Molecular Diagnosis of Genetic Diseases

377 Pages·2002·4.723 MB·English

by  Rob Elles

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Optimizing PCR for Clinical Diagnosis
Michael P. Bulman
1. Introduction
The polymerase chain reaction (PCR) has rapidly become an essential tool
within the diagnostic laboratory. Therefore,it is crucial when setting up a new
PCR-based test to ensure that the PCR reaction is carefully designed to be as
robust and reliable as possible. Usually,little optimization is required. However,
there are some instances when a particular region of DNA proves difficult to
amplify by PCR. A number of factors are important to consider when choos-
ing PCR conditions,and these are discussed in this chapter.
2. Materials
Analytical-grade reagents should be used at all stages, unless otherwise
stated.
2.1. PCR Reaction Buffers
As an alternative to the buffer supplied by the manufacturer,use of either of
the following buffers may be beneficial in the PCR.
2.1.1.Buffer “A” 10X
1. 670 mMTris-HCl (pH 8.0 at 25°C).
2. 166 mMammonium sulfate.
3. 37 mMmagnesium chloride.
4. 67 µMethylenediaminetetraacetic acid (EDTA).
5. 0.85 mg/mL bovine serum albumin (BSA).
Filter-sterilize and store in 1-mL aliquots at –20°C.
This is a particularly robust PCR buffer,which provides a good yield of prod-
uct,and is excellent regardless of the quality of the DNA template.
From:Methods in Molecular Medicine, vol.92:Molecular Diagnosis of Genetic Diseases, Second Edition
Edited by:R.Elles and R.Mountford ©Humana Press Inc., Totowa, NJ
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2 Bulman
2.1.2.Buffer “R” 20X
1. 1.0 MTris-HCl (pH 9.0 at 25°C).
2. 400 mMammonium sulfate.
3. 30 mMmagnesium chloride.
Filter-sterilize and store in 1-mL aliquots at –20°C.
This buffer is useful for templates that are difficult to amplify,such as those
with GC-rich tracts,and for longer templates (800 basepairs [bp] + in size).
2.2. Deoxynucleotide Triphosphates (dNTPs)
Purchase individual dNTPs as 100 mMstocks (e.g.,Roche Diagnostics UK
Ltd,Lewes,UK; dNTP set #1969064). Make a 2.5-mMworking stock by com-
bining 2.5 µL of each dNTP and adding 90µL of water. Store dNTPs in small
aliquots at –20°C and avoid multiple freeze-thaw cycles, as dNTPs are prone
to degradation (especially for multiplex PCR). The final concentration of each
dNTP in the PCR reaction is usually 200–250 µM. To increase the speci-
ficity/fidelity of the PCR, decrease the concentration of dNTPs with a corre-
sponding decrease in MgCl concentration.
2
2.3. Magnesium Chloride (if required)
10X “A” and 20X “R” buffers already contain magnesium at 37 mM and
30 mM respectively (to give a final concentration in the PCR reaction of
3.7 mMand 1.5 mM,respectively). However,for those 10X buffers that do not
contain magnesium, this is usually supplied as a 25–50-mMstock and should
be used at 1.5–5.0 mMfinal concentration in the PCR. Mix the magnesium thor-
oughly prior to addition to the PCR mix.
2.4. Oligonucleotide Primers
The final concentration in a PCR reaction should be between 0.1 µM and
0.5 µMof each primer. Primer concentrations that are too high may lead to mis-
priming in the reaction. Conversely,primer concentrations that are too low may
not give good yields of product.
2.5. Polymerases
There are many suppliers and varieties of heat-stable Taqpolymerases on the
market. (See http://www.alkami.com/methods/refpoly.htm and/or http://
www.neb.com/neb/frame_tech.html for information of most of those avail-
able.) The most frequently used enzyme is Taqpolymerase. This enzyme does
not have a 3′–5′exonuclease activity,which has two consequences: it exhibits
a non-template addition of usually an adenine base at the 3′end of the product
(1); secondly, the lack of the 3′–5′ exonuclease activity means that Taq
does not correct for the incorporation of mismatched bases—it has no “proof-
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Optimizing PCR for Clinical Diagnosis 3
reading”activity (2–5). Therefore,for those techniques such as PCR-based site-
directed mutagenesis where a reduced error rate is crucial,it is important to use
a proof-reading enzyme such as Pfu from Stratagene (La Jolla, CA) (6) or
Vent™ polymerase from New England Biolabs (Hitchen, Hertsfordshire, UK)
(7). For the vast majority of applications,however,Taqis perfectly adequate.
2.6.Template DNA
Dilute good-quality genomic DNA in TE or deionized water to approx
25–50 ng/µL and use 2.5-µL in a 25-µL reaction.
3. Methods
Unfortunately,there is no single set of conditions that can be applied to all
PCR amplifications. Factors such as primer sequence, product length, and
primer annealing temperature will differ for each assay. For the reliable ampli-
fication of a specific target, the optimal conditions for PCR will be found
empirically. However,a well-designed PCR reaction should work with little or
no optimization necessary.
3.1. Design of PCR Primer Pairs
The selection of the correct pair of primer sequences for the PCR reaction
may be the most critical parameter for successful PCR. The primer set must
hybridize efficiently to the target sequence with as little hybridization as pos-
sible to other sequences also present in the sample. Poorly designed primers
may result in the synthesis of little or no product as a result of “primer-dimer”
formation and/or nonspecific amplification.
3.2. Primer Length
In general, oligonucleotides between 20 and 30 bases are sufficiently
sequence-specific for complex genomes,provided that the annealing tempera-
ture is optimal.
3.3. GC Content
Ideally,primer sequences should be designed to have a GC content between
45% and 55%. Stretches of poly C or poly G should be avoided, as these can
promote nonspecific annealing. Similarly,stretches of poly A or poly T should
also be avoided,as these may open up stretches of the primer-template complex.
3.4. Melting Temperature (T )
m
As a starting point,the annealing temperature for a primer pair is calculated
as 5°C below the estimated T . Ideally the primers should closely match each
m
other in their melting temperatures,or amplification efficiency will be reduced
and may even lead to the failure of the PCR.
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4 Bulman
A rough and ready way to calculate T for primers <20 bp is (8):
m
T = [4(G+C) + 2(A+T)]°C (1)
m
To calculate T for primers >20 bp use (9):
m
T = 62.3°C + 0.41°C (%G-C) – 500/length (2)
m
Online T calculators such as that found at http://alces.med.umn.edu/
m
rawtm.html are also extremely useful.
3.5. General Comments on Primer Design
Alternatively,use a free web tool such as Primer3(available at http://www-
genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi) or a commercially avail-
able program such as Oligo 6 primer analysis software (Molecular Biology
Insights Inc.,Cascade,CO; http://www.oligo.net) to help design primers.
In conclusion, an “ideal” primer will have a 50% GC content with a
near random nucleotide composition, and will be 20–25 bp long resulting in
a T of 56–62°C. However, primers can only be designed from the available
m
sequence,and sometimes primer design is tricky. Compromise is the key,and
it is very unusual to be unable to design a primer pair that will work after some
optimization.
3.6. Standard PCR Reaction
A standard 25µl PCR reaction contains the following components:
Component Volume
10X buffer 2.5 µL
dNTP mix 2.5 mMeach 2.5 µL
5 µMprimer mix 5.0 µL
Taq polymerase (5 U/µL) 0.05–0.2 µL
DNA template (~25–50 ng/µL) 2.5 µL
Sterile deionized water to 25 µL
It is important to mix all components thoroughly after thawing prior to
assembly of the PCR master mix.
Note:It is essential to include a water “negative control”for each PCR setup,
otherwise contamination in the reaction components is not apparent.
3.7. PCR Program
3.7.1.Denaturing Temperature
This is normally 94–95°C for 30 s to 1 min per cycle after an initial 3–5 min
incubation at 95°C. It is important to keep the denaturation temperature/
time to a minimum,since Taqpolymerase may lose activity in repeated cycling.
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Optimizing PCR for Clinical Diagnosis 5
3.7.2.Annealing Temperature
See Subheading 3.4. to estimate the T and anneal at 5°C below this
m
temperature.
3.7.3.Elongation Temperature and Time
The elongation temperature is normally 70–72°C, and the elongation time
depends on the size of the final size of the PCR product. Generally, 30 s to
2 min is sufficient for most PCR reactions; however, for larger products, a
general rule is to extend for 1 min per kilobase of product size.
3.7.4.Cycle Number
The standard number of cycles necessary for efficient amplification is
25–40 cycles. Increasing this to >40 cycles does not generally increase the rel-
ative amount of PCR product because of the plateau effect,in which the expo-
nential rate of product accumulation in the later stages of PCR is attenuated (10).
3.7.5.Final Extension
Program one final extension cycle for 7–10 min at 72°C to fill in any incom-
plete polymerization. Then cool the reaction to 4–18°C.
3.7.6.Thermal Cyclers
Many thermal cyclers are available. Some of these are compared at http://
www.biocompare.com/molbio.asp?catid=33. The author uses the 0.5-mL
GeneAmp® PCR System 9700 (Applied Biosystems [ABI], Foster City, CA)
and the MJ Research DNA Engine Tetrad™Thermal Cycler (Genetic Research
Instrumentation Ltd,Braintree,Essex,UK).
4. Notes
Most manufacturers of PCR reagents and equipment have excellent web
sites with useful online guides that can often be downloaded as PDFs (e.g.,
Qiagen at http://www.qiagen.com/literature/pcrlit.asp; ABI at http://www.
appliedbiosystems.com/support/techtools/; Invitrogen at http://www.invitrogen.
com/content.cfm?pageid = 4155&cfid = 6398854&cftoken = 97182355).
4.1. Hot Start PCR
PCR hot start can be used to increase the reaction sensitivity, reduce non-
specific products in the PCR,and increase the PCR yield. The simplest way to
set up hot start PCR is to use one of the chemically modified Taqpolymerases
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6 Bulman
such as AmpliTaq Gold (ABI). For details,seehttp://www.appliedbiosystems.
com/products/productdetail.cfm?ID = 104 or Platinum Taq (Invitrogen Ltd.,
Paisley,UK).
4.2. Enhancers for PCR
It may be beneficial to use one or more additives to increase the yield,
specificity, and consistency of the PCR reaction. A variety of such agents are
available, including dimethyl sulfoxide (DMSO), dimethyl formamide,
betaine (N, N, N-trimethylglycine = [carboxymethyl] trimethylammonium)
formamide, 7-deaza-2′-deoxyguanosine (7 deaza GTP), non-ionic detergents
(e.g., Triton X-100, Tween 20, and Nonidet P-40), BSA, urea, and glycerol.
These additives are believed to lower the T of the target DNA. A helpful
m
discussion of the benefits of the most useful additives can be found at
Rob Cruickshank’s PCR additive page at http://taxonomy.zoology.gla.ac.uk/
~rcruicks/additives.html
Two of the most commonly used PCR additives are the organic solvents
DMSO and betaine. Both of these are particularly useful for GC-rich templates
by acting as helix destabilizers. DMSO is included in the PCR reaction at a final
concentration of 5–10% (v/v), although DMSO at 10% and higher has been
shown to reduce the activity of Taqby up to 50% (10,11). Betaine is used at a
final concentration of 1 M (from a 5-M stock in water), and is often included
in commercial PCR kits as an “unidentified”additive.
Recently, a number of novel potent PCR enhancers have been discovered,
and the most effective of these is tetramethylene sulfoxide (12). Use of low-
mol-wt compounds such as this has been shown to be more beneficial in the
amplification of high GC-rich templates than DMSO and betaine.
4.3. PCR Mixture for Difficult to Amplify Templates
Compound Volume
10X PCR buffer 2.5 µL
Modified dNTP mix* 1.0 µL
5 µMprimer mix 5.0 µL
1 mM7-deaza GTP 1.25 µL
DMSO 1.25 µL
5 Mbetaine 5.0 µL
Amplitaq Gold™ 0.25 µL
DNA (25 ng/µL) 5.0 µL
Sterile deionized water To 25 µL
*Modified dNTP mixture contains:5 µL each of dTTP,dCTP,and dATP plus 3.8 µL of
dGTP made up to 100 µL with water (100-mMstocks of each dNTP).
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Optimizing PCR for Clinical Diagnosis 7
Cycling conditions:
1. 95°C*12 min 1 cycle
2. 95°C*11 min}
3. X°C*11 min 40 cycles
4. 72°C*12 min
5. 72°C*10 min 1 cycle
*Annealing temperature varies depending on the primer set.
4.4. Multiplex PCR
This is a demanding technique often used to amplify several PCR products
in a single reaction. Extensive optimization is often required to produce a robust
and reliable PCR reaction as with multiple primer pairs in a single-tube reac-
tion, which increases the likelihood of primer-dimer and other nonspecific
products that may interfere with the amplification of the products required. An
extensive troubleshooting guide for multiplex PCR can be found at http://www.
info.med.yale.edu/genetics/ward/tavi/Trblesht.html.
References
1. Clark,J. M. (1988) Novel non-templated nucleotide addition reactions catalyzed
by procaryotic and eucaryotic DNA polymerases. Nucleic Acids Res. 25,
9677–9686.
2. Tindall,K. R. and Kunkel,T. A. (1988) Fidelity of DNA synthesis by the Thermus
aquaticus DNA polymerase. Biochemistry9,6008–6013.
3. Krawczak, M., Reiss, J., Schmidtke, J., and Rosler, U. (1989) Polymerase chain
reaction:replication errors and reliability of gene diagnosis. Nucleic Acids Res.25,
2197–2201.
4. Kwok, S., Kellogg, D. E., McKinney, N., Spasic, D., Goda, L., Levenson, C.,
et al. (1990) Effects of primer-template mismatches on the polymerase chain reac-
tion:human immunodeficiency virus type 1 model studies. Nucleic Acids Res.25,
999–1005.
5. Eckert, K. A. and Kunkel, T. A. (1991) DNA polymerase fidelity and the poly-
merase chain reaction. PCR Methods Appl.1,17–24. Review.
6. Lundberg,K. S.,Shoemaker,D. D.,Adams,M. W.,Short,J. M.,Sorge,J. A.,and
Mathur, E. J. (1991) High-fidelity amplification using a thermostable DNA
polymerase isolated from Pyrococcus furiosus. Gene1,1–6.
7. Mattila,P.,Korpela,J.,Tenkanen,T.,and Pitkanen,K. (1991) Fidelity of DNA syn-
thesis by the Thermococcus litoralis DNA polymerase—an extremely heat stable
enzyme with proofreading activity. Nucleic Acids Res.25,4967–4973.
8. Suggs, S. V., Hirose, T., Miyake, E. H., Kawashima, M. J., Johnson, K. I.,
and Wallace,R. B. (1981) Using Purified Genes,in ICN-UCLA Symp. Dev. Biol.
23,683.
9. Bolton,E. T. and McCarthy,B. J. (1962) Proc. Natl. Acad. Sci. USA48,1390–1397.
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10. Innis,M. A. and Gelfand,D. H. (1990) Optimisation of PCRs,in PCR Protocols
(Innis,Gelfand,Sninsky and White,eds.),Academic Press,New York,pp. 3–12.
11. Gelfand,D. H. and White,T. J. (1990) Thermostable DNA polymerases,in PCR
Protocols(Innis,Gelfand,Sninsky and White,eds.),Academic Press,New York,
pp. 129–141.
12. Chakrabarti, R. and Schutt, C. E. (2002) Novel sulphoxides facilitate GC-rich
template amplification. BioTechniques32,866–874.
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Current and Emerging Techniques
for Diagnostic Mutation Detection
An Overview of Methods for Mutation Detection
Claire F.Taylor and Graham R.Taylor
1. Mutation Detection: An Introduction
This chapter provides a broad overview of the range of mutation detection
techniques that are now available.
For the purposes of this chapter, a mutation can be defined as a sequence
change in a test sample compared with the sequence of a reference standard.
This definition implies nothing about the phenotypic consequences (e.g.,path-
ogenicity) of a mutation. A polymorphism may be defined as a mutation that
occurs in a substantial proportion (>1%) of a population and is tacitly assumed
to be non-pathogenic, although the true pathogenicity may be unknown. A
polymorphism has also been defined as a Mendelian trait that exists in the pop-
ulation,with the frequency of the more rare of the two alleles greater than 1–2%
(1). If we accept that DNA sequence is a Mendelian trait,then the two defini-
tions of polymorphism are the same.
The detection of a single base change in the human genome requires a
signal(cid:1)background ratio of 1(cid:1)6 × 109—a formidable task. To achieve such
selectivity in the field of electronics would require amplification and noise
reduction,and it is no surprise that analogous processes are found in molecu-
lar genetics—for example, amplification by the polymerase chain reaction
(PCR) and noise reduction by the stringent annealing of probes and primers.
Mutation detection techniques can be divided into techniques that test for
known mutations (genotyping) and those that scan for any mutation in a par-
From:Methods in Molecular Medicine, vol.92:Molecular Diagnosis of Genetic Diseases, Second Edition
Edited by:R.Elles and R.Mountford ©Humana Press Inc., Totowa, NJ
9