Table Of Content1
PCR Methodology Applied to Genetic Studies
of Lipoprotein Metabolism and Atherosclerosis
Shelley A. Cole and James E. Hixson
1. Introduction
During the 1980s molecular genetics became widely applied to hpoprotein
research. The cloning of candrdate genes for atherosclerosis (apohpoprotems,
lipid-processing enzymes, lrpid transfer proteins, and hpoprotem receptors)
allowed the identrficatron of mutattons responstble for rare dysltpopro-
teinemtas, as well as more common genettc variants. The cloned candidate
genes were used as probes on Southern blots to find polymorphtc nucleottde
substituttons at restriction enzyme sates( restrtction fragment-length polymor-
phisms or RFLPs) that could be used as genetic markers m linkage and asso-
clatron studtes. Such molecular-genetic studies have shown that genetic
variation 1sa component m mdivrdual variation m lipoprotein metabohsm and
rusk of atherosclerosts.
Southern blotting IS a laborrous and time-consummg method to genotype
large groups of indtviduals for genetic studies. With the advent of the poly-
merase chain reaction (PCR) to selectively amphfy specific DNA sequences
(I), there came a much faster way to type indrviduals for genetic mutations
involved m atherosclerosis, and to type polymorphic markers at candidate
genes. Though most widely applied to RFLPs at first, PCR was soon applied to
detect nucleotide substitutions and other polymorphrsms that were not associ-
ated with changes in restriction enzyme sttes. Although PCR has Impacted all
areas of molecular-genetic studies of lipoprotem metabolism mcludmg clon-
mg, sequencing, and gene expression, this chapter will concentrate on the
application of PCR m genetic-marker studies as a method to study the effect of
inter-indrvtdual genetic varration on lipoprotein metabolism and atherosclerotic
From Methods in Molecular B/o/ogy, Vol 110 Llpoprotem Protocols
Edlted by J M Ordovas 0 Humana Press Inc , Totowa, NJ
7
2 Co/e and Hixson
risk. Though not an exhaustrve presentation, it will include an overview of the
basic approaches afforded by the PCR method. For a more detailed discussion
of the PCR process itself, the reader is referred to the many excellent publica-
tions dedicated to this subject (i.e , refs. 2 and 3).
2. Materials
2.1, Equipment and Supplies
1, Thermal Cycler (see Note 1).
2 Vertical electrophoresis apparatus (for acrylamide-gel electrophoresls, see
Note 2)
3. Horizontal electrophoresis apparatus (for agarose-gel electrophorests, see
Note 3)
4 Temperature-gradient gel electrophoresis (TGGE) apparatus (Biometra, Got-
tmgen, Germany or TGGE System, Diagen GmbH, Hilden, Germany)
5. Gradient maker (Bto-Rad Laboratories, Hercules, CA).
6 Dot- or slot-blottmg apparatus (Mmlfold I or II Mamfold, S&S, Keene, NH)
7 Ultraviolet transillummator (UVP, Upland, CA, or UVP, Cambrtdge, UK)
8 Recording camera (Gel Dot 1000 Video Gel Documentation System, Bto-Rad
Laboratories, or Foto/System 1000 Camera System, Fotodyne, Hartland, WI).
9. UV chamber (GS Gene Lmker, Bio-Rad Laboratories)
10 Stertle thin-wall 0 5-mL microfuge tubes
11 96-Well Microtiter dishes.
12 Type 57 high-speed Polaroid film.
13 Whatman 3MM chromatographtc paper.
14. Hybond N filters (Amersham Life Science, Arlington Heights, IL, or Amersham
Internattonal plc, Little Chalfont, Bucks, UK).
15. BtoMax MS mtenstfymg screen (Kodak Scientific Imaging Systems, Rochester, NY).
16. Autoradiographic film (Kodak XAR-5, Kodak Scientific Imaging Systems or
Beta-max from Amersham).
2.2. Reagents
2.2.7. Restriction-Enzyme Cleavage of PCR Products
to Detect RFLPs and Protein lsoforms
1 Synthetic olrgonucleotrdes (primers) flanking the sequence to be amplified
(see Note 4).
2. Tuq DNA polymerase: Thermus aquatlcus DNA polymerase or AmplitaqTM
(Perkm Elmer Cetus, Norwalk, CT). Tuq DNA polymerase is currently available
from several other manufacturers.
3 Mixture of dNTPs (dGTP, dATP, dTTP, dCTP each at 2.5 mM) Most suppliers
provide premix nucleotldes; alternatively, the workmg mixture can be prepared
by mixing equal volumes of a 10 mM solution of each of the four dNTPs
4 5X Tuq buffer (or 5X PCR buffer), whtch includes 250 & KCl, 50 mM Tris-
HCl, pH 8 3, and 7 5 mM MgCl* Thts buffer can also be prepared without the
PCR Methodology 3
MgC12. In that case, the optimal amount of MgCl, should be added separately as
suggested in Notes 5 and 6.
5 MgCl, (see Notes 5 and 6)
6 Dlmethyl sulfoxlde (DMSO). Filter-stenhze.
7 Light mineral 011 (see Note 7)
8. Suitable restriction endonuclease enzymes and buffers: Suppliers generally ship
the enzymes with optimized digestion buffers and they should be used as mdl-
cated by the manufacturer. AlternatIvely, One-Phor-All Buffer (Pharmacla
Biotech, Piscataway, NJ) can be used with a wide range of reactlons.
9 Appropriate gels either agarose or acrylamide for checking the reactlon and the
DNA fragments (see Note 8)
10 5 mg/mL Ethldmm bromide (EtBr)* 500 mg EtBr (Sigma E-875 l), add ddH,O to
100 mL The working solution of EtBr (1 pg/mL) should be prepared by diluting
the stock solution with water or gel buffer.
11 10X TBE buffer. 0 90 MTns-borate, pH 8 0,0.02 M ethylenedlamme tetra-acetlc
acid EDTA). Dilute the stock to 1X TBE or 0 5X TBE for use
12 20X TAE buffer. 0 8 M Tns, 0.4 M NaOAc, and 0 04 M Na2EDTA, and glacial
acetic acid to pH 8 3 m ddH,O Mix 96.9 g Tris base, 32 8 g NaOAc-3H,O and
14.9 g Na*EDTA Dissolve m approx 700 mL of ddH*O, adjust the pH to 8.3 with
glacial acetlc acid, and bring to 1 L with ddH20. To prepare 1X TAE, &lute 1:20
with ddH*O. This buffer can be mltlally sterilized by autoclaving and it can be
stored at room temperature. If needed, it can be sterilized agam by filtration
through a 0 22-w filter
2.2.2. Gel Analysis of Physical Properties
that Distinguish Allelic Substitutions
in PCR Products Temperature Gradient Gel Electrophoresis (TGGE),
Denaturing-Gradlent Gel Electrophoresis (DGGE),
and Single-Stranded Conformation Polymorphism (SSCP)
In addition to those reagents listed in Subheading 2.2.1., the followmg
specific reagents are used for TGGE, DGGE, and SSCP.
1 Acrylamide (electrophoresls grade)
2. N,N’Methylenebisacrylamlde (electrophoresis grade).
3. 10% (w/v) Ammonium persulfate: preferably made fresh prior to use. Alterna-
tively, it can be kept at 4°C for up to 1 wk.
4. TEMED (N,N,N’,N’ Tetramethylenediamme). Store at 4°C.
5 SDS
6 Urea
7 [Y-~~P]ATP 5000 Cl/mm01 (Amersham)
8. [Y-~~P]ATP: 3000 Wmmol.
9 [a-32P]dCTP. >3000 Wmmol.
10 T4 DNA polynucleotlde kmase (10,000 U/n&, New England Blolabs, Beverly, MA)
4 Cole and Hixson
11 T4 buffer, 50 mM Tris-HCl, pH 7 510 mMMgSO,, 5 mA4 DTT, 50 pg/mL bovine
serum-albumm (BSA)
12 2X TGGE loading buffer 40% sucrose.
13. Glycerol.
14 Formamide dye. 950 mL/L formamide, 20 mmol/L EDTA, 0 5 g/L bromophenol
blue, 0 5 g/L xylene cyan01 FF.
15. TGGE gel support films (Diagen GmbH, Hrlden, Germany)
2.2.3. Typing Known Nucleotide Substitutions Using PCR
and Hybridization with Allele-Specific Oligonucleotides (ASO)
In addition to those reagents listed in Subheading 2.2.1., the followmg
specific reagents are used for hybridization with allele-specific oligo-
nucleotides (ASO).
1 5X SSPE. 0.75 M NaCl, 50 mMNaH2P0,, 5 mA4 EDTA, pH 7 4
2 50X Denhart’s 1 g F~coll, 1 g polyvmylpyrohdone, 1 g BSA, and ddHzO to 100
mL Pass through a Nalgene filter and keep m refrigerator
3 20X Standard salme-citrate (SSC): 17 53 g NaCI, 8.82 g sodmm citrate Dissolve
m approx 80 mL ddHzO, adjust pH to 7 0 wrth hydrochloric acid (HCl), and
bring final volume to 100 mL. To prepare 1X SSC ( 15 mM NaCI, 1.5 mA4 Na
citrate), dilute 1.20 with dHzO.
2.2.4. Batch Analysis of PCR Products
1 Gelatin
2. Nomdet P-40
3. Tween-20.
2.2.5. PCR-Based Solid-Phase Methods
for Genotyping of Known Nucleotide Substitutions
1. 5’-Biotinylated primers (see Note 4).
2. 5% (w/v) Suspension of avidm-coated polystyrene particles
3. Phosphate buffered salme (PBS)* 140 mMNaC1,2 6 mMKC1, 10 mMNa2HP04,
1.8 mA4 KH2P04, pH 7 4. Store at 4°C.
4. Sequencing kits (1 e , Sequenase, US Brochemicals, Cleveland, OH)
3. Methods
3.7. PCR for Detection of Nucleotide Substitutions
as Genetic Markers for Linkage and Association Studies
For the detection of polymorphic nucleottde substitutions, PCR is an
efficient process where results on many samples can usually be obtamed in a
single day. However, the DNA sequence of the gene region containing the
polymorphism must be known. This sequence is used to design synthetic oh-
gonucleottde primers that flank the polymorphtsm. PCR involves multtple
PCR Methodology 5
cycles of denaturation of the template DNA, annealing of the primers to the
template, and extension wtth Tuq DNA polymerase to amplify the regron
flanked by the primers Several different procedures can then be used to detect
the presence or absence of the nucleotide substitution m the amplified frag-
ment. For those that change restriction-enzyme sites, the amplified fragment is
digested with the appropriate restriction enzyme and electrophoresed to deter-
mme the fragment sizes. For those substitutions that do not alter restrtctton-
enzyme sues, several methods can be used to detect the presence or absence of
the change. In some Instances, restriction sites can be created to distmgutsh the
nucleotide substitution by using a mismatched primer (4). PCR products can
be probed under stringent condttions with AS0 that only remain hybridized tf
there are no sequence mismatches. Other techniques rely on the altered elec-
trophorettc mtgration of DNA with different base compositions. Newer tech-
nologies are also discussed that depend on the initial amplificatton of the region
of interest by PCR.
3.1.1. Restrrctlon-Enzyme Cleavage
of PCR Products to Detect RFLPs and Protein lsoforms
One of the first apphcattons of PCR m lipoprotein research was to type pre-
viously identified genetic markers. When a polymorphic nucleotide substitu-
tion alters a restriction-enzyme site m a PCR product, amplified DNA samples
will show an RFLP on digestion with the enzyme and subsequent gel electro-
phoresis and staining with ethidmm bromide. If the restrtction satei s absent in
both alleles of a DNA sample, there will be one fragment on the gel corre-
sponding m size to the uncut PCR product. If the restriction site is present m
both alleles, there will be two fragments whose sizes are determined by the
position of the enzyme-cleavage site m the amplified fragment. If the DNA
sample is heterozygous, both uncut and cut fragments will be present
When protein polymorphisms are caused by nucleotide substttutions that
also alter restrtctton-enzyme sites, they can be typed at the DNA level by
restrtction-enzyme tsoform genotyping (restriction isotyping) (5). Restriction
tsotypmg provides a rapid method of typing the common apohpoprotem E (apo
E) tsoforms (E2, E3, E4) that are important determinants of plasma ltptd levels
(6). Synthetic ollgonucleotides are used to amplify apo E gene sequences that
span ammo acid postttons 112 and 158. The amplification products are digested
with HhaI and subjected to electrophorests on polyacrylamide gels. Each of
the isoforms is unambtguously distingutshed by a unique combination of HhaI
fragment sizes.H haI cleaves at G CGC encoding Argt12 (E4) and Arg158 (E3,
E4), but does not cleave at G TGC encoding Cys’12 (E2, E3) and cyst58 (E2).
Mutations m apo A-IV are responsible for isoforms that have effects on lipid
and lipoprotein levels (7), and that may be associated with differential response
6 Cole and Hixson
to diet (8,9). Restrictton isotypmg can also be used to dlstmgulsh apo A-IV
lsoforms at the DNA level (10). In this instance, a mismatched primer IS used
m the PCR reaction to introduce a PvuII restriction site when the primer amph-
fies an apo A-IV-2 allele, but not an apo A-IV-1 allele. An alrquot from the
same PCR reaction can be used to type the Thr347+Ser polymorphism by
digestion with Hi&.
3.1 .l .l. BASIC PCR PROTOCOL
The protocols descrtbed m this chapter include one or more steps requiring
DNA amplification. To simplify the description of specific protocols, we
delineate below a basic PCR protocol. It should be noted that each laboratory
may require slight modifications depending on the DNA template characteris-
tics, DNA quality, source of reagents, and equipment available, to name some
of the factors that may affect the PCR reaction (see Notes 5 and 6).
The PCR reaction IS usually performed m volumes ranging from 5-l 00 $.
Because of the small volumes used per reaction, we recommend making a cock-
tall with all the reaction components (allow for a slight excess)w ith the excep-
tion of the DNA template:
To prepare a cocktail for 10 PCR reactions of 100 $ each, mix the follow-
ing components:
1 200 $5X PCR buffer
2 80 pL dNTPs (each at 2 5 mM) This results m a final concentration of each
dNTP m the reaction of 200 w.
3 40 & primer pair (each primer at 25 pM. The primer solution 1sp repared by
mixing 1: 1 mixture of the 50-mprlmer solutions) This provides a final concen-
tratlon of 1 pmol/pL. of each primer m the reactlon mixture
4 20 pL Taq polymerase
5. 650 pL ddH20 (some reactions require additional reactlon components, such as
DMSO or additional MgC12, to achieve optimal results. This is usually specified
in the reaction conditions. In those cases, the volume of the addltlonal compo-
nent should be substrated from that of ddH20 to mamtam the final concentration
of all other reagents) Keep the cocktail on ice
To perform the PCR reaction, follow the following steps.
1 Pipet 100 pL of the cocktail mto a sterile 0 5-mL Eppendorf tube (the amounts
described should be scaled down to fit other usual reactlon volumes, i e , 50,
2075 a).
2 Add 1 p.L of DNA (contammg about 100 ng genomlc DNA).
3 Mix and quick spin to bring the reaction mixture to the bottom of the tube
4. If the thermal cycler does not have a hot bonnet, overlay the reaction mixture
with one drop of mineral oil
PCR Methodology 7
A2A2 A7A2 AlA
4 255 bp
785 bp *
70 bp +
FORWARD PRIMER REVERSE PRIMER
- -
4
7. Avall 785 I
255 bp
Fig. 1. PCR-based typing of the AvaII RFLP in exon 13 of the LDL receptor gene.
The schematic below the gel shows the portion of the LDL Receptor gene that is
amplified for typing of the AvaII RFLP in exon 13 (primers shown by horizontal
arrows, polymorphic AvuII site shown by a vertical arrow). The sizes of the uncut PCR
product and the AvaII fragments are given below the map. The gel shows typing of
DNA samples for each genotype (fragment sizes shown adjacent to the gel).
5. Cap the tubes and place them in the temperature block, making sure that each
tube makes good contact with the heater walls.
6. Program the thermal cycler for the reactions conditions (see Note 6): Denatur-
ation: 93-94°C for 1.5 min; Annealing: 5065°C for 2 min; Extension: 72°C
for 2 min.
Below are presented some specific examples where the PCR technique is
used to detect polymorphisms that may be relevant as genetic markers. (Primer
sequences, PCR, and cutting conditions for other genetic markers frequently
studied in lipid research are presented in Appendix 1.)
3.1 .1.2. DETECTION OF THE AvAII RFLP OF THE LDL RECEPTOR
Using PCR amplification, we have developed a protocol to detect the AvaII
RFLP in exon 13 of the LDL receptor (12). The results are shown in Fig. 1.
We used published sequence data (13) to design primers to amplify the region
containing the RFLP.
1. Prepare the reaction mix as described in Subheading 3.1.1.1. (see Note 5), using
the following primers: forward primer from exon 13 (5’-CAGTGCCAA
CCGCCTCACAGG-3’) and reverse primer from exon 14 (5’-CCTCTCACA
CCAGTTCACTC-3’). Use DMSO in the reaction at a final concentration of
10% (see Note 5).
8 Cole and Hixson
2 Add 0.5-1.0 1.18g enomlc DNA.
3. Set the thermocycler to the following condltlons (see Notes 6 and 9): denatur-
ation: 94’C for 1 mm, annealmg and elongation, 64’C for 1 5 mm Use 30 cycles
of amplification.
4 After successful amphficatlon, digest the 255-bp amplification product with
AvaII (see Note 10) by addmg 4-8 U of the restrlctlon enzyme directly to the
tube containing the PCR reaction (see Note 11)
5 Incubate the reaction for 2-6 h at 37’C.
6 Analyze the results of the dlgestlon on a 1 5% agarose gel (see Note 12) The
digestion of the amplification product with AvaII produces 185- and 70-bp frag-
ments when an mdivldual is homozygous for presence of the site (designated
A2A2), a 255-bp fragment if an mdlvidual is homozygous for absence of the site
(designated AlAl), and all three fragments in heterozygotes (AlA2)
3.1.2. Gel Analysis of Physical Properties that Distinguish Allelic
Substrtutions in PCR Products (TGGE, DGGE, and SSCP)
Polymorphic nucleotide substitutions can be detected m PCR fragments even
when they do not alter a restriction site. In TGGE, a double-stranded PCR
fragment dlssoclates into a partially single-stranded fragment at a temperature
specific for its DNA sequence, thus slowing its migration through a denaturing
gel (19). Sequence differences as small as a single basepalr substitution are
distinguished by distinct denaturatlon temperatures and migration to different
positions in the gel. TGGE 1sa simplified version of DGGE, m which the tem-
perature of the gel remains constant throughout electrophoresls, but the gel
contains a gradient of denaturant (28). In SSCP analysis (20), radlolabeled
primers are used to amplify fragments that are denatured, electrophoresed
through nondenaturmg gels, and exposed to X-ray film. Polymorphic smgle-
stranded fragments assume distinctive conformations and migrate differently
owing to the substttutlons m their sequence (20). These techniques are simple
and sensitive methods able to detect most nucleotide substitutions.
The basic principles of primer design and optimization of the PCR reaction
are the same as previously described for RFLP analysis. The primers should be
chosen to amplify DNA fragments from 300-500 bp in length. In some cases,
the primers may include GC-rich segmentso n their 5’ ends called “GC-clamps,”
which improve the detection of polymorphisms by altering conditions for
fragment dissociation (21).
The TGGE apparatus uses a standard vertical-gel electrophoresis chamber,
or an apparatus specially designed for this purpose, with a denaturing poly-
acrylamlde gel contaimng urea and formamide. A temperature gradient is
applied either perpendicular or parallel to the direction of electrophoresls. To
establish the temperature gradient, aluminum plates with internal channels that
circulate cooled and heated water are attached to the surfaces of the glass plates
PCR Methodo/ogy 9
of the vertical polyacrylamrde gel (19). For a more detailed descrrptron of this
technique, see ref. 22
To type polymorphisms using TGGE, it is first necessary to determine the
temperature range and condrtrons for denaturtng the amplified fragment. This
is carried out using a denaturing 6.5% polyacrylamide gel (37.5:1,
acrylamrde:bis-acrylamide; 0.8 mm thick) with one preparative well spannmg
the width of the gel.
1 Prepare two acrylamide stock soluttons, one with 100% denaturant (40%
formamide, 7 M urea), and one wtthout denaturant The final volume percentage
of denaturant m the gel (usually 40-60%) IS determmed by the proportion of the
denaturing acrylamtde solutron that 1s added to the nondenaturmg solutton In
thts way, only two solutions need to be prepared and mtxed to generate a range of
gels differing by their final denaturant concentrations
2 Establish a wide temperature gradient perpendicular to the dtrectton of electro-
phoresis, with cold temperature (I e., 16’C) on the left of the gel and hot tempera-
ture (1 e., 46°C) on the right of the gel
3. MIX the PCR reaction (100 pL volume) of the desired fragment with an equal
volume of 2X TGGE loading buffer (40% sucrose), and load into the prepara-
tive well
4 Carry out the electrophorests in 0.5X TBE (70-80 V for 22 h).
5 Stain the gel using EtBr to reveal a sigmoidally shaped curve that results from
denaturatton of the double-stranded fragment into more slowly migrating
single-stranded structures. The narrow temperature range correspondmg to the
posmon of the mflectton pomts of the sigmoidal curve represents the denatur-
mg temperature of the fragment This temperature range, along wtth the per-
cent denaturant used m the gel, IS used to tdenttfy and type polymorphtsms m
the fragment
Once the denaturing condttions of the PCR fragment have been determmed,
genomrc DNA samples can be routmely screened for the presence of
polymorphisms.
1 Amplify the fragments of interest from DNA samples m PCR reactions (20 pL
volume)
2 Mix the amplificatton product with an equal volume of 2X TGGE loading buffer
3 Load each sample mto separate wells on a 6.5% polyacrylamtde gel havmg the
appropriate denaturant concentratron, determined as aforementtoned The
alummum plates are used to establish a temperature gradient parallel to the dtrec-
tton of electrophoresrs (cooler on top, warmer on the bottom) using the narrower
temperature range as determined from the perpendicular gradient gel prevtously
descrtbed.
4. Stain with EtBr. Polymorphtsms are resolved because each allele will denature at
slightly different temperatures and migrate to distinctive positions during
electrophorests
10 Cole and Hixson
3.1.2 1. IDENTIFICATION OF POLYMORPHISMS
IN THE ANGIOTENSINOGEN (AGT) GENE USING TGGE
Recent studies have identified polymorphisms in AGT that are associated
with essential hypertension in humans (for review, see ref. 23). One of these,
the Met235+Thr substttution m exon 2 of AGT, was originally identified using
SSCP (24). We developed a TGGE assay to type the Met235+Thr substitution,
and identified four other variants m exon 2 of AGT (25). Two of these, a
Tyr24*+Cys ammo actd substitutton and a T+C nucleotide substnution, had
been previously characterized (24), whereas two new variants were identified
m a US black populatton (Thr209+Ile and Leu2i’+Arg).
To amplify exon 2 of the AGT gene use the following protocol:
1 MIX 19 & of the standard PCR reaction mixture (containmg the followmg
primers forward prtmer 5’-GATGCGCACAAGGTCCTGTC-3’ and reverse
primer. 5’-GCCAGCAGAGAGGTTTGCCT-3’) with 0 5 pg of DNA in 1 pL of
ddHzO or the appropriate DNA storage buffer.
2. Carry out the amphficatlon usmg the following condmons Imttal denaturatron at
95°C for 5 mm followed by 30 ampbfication cycles (60°C for 1 min, 70°C for
2 mm, and 95°C for 1 mm)
3 Load the PCR products onto a denaturmg polyacrylamtde gel (5 6 A4 urea, 32%
formamrde) with a temperature gradient of 33°C at the top of the gel and 41 “C at
the bottom
4 Electrophorese the gel at 70 V for 17 h
5. Stam the gel wrth EtBr and photograph under UV light
3.1.2.2. TGGE ANALYSIS OF ENDOTHELIAL CELL
LEUKOCYTE ADHESION MOLECULE 1 (ELAM-1)
ELAM- 1 (also called selectm E) is an adhesion molecule that mediates bmd-
ing of circulatmg leukocytes to endothehal cells m the arterial wall. Figure 2
shows the results of TGGE analysis of intron 2 of the gene encoding ELAM- 1.
PCR primers were destgned usmg published sequence data from exons 1
and 2 (26) to amplify the 631-bp intron (forward primer, 5’CAGTTTCTC
TGAGCTCTCACTTTGG-3’; reverse primer, 5’- CCAGGATCCACTCTC
TTTAATGA-3’).
1. Mix 19 pL of the standard PCR reaction mixture (contammg the primers mdt-
cateda bove) with 0.5 ug of DNA in 1 & of ddHzO or DNA storage buffer (27)
2. Carry out the PCR reaction using the following condltlons denaturatton for
5 mm at 95’C, followed by 30 cycles of denaturatton at 95°C for 1 mm, annealing
at 56°C for 1 mm, and elongatton at 70°C for 1 mm.
3 To determine the denaturatton temperature, electrophorese the PCR fragment at
70 V for 22 h m a 6 5% denaturing polyacrylamide gel (40% denaturant) with a
temperature gradient (16-46”(Z) perpendicular to the direction of electrophoresrs.
4. Stam using EtBr.
