Vascular Disease. Molecular Biology and Gene Transfer Protocols

404 Pages·1999·3.247 MB·English

by  Andrew H. Baker

Mutation Detection by PCR-SSCP Analysis 3
1
Detection of Mutations and DNA Polymorphisms
in Genes Involved in Cardiovascular Diseases
by Polymerase Chain Reaction–Single-Strand
Conformation Polymorphism Analysis
Shu Yeand Adriano M. Henney
1. Introduction
Over the last 15 years, there has been remarkably rapid progress in defining
the molecular basis of inherited disorders. Many disease genes (the majority of
which are genes responsible for monogenic Mendelian diseases) have now been
identified, predominately through linkage analysis and positional cloning
approaches. With the continuing expansion in this research area, the number of
genes to be screened for disease-causing mutations will continue to increase,
especially as there are now worldwide efforts aiming to identify the gene
lesions that contribute to complex diseases, such as hypertension, diabetes
mellitus, and coronary artery diseases, each of which involves many suscepti-
bility genes.
Disease-causing mutations can be broadly classified into two groups: those
causing a significant change in chromosome or gene structures (e.g., large
deletions, insertions, and rearrangements) and those involving only one or a
few nucleotides (e.g., point mutations, and small deletions and insertions) (1).
The former group of mutations can be detected using, for example, cytogenetic
techniques, pulsed field gel electrophoresis, and Southern blotting. Detection
of the latter group of mutations, however, require different methodologies.
DNA sequencing will be the ultimate technique for identifying such mutations.
However, despite automation, sequencing remains a relatively slow procedure
and is not cost-effective. Therefore, a number of different mutation detection
techniques have been developed, such as ribonuclease A cleavage analysis and
From:Methods in Molecular Medicine, vol. 30: Vascular Disease: Molecular Biology and Gene TherapyProtocols
Edited by: A. H. Baker © Humana Press Inc., Totowa, NJ
3
4 Ye and Henney
chemical cleavage analysis, both of which involve cleavage of heteroduplex
molecules at the site of mismatched base pairs resulting from a point muta-
tion; denaturing gradient gel electrophoresis and temperature gradient gel
electrophoresis, which assess the differences in the melting point of hetero-
duplex molecules; and single-strand conformation polymorphism analysis
and heteroduplex analysis (seeChapter 2), which rely on the differences in gel
electrophoretic mobility between wild-type and mutant DNA molecules (1,2).
Of these different techniques, single-strand conformation polymorphism
(SSCP) analysis, originally developed by Orita et al. (3,4), is currently the
most widely used method for mutation detection. It relies on the fact that,
under nondenaturing conditions, single-stranded DNA adopts a folded con-
formation that is stabilised by intrastrand interactions. Because DNAs with
different nucleotide compositions may adopt different conformations, the
electrophoretic mobility of a single-stranded DNA fragment in a non-dena-
turing polyacrylamide gel will depend not only on its size but also on its
nucleotide composition. To search for mutations in a given DNA sequence,
polymerase chain reaction (PCR) is first carried out using DNA templates
from different individuals under study (seeSubheading 3.1.). The PCR prod-
ucts are then denatured to separate the two single strands, and fractionated by
nondenaturing polyacrylamide gel electrophoresis (see Subheadings 3.2.and
3.3.). Where mutations exist, the PCR products are expected to migrate at
different speeds. The different mobility patterns are detected by autoradiog-
raphy (Fig. 1).
2. Materials
2.1. Amplification of Target Sequences by PCR
1. 25–250 ng/mL of genomic DNA.
2. Forward and reverse PCR primers: 20mer oligonucleotides, dissolved in distilled
water or TE at a concentration of 1 µg/µL, store at –20°C.
3. 2 mM dNTP mix, store at –20°C.
4. 10 mCi/mL [α-32P] dCTP or [α-33P] dCTP. Caution: follow local rules for han-
dling, storage, and disposal of radioactivity.
5. 10× PCR buffer: 500 mMpotassium chloride, 100 mMTris-HCl, pH 8.3, 0.01%
w/v gelatin.
6. 25 mM magnesium chloride.
7. Taq DNA polymerase, store at –20°C.
8. Mineral oil.
9. Agarose.
10. 10×TAE buffer: 400 mMTris-HCl, 10 mMEDTA, adjust to pH 8.0 with glacial
acetic acid.
11. 6×sample loading buffer: 15% (w/v) Ficoll-400, 0.05% (w/v) bromophenol blue,
0.05% (w/v) xylene cyanol.
Mutation Detection by PCR-SSCP Analysis 5
Fig. 1. Single-strand conformation polymorphism (SSCP) analysis. The sequence
to be screened for mutations is amplified by PCR using DNA templates from different
individuals. The two DNA strands of the PCR products are then separated by heating.
Single-stranded DNA molecules with a point mutation (marked (cid:1)on the sense strand
and (cid:2)on the antisense strand) have different conformations as compared with single
stranded DNA molecules of the wild-type (marked (cid:3)on the sense strand and (cid:4) on the
antisense strand). Denatured PCR products are subjected to native polyacrylamide gel
electrophoresis. Because of the different conformations, single stranded DNA mol-
ecules deriving from the mutant and wild-type have different mobility. The different
mobility patterns are detected by autoradiography. SS, single-strand; DS, double-
strand; HD, heteroduplex (reannealed double-stranded DNA: one strand from the wild-
type and the other from the mutant).
12. Ethidium bromide: dissolved in distilled water to 10 mg/mL. Caution:Ethidium
bromide is a suspected carcinogen.
13. DNA size marker: e.g., 1 kb ladder (Gibco BRL, Grand Island, NY), store at –20°C.
14. Thermal cycler.
15. Horizontal gel electrophoresis apparatus.
16. UV transilluminator.
2.2. Nondenaturing Polyacrylamide Gel Electrophoresis
1. 49% (w/v) acrylamide stock solution: 49% (w/v) acrylamide and 1% (w/v)
bisacrylamide, store at 4°C.Caution:unpolymerized acrylamide is a neurotoxin;
wear gloves.
2. 10× TBE buffer: 89 mM Tris-borate, 2 mM EDTA, pH 8.3.
3. 20% (w/v) ammonium persulphate: freshly prepared with distilled water.
4. NNN'N'-tetramethylethylenediamine (TEMED).
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5. Glycerol.
6. 2% dimethyldichlororosilane. Caution: used in fume hood cabinet.
7. 0.1% (w/v) SDS in 10 mM EDTA.
8. 2×formamide loading buffer: 95% formamide, 20 mM EDTA, 0.05% (w/v) bro-
mophenol blue and 0.05% (w/v) xylene cyanol, store at –20°C.
9. Detergent (e.g., Alconox, Alconox plc, New York, NY).
10. Whatman 3MM filter paper.
11. Plastic wrap, e.g., Saran Wrap.
12. X-ray films, e.g., Hyperfilm MP (Amersham, UK).
13. Vertical polyacrylamide gel electrophoresis apparatus with approx 30 cm (width)
by 40 cm (length) glass plates, and 0.4 mm (thick) spacers and shark’s-tooth comb.
14. Gel dryer.
3. Methods
Preparation of the PCR reactions takes 1–2 h; PCR amplification 2–3 h;
preparation of agarose checking gel, sample preparation, loading and running
another 2–3 h. All these can be carried out on d 1. In addition, the nondenaturing
polyacrylamide gel(s) can be prepared (it takes approx 1 h) during PCR ampli-
fication, and run at room temperature overnight. On d 2, more nondenaturing
polyacrylamide gel(s) can be prepared, and run at 4°C for several hours.
3.1. Amplification of Target Sequence by PCR (see Notes 1 and 2)
When setting up multiple PCR reactions, prepare a premix containing all
reagents listed below (scaled up correspondingly) except template DNA, and
dispense 23 µL aliquots into microcentrifuge tubes each containing 2 µL of
DNA sample. In parallel with PCR reactions of tested samples, set up the fol-
lowing controls:
a. PCR negative control: a PCR reaction without template DNA.
b. SSCP positive control: a PCR reaction with DNA from an individual known
to carry a mutation in the target sequence, if available.
1. For each PCR, set up the following 25 µL reaction in a microcentrifuge tube (or a
microtiter plate): 2.0 µL template DNA (0.025–0.25 µg/µL), 0.2 µL forward
primer (1 µg/µL), 0.2 µL reverse primer (1 µg/µL), 2.5 µL 2 mM dNTP, 0.3 µL
[α-32P] dCTP (10 µCi/µL), 2.5 µL 10×PCR buffer, 1.5 µL 25 mM magnesium
chloride (seeNote 3), 0.2 µmL Taq DNA polymerase (5 U/µL) (to be added last),
15.6µL sterile distilled water.
2. Mix well, and overlay each solution with 30 µL of mineral oil.
3. Place the tubes in a thermal cycler, and program it to perform the following
cycling (see Note 1): Initial step: 94°C for 3 min; 30 cycles of: 94°C for 30 s
(denaturation), 55°C for 1 min (annealing), 72°C for 1 min (extension); final
step: 72°C for 10 min.
4. Prepare a 1.5% (v/w) agarose gel with 1×TAE buffer and 0.5 µg/mL ethidium
bromide.
Mutation Detection by PCR-SSCP Analysis 7
5. Take a 5-µL aliquot from each PCR reaction, and mix it with 1 µL of 6×sample
loading buffer.
6. Load the mixtures, as well as a DNA size marker, onto separate wells in the
agarose gel.
7. Run the gel in 1×TAE buffer until the bromophenol blue tracking dye is approx
5 cm away from the wells.
8. Observe the gel on a UV transilluminator.
9. Proceed to nondenaturing polyacrylamide gel electrophoresis (Subheading 3.3.)
or store PCR products at –20°C.
3.2. Preparation of Nondenaturing Polyacrylamide Gel (see Note 4)
1. Clean two glass plates (first wash thoroughly with detergent and tap water, rinse
with distilled water, and dry, then wipe with absolute ethanol).
2. Treat one side of one of the plates with dimethyldichlorosilane (in a fume hood
cabinet, pipet approx 5 mL of 2% dimethyldichlorosilane onto the plate surface
and spread evenly over the entire surface with a Kimwipe tissue). Leave the plate
in the fume hood cabinet until dry.
3. Place the two plates together with the dimethyldichlorsilane-treated surface fac-
ing inward. Insert two 0.4-mm-thick spacers, one on each side. Seal the sides and
bottom with tape.
4. Prepare a 4.5% nondenaturing acrylamide gel mix (see Note 5): 9 mL 49%
acrylamide stock solution, 10 mL 10× TBE buffer, 91 mL distilled water. Mix
well. Add 100 µL of 20% ammonium persulfate and 100 µL TEMED; 5% or
10% glycerol may be added in the gel mix (seeNote 6).
5. With the plate tilted from the horizontal, slowly inject the acrylamide mix into
the space between the plates using a 50-mL syringe without forming air bubbles.
Insert Shark’s-tooth comb with the flat side facing downward, and clipped in
place to form a flat surface at the top of the gel.
6. Let the gel set.
7. Between 2 and 24 h after the gel is poured, remove the clips, tape, and comb.
8. Fix the plates in a vertical electrophoresis apparatus.
9. Add 1× TBE buffer to the top and bottom tanks.
10. Using a pipet, flush the flat gel surface with TBE buffer.
11. Reinsert the comb with teeth downward and just in contact with the gel surface.
3.3. Sample Preparation and Electrophoresis
1. Dilute PCR products 5–20 folds (depending on the efficiency of PCR reaction)
with 0.1% SDS/10 mMEDTA (omit this step if using 33P instead of 32P in PCR
reaction) (seeNote 7).
2. Transfer a 5-µL aliquot of the diluted sample (or undiluted PCR products if
using33P) into a fresh tube containing 5 µL of 2×formamide loading buffer, and
mix gently.
3. Heat at 95°C (e.g., on a heating block or a thermal cycler) for 3 min.
4. Snap chill on a ice/water mixture.
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5. Load 3 µL of each sample onto the nondenaturing polyacrylamide gel. Also load
3µL of an undenatured (unheated) sample.
6. Connect the electrophoresis apparatus to a power supply, and carry out electro-
phoresis at a constant current of 30 mA at 4°C for 3–6 h (for gels without glyc-
erol) or 15 mA at room temperature for 12–16 h (for gels containing glycerol)
(seeNote 8).
7. Disconnect power and detach plates from the electrophoresis apparatus.
8. Place the plates on a flat surface and insert a spatula into the space between the
two plates and carefully pry them apart.
9. Lay a sheet of Whatman 3MM paper on the gel, press gently, and carefully lift up
the 3MM paper to which the gel has adhered.
10. Turn the 3MM paper over and cover the gel with plastic wrap.
11. Dry the gel at 80°C in a gel dryer for 1–2 h.
12. Expose an X-ray film to the gel for several hours to days at room temperature
without intensifying screens.
3.4. Data Interpretation
Typically, each DNA fragment deriving from a wild-type or mutant homozy-
gous sample produces three bands, two corresponding to the two different
single-strandedDNA molecules and the remainder corresponding to the double-
stranded. Usually the fastest migrating band represents the double-stranded DNA,
but there are exceptions. Corunning an undenatured sample helps to identify the
position of the double-stranded DNA. In some cases, there are more than three
bands for each fragment, presumably because a same single-stranded DNA can
adopt more than one conformation. Although DNA fragments from wild-type
and mutant homozygous samples have the same number of bands, the positions
of the bands corresponding to one or both single-stranded molecules differ. A
heterozygous sample, in contrast, will have all bands of a wild-type and all bands
of a mutant homozygote. In addition, the double-stranded DNA from a heterozy-
gous sample sometimes produces two or three bands, respectively, representing
the fast migrating homoduplex band and one or two slowly migrating heterodu-
plex bands. Figure 2 shows a typical SSCP autoradiograph.
SSCP analysis can only indicate that there are sequence variations within
the DNA fragment being studied. It does not reveal the position and nature of
the mutations. To obtain such information, DNA sequencing is required. PCR
products used for SSCP analysis can be used as templates in DNA sequencing
(5). Alternatively, DNA in mutant bands on SSCP gels can be recovered,
reamplified by PCR, and used as templates in sequencing analysis (6,7).
Fig. 2. Autoradiograph of SSCP analysis. A 433 bp sequence in the stromelysin
gene promoter was PCR amplified. The amplicon was cleaved into two fragments,
sized 181 bp and 258 bp. respectively, with restriction endonuclease EcoRI. The
Mutation Detection by PCR-SSCP Analysis 9
digests were denatured and then subjected to nondenaturing polyacrylamide gel elec-
trophoresis. Shown in the figure are the two single-strands (SS) and double-strand
(DS) of the 181 bp fragment, and the DS of the 258 bp fragment. Both SSs of the 181
bp fragment in lanes 1, 3, 4, 5, and 6 migrate more slowly than those in lanes 2, 8, and
9. Both fast and slowly migrating bands of the two SSs of the 181 bp are present in
lane 7. Also seen in lane 7 is an extra band immediately above the DS of the 181 bp
fragment, which represents the formation of heteroduplex (HD). DNA sequencing has
revealed that the variation in mobility of single-stranded DNA is due to a single nucle-
otide difference. Samples 1, 3, 4, 5, and 6 are wild-type homozygotes, samples 2, 8,
and 9 are mutant homozygotes, and sample 7 is a heterozygote.
10 Ye and Henney
4. Notes
1. The fidelity and efficiency of PCR reactions are affected by a number of factors,
such as the amount of template DNA, the amount and melting temperature (Tm)
of the primers, Mg2+concentration, annealing temperature, and cycling number.
(8). PCR conditions should therefore be optimised individually for each set of
primers, and the conditions described inSubheading 3.1.can be used as a start-
ing point for optimization. Because nonspecific bands complicate the interpreta-
tion of SSCP results, it is worth making the efforts to optimize the PCR conditions
so that there are only minimal spurious products (ideally there should be only a
single major band on an agarose checking gel).
2. The ability to detect mutations decreases with increasing fragment length.
Estimated sensitivity approx 90% for 100–300 bp fragments, but drops signifi-
cantly for fragments over 300 bases (67% for 300–450 bp fragments) (9–12).
Therefore, DNA fragments between 100 and 300 bases are used. If the PCR
amplicon is too long, it can be cleaved into smaller fragments with suitable
restriction endonucleases prior to denaturation and polyacrylamide gel electro-
phoresis(13).
3. If there are significant nonspecific bands, reduce the Mg2+ concentration and/or
the number of amplification cycles, and/or increase the annealing temperature.
If, on the other hand, the expected PCR product cannot be seen, increase the
Mg2+ concentration and/or number of amplification cycles, and/or reduce the
annealing temperature. In some difficult situations, “hot start” or “touch down”
PCR might be preferable.
4. SSCP analysis can also be carried out using smaller polyacrylamide gels, although
the sensitivity is likely to decrease. It has been reported that mutations can be
detected using 9% mini-gels (0.75 mm × 6 cm × 8 cm) (14,15). In addition to
autoradiography, other methods, such as silver staining(6,15), ethidium bromide
staining(16), and fluorescence labeling (17–19), have been applied successfully
to detect DNA bands in SSCP analysis.
5. The ratio of acrylamide to bisacrylamide determines the percentage of crosslinking.
A ratio of 49:1 is commonly used for SSCP.
6. In some cases, the addition of 5% or 10% glycerol in the gel increases mobility
shift (3). Gels containing glycerol tend to produce somewhat diffused bands.
7. A total of 40 samples (including tested samples, and positive and negative con-
trols) can be loaded onto a 30-cm-wide gel, and two or even more gels can be
run at once. Therefore, 70 samples can be analyzed within two days, although
autoradiographs may not be ready for another day or two, depending on the
strength of signals.
8. Some mutations are detected more readily at room temperature, others at 4°C
(20). Therefore, usually each DNA fragment is analyzed on at least two different
conditions. A useful combination is a glycerol containing gel run at room tem-
perature and a gel without glycerol run at 4°C(3,4).
Mutation Detection by PCR-SSCP Analysis 11
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