylene cyanol).