protocols in human molecular genetics

CHAPTER 1
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:
l
Length: 18-30 bp. Shorter and longer primers may, however, work well.
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
.

.
.
.
.
l
.
.
.
.
.
.
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.
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).
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
Getting Started in PCR
3. O.lMMgCl,, 100 mL.
4. 0.2% Gelatin (Difco), 100 mL.
3
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
5.1. Denuturation (94°C)
5
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.
2.
3.
4.
5.
6.

7.
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.
Wear gloves when making up solutions; avoid touching the inside of the
tube cap.
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) .
Ahquot reagents and reaction buffers, and use each ahquot only once. See also
Sections 2 and 3.
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.
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.
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
Table 3
Troubleshooting in PCR
7
Problem Causes Remedies
No detectable product
after repeated at-
tempts
Multiple bands on
agarose gel of ampli-
fied product
Continuous “smear” of
amplified product on
agarose gel
Predominance of very
high mol wt amplified
product
“Primer dimer’%
Inadequate melting of

DNA template
Target sequence too rare
Annealing temperature
too high
CC content of target
sequence too high
Primers anneal to each
other (primer dimer)
or to themselves
“Overamplificauon”
Primers too short or
degenerate
Concentration of dNTPs
or of enzyme too high
Annealing temperature
too low for CC
content of primers
“Overamplification”
Reamplification of
primary amplified
product
Complementanty
between 3’ ends
of pnmers
Increase ume in
denature step
Increase number of
cycles (up to 60)
Lower temperature by
5°C

Try 10% DMSO in
reaction
See note a
Reduce number of
cycles; reduce exten-
sron time
See no& a
Reduce either by 2-10x
Raise annealing tempera-
ture by 5°C
Reduce number of cycles
Gel-purify primary
product before
reamplifcauon
See note a
p
In each case, the
remedy 1s to increase the 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.
1.
6.
7.
8.
9.
10.
11.
12.
13.
References
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

Mezei, L. M. (1990) Effect of oil overlay on PCR amphficauon, m Amp2ajicatron.s Perkm-
Elmer, Norwalk, CT, vol. 4, p. 11.
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
Sarkar G. and Sommer, S. S. (1990) Shedding light on PCR contammauon. Nature
343,27.
Kitchin, P. A., Szotyori, Z., Fromholc, C , and Almond, N (1990) Avoidance of false
positives. Natun 344,201.
Kwok, S. and Higuchi, R. (1989) Avordmg false positives wnh PCR Nature339,237,238
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.
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
Crmmo, G. D., Metchette, K., Isaacs, S. T., and Zhu, Y. S. (1990) More false positive
problems. Nature 345, ‘7’73,174.
CHAFTER 2
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.
1.
2.
3.
4.
5.
6.
7.
a.
2. Materials (see Note 1)
Ribonuclease inhibitor (RNasin; Pharmacia, catalog no. 27-0815-01;
27,000 U/mL).
Random hexamer primers (pd(N)s, Pharmacia, catalog no. 27-2166-01;
at 5 mg/mL) .
5x POL buffer (250 mMTrisHC1, pH 8.3 at 3’7”C, 40 mM MgCl,, 150
mMKC1; 50 mMdithiothreito1 [D’IT]).
Deoxyribonucleotide triphosphate mixture (dNTPs; mixture of 2.5 mM
each of dATP, dTTP, dCTP, and dGTP) .
Reverse Transcriptase (M-MuLV, Pharmacia catalog no. 27-0925
02;12,000 U/mL).
Modified T7 DNA polymerase (SequenaseTM; usually supplied at 12.5

U&L from United States Biochemicals [USB] catalog no. 70722).
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.
Sequencing stop solution (STOP; 95% formamide, 20mMEDTA, 0.05%
bromophenol blue, 0.05% xylene cyanol).
b
Total RNA
Random Primers
Reverse Transcrlptase Total RNA
ltt Strand CDNA

E

Fl


-
Seauence
*
w
4
x
l
Dldeoxynucleotlde
Seauenclng
b w
b

f
Alkali.
-A / s
PCR
Ethanol pptn 1 st Strand cDNA
Ampl If led cDNA
Seauenclng
Primer Excess of
Single Strands
Template
Pig. 1. Schematic dmgram of the strategy for direct DNA sequencing of PCR-amphfied cDNA. Total cellular RNAis copied by
randomly primed reverse transcription, the RNA is hydrolyzed by alkali, and the specific cDNA is amphfied by PCR. An aliquot
of the PCR product 1s used in a single-strand-producing reactron (SSPR) that has a single oligonucleotrde primer. The single-
stranded mixture is sequenced usmg a 5’-end-labeled primer and dldeoxynucleotide termination.
12 Gibbs, Nguyen, and Caskey
9. Reverse-transcription hydrolysis solution (RTH):
O.'7M
NaOH, 40 mM
EDTA.
10. ZMammonium acetate, pH 4.5.
11. 10x PCR buffer: 67 mMMgCl,, 166 mM (NH,) $O,, 28 mM2-mercapto-
ethanol, 68 l.tMEDTA, 670 mMTris-HCl; pH 8.8 at 25°C.
12.
Tuq
DNA polymerase (Pet-kin-Elmer/Cetus).
13. 75Mammonium acetate.
14. [32P] yTP (6000 Ci/mmol).
15. T4 polynucleotide kinase.
16. 10x kinase buffer: 100 mMTris-HCl, pH 7.6; 100 mMMgC12; 100 mM
DTT.

17. NENsorb@ columns (New England Nuclear/Dupont) .
18. TE buffer: 10 mMTris-HCI, pH 7.0; 1 mMEDTA
3. Methods
3.1. Reverse lknscription Reactions (see Notes 2,3)
1. Mix the following on ice: 0.5-5.0 l,tg total cellular RNA, 0.5 FL RNasin;
2.0 ltL pd(N)s primers; 4 l.tL of 5x POL buffer; and H,O (treated with
diethylpyrocarbonate) to 15.5 l.l.L.
2. Heat at 95’C for 1 min, chill on ice, and pulse/spin in a microfuge.
Then add, at room temperature, 2.0 ltL of dNTPs, 0.5 ltL of RNasin, and
1.0 l.tL of reverse transcriptase.
3. Incubate at 37°C for 1 h.
4. Add 30 yL of RTH solution, mix gently, and incubate at 65°C for 10 min.
5. Add 5 l.tL of 2Mammonium acetate (pH 4.5), mix, add 130 ltL of etha-
nol, and chill at -2O’C for at least 4 h (preferably overnight). Then spin,
wash in 70% ethanol, wash again in 100% ethanol, and dry.
3.2. Polymerase Chain Reaction (see Note 4)
1. Mix 5-10% of the product of one cDNA-synthesis reaction with 50 pmol
of each PCR primer (seeNote 9) in a total vol of 50 l.tL containing 5 lt,L of
10x PCR buffer, 1.5 mMof each dNTP (3 l.tL of 25 mM mixture) and
10% DMSO. (This buffer is a slight modification of that described by
Kogan et al. [IO].)
2. Heat to 94°C for 5 min and centrifuge for 5 s.
3. Add 2.5 U of
Tq
DNA polymerase, mix gently, and overlay with mineral oil.
4. We typically perform 23-28 cycles of DNA polymerization (68”C, l-3
min), denaturation (94’C, 30 s), and annealing (37-65’C, 30 s). The
optimum annealing temperature must be determined empirically In
initial reactions, allow at least 1 mm of extension/500 bases.
Direct Sequencing of DNA

13
5.
The final incubation at 68°C is extended for 7 min.
6. Remove the sample from under the oil.
3.3. Single-Strand-Producing Reactions (SSPRs)
(see Notes 538)
1.
2.
3.
Take 1 yL of the PCR product to initiate a second PCR that is identical
to the first except that only one primer is used. Use a primer that is
opposite in sense to the sequencing primer that will be employed.
Perform the same number of cycles of the SSPR as was used for the
initial PCR. Use the same cycling temperatures, but double the length of
the annealing and polymerase extension times.
Dilute the reaction with an equal volume of Hz0 and add an equal vol-
ume of 7.5Mammonium acetate, mix, add 2.5 vol of ethanol, chill for 15
min at -70°C (or overnight at 4*C), and spin for 30 min in a microfuge.
Repeat the ammonium acetate precipitation. Wash with 70% ethanol,
again with 100% ethanol, and dry to completion under vacuum. Dis
solve the pellet in 10 ltL of Hz0 immediately before use in the DNA-
sequencing reaction
3.4. Radiolabeling the DNA Sequencing Primer
(see Note 9)
1.
2.
1.
2.
3.
4.

5.
Kinase reactions contain 20-50 pm01 of primer, 50-70 l.tCi of [32P]
yATP (6000 Ci/mmol), 30 U of T4 polynucleotide kinase, and 5 uL
of 10x kinase buffer in a total vol of 50 p.L. Reactions proceed at 37OC
for 45 min.
Purify the labeled primer by passage through a NENsorb@ column.
Dry the product to completion, and then resuspend in 12 l,tL of H,O
immediately before use.
3.5. DNA-Sequencing Reactions (see Notes 1 O-13)
Add 5.0 l,tL of single-strand DNA template to 3 yL of labeled primer and
2.0 yL of 5x POL buff er, in a standard 1.5mL microcentrifuge tube.
Heat to 95°C for 10 min.
Centrifuge for 5 s to bring down condensation.
Dispense 2.5~p.L aliquots of the primer template mixture into four ap-
propriately labeled tubes (IT, lC, lG, 1A). Do this step on the bench,
i.e., at room temperature.
Add 2.0 l.tL of the appropriate dideoxy-terminator/Sequenasem mix (see
above) to each of the four tubes and place immediately at 50°C. Incubate
for 10 min.
14
Gibbs, Nguyen, and Caskey
6. Centrifuge for 5 s to bring down condensation and add 3.0 FL of STOP
solution.
7. Heat to 80% for 2 min and analyze by electrophoresis and autoradiogra-
phy (see Chapter 3).
4. Notes
1. Recommended manufacturers: These recommendations are not meant
to imply that only the specified manufacturer products can be used.
2. Synthesis of the first cDNA strand: Syntheses of cDNA have been per-
formed from poly(A+) RNA, total cellular RNA or crude cell extracts

(1 I, 12). We always prepare total cellular RNA by the guanidinium method
(13), which is convenient when a relatively small number of samples are
to be analyzed. There is no need to prepare poly(At) RNA, although if
you already have some it works fine.
There are at least three methods for priming, the synthesis of cDNA
random priming, oligo (dT) priming, and specific oligimer priming.
Priming with a specific oligimer has been avoided, since the resulting
cDNA cannot be used as a template for PCR of other DNAfragments. In
addition, the conditions for annealing of a specific oligimer must be
stringently controlled. There seems to be little difference in performance
among the nonspecific priming methods, although oligo (dT) has the
theoretical disadvantage of less efficient coverage of the 5’ end of the
message. Thus, the random hexamers offer the advantages of a simple
protocol that yields a product that can be used for amplification in mul-
tiple PC%. Note that controlled synthesis of a second cDNA strand is
unnecessary. However, including the alkaline hydrolysis step after the
cDNA synthesis improves the quality of the final product as determined
by agarose gel electrophoresis.
3. Contamination: One of the most pernicious problems associated with
the extreme sensitivity of PCR is the potential for false amplifications as
a result of contamination of the reactions by minute amounts of DNA.
The most common source of contamination is the products of previous
PC%, and the best solution to the problem is extreme caution when
handling the PCR reagents. To check for contaminants, a negative con-
trol reaction without any DNA template should always be run in parallel
with any PCR. In the case of cDNA amplifications, two excellent negative
controls are the omission of reverse transcriptase in the cDNA synthesis
step, and alkaline hydrolysis of the RNA before the beginning of the
procedure. Neither of these reactions should yield a PCR product.
Direct Sequencing of DNA 15

A further source of contamination in cDNA amplifications is caused
by the presence of genomic DNA The simplest way to overcome this
problem is to choose PCR priming sites that are separated by large in-
trons so that only the spliced RNA sequences will be amplified. If the
DNA and cDNA amplifications cannot be distinguished by primer posi-
tioning, then extra care should be taken during the preparation of the
RNA to avoid collecting DNA Consider DNAse treatment of the RNA
only as a last resort.
4. Optimal PCR buffers: At least two PCR buffer systems are in common
use at this time. We have had most experience with the DMSO-contain-
ing buffer described above (IO), but the buffer recommended by the
Cetus Corporation (2.5 mMMgClz, 200 l.tMdNTP, 50 mMKCl,200 pg/
mL gelatin, and 10 mMTris-HCl at pH. 8.4) (14) works at least as well
under most circumstances. The “Cetus buffer” has the potential disad-
vantage that the concentration of some of the ingredients may need to
be carefully optimized to ensure most efficient and specific amplifica-
tion. However, the “Cetus buffer” has the advantage that it is more likely
to be compatible with subsequent procedures used to analyze the PCR
products. This is sometimes evident when collecting PCR products by
ethanol precipitation, when material other than DNA is sometimes
pelleted from the DMSO-containing buffer (i.e., salt and protein).
Whatever final PCR protocol is chosen, it is important that a high level
of specificity is achieved in the amplification. PCRs that contain multi-
ple species when analyzed by agarose gel electrophoresis usually do not
sequence well.
5. Separate vs simultaneous amplification and single-strand production: A
key step in the analysis is the generation of a single strand by asymmetric
priming in a PCR-like reaction. As described in the original report of the
method, a single PCR is performed with different amounts of each primer
(7). Initially there is an exponential increase in the amount of the de-

sired fragment, and then, as one primer is exhausted, the second primer
continues to produce single strands. We separate the two reactions,
doing one PCR to generate plenty of double-stranded material, and
then taking aliquot of the product to initiate a second reaction that con-
tains only one primer. This is more cumbersome, but in our hands makes
for more reliable results, presumably because the amount of double-
stranded material is relatively constant when the singlestrand produc-
tion process begins.
The two-step procedure also has the advantage that the success of the
initial PCR can be monitored and that the PCR can be used to seed
16 Gibbs, Nguyen, and Caskey
multiple SSPRs. There is no need to return to the cDNA-synthesis prod-
ucts in each case. Increasing the distance between the primer used to
generate singlestranded DNA template and the DNA-sequencing primer
can diminish the signal from the sequenced products; however, primers
as far as 4 kb apart have functioned reliably.
6. Intermediate steps between PCR and SSPRs: When initiating the SSPRs,
it is not necessary to purify the products of the first reaction by phenol
and NENsorb@ chromatography, as has been previously described (15).
Instead, the second reaction can be initiated by simply taking a small
aliquot of the first PCR (11%) without any purification. If more than
1% is used, then the products of the second reaction might not se-
quence well. If the SSPRs cannot be made to work this way, then try the
phenol/NENsorb@-affinity-column approach.
a. Dilute the PCRwith an equal vol of H,O.
b. Extract with an equal vol of phenol (saturated with TE buffer).
c. Reextract the phenol phase with an equal vol of fresh H,O.
d. Remove all traces of phenol with ether.
e. Remove all traces of ether.
f. Passage the DNA through a NENsorb@ column, eluting with

50% methanol.
g. Lyophilize and resuspend in 50 uL of H,O; use l-2 l.tL for SSPR
7. Agarose gel electrophoresis of SSPR products: In most cases, the analysis
of SSPR products by agarose gel electrophoresis reveals a band at the
position of the double-stranded fragment, and a faster-migrating band
representing the single-stranded material (Fig. 2). At high agarose con-
centrations (~1%) or when the single strands have an unusual second-
ary structure, the single-strand band is sometimes at a position of higher
mol wt. Not infrequently, multiple bands are seen, which may reflect the
presence of many different secondary structures in the single strands or
may be attributable to the internal priming of the double-stranded tem-
plate during SSPR. All different types of SSPR product can sequence
well, but there is a loose correlation between the complexity of the aga-
rose gel morphology and the failure to sequence. In general, it is the
“cleanliness” of the initial PCR that is more important than the agarose
gel pattern of the SSPR product.
8. Buffers for SSPR: Either of the PCR buffer systems described above can
be used for the SSPRs. However, when using the DMSO-containing buffer,
it is particularly important that there is no carry-over of salt or protein
into the pellet to be used for the DNA sequencing. Therefore, we have a
preference for the use of the buffer recommended by the Cetus Corpo
Direct Sequencing of DNA 17
Pig. 2. PCR amplification of hypoxanthine phosphoribosyltransferase (HPRT) cDNA
and production of single strands. A 920-b fragment containing the human peptide-
coding region was amplified from cDNA as described in the text, using the specific
oligonucleotide primers #365 (5’- CCG CCC AAA GGG AAC TGA TAG TC -3’) and
#863 (5’- CTT CCT CCT CCT GAG CAG TCA G -3’). Single strands were generated
from the PCR products as described in the text. Lane M. W., mol wt markers; Lane 1,
PCR product; Lane 2, SSPR product using oligimer #365; Lane 3, SSPR product us-
ing oligimer #863. The faster-moving bands represent the single-stranded fragments.

ration for the SSPRs, except when we find that a particular primer set
functions much better in the DMSO-containing buffer. In that case, the
DMSO-containing buffer is used in the SSPR, but great care is taken to
avoid salt or protein coprecipitation.
9. Sequencing primers: The DNA-sequencing primers are routinely con-
structed as 18mers. The use of end-labeled primers that are comple-
mentary to sequences between the PCR primers that were first used for
amplification enables greater specificity, since nonspecific PCR contami-
nants will not be primed during the sequencing. However, the PCR prim-
ers usually can be used as the sequencing primers if the initial PCRs
appear homogeneous when assayed by agarose gel electrophoresis. This
is a great advantage, since it obviates the need for the construction of
additional oligimers. To ensure that the PCR primers can be used for
sequencing, we find it necessary to (a) use the minimum amount of
primer in the initial PCR (as little as 5 pmol of each primer) and (b)
perform the minimum number of PCR cycles that produce a visible
band on an agarose gel from 10% of the reaction products (as few as 23
for rare cDNAs or unique human gene sequences). Thus, when trouble-
18 Gibbs, Nguyen, and Caskey
shooting a reaction in which the PCR primers will not give a good se-
quence, and when it is not desirable to synthesize a new oligomer, the
amount of PCR primer and the number of cycles in the initial reactions
should be titrated downward.
10. Sequencing buffer: The sequencing buffer that we prefer is the reverse-
transcription buffer (see POL buffer, above) and not the usual mixture
recommended by United States Biochemicals. The POL buffer has a
lower ionic strength and, in our hands, gives a cleaner sequence.
11. SequenaseTM vs reverse transcriptase or Tuq: Reverse transcriptase and
Tuq DNA polymerase have each been used for direct DNA sequencing.
We have not had success with reverse transcriptase, although others re-

port good results (5). We have had no experience with Taq, but note that
others report the superiority of that enzyme (16). Tuq sequencing is ex-
pensive, both because of the cost of the enzyme and because of the high
concentrations of nucleotides that must be used. We have not encoun-
tered a region of DNA secondary structure that could not be resolved by
T7 DNA polymerase sequencing at .50X, and believe that the only ad-
vantage of Taq will be in the coupling of PCR to the sequencing by fully
automated protocols (15).
12. Sequencing reaction temperature: A most important feature of this pro
tocol is the temperature of the sequencing reactions. In our hands the
results from reactions at 50°C are spectacularly better than those from
reactions at 37OC (see Fig. 3).
13. Automated DNA-sequencing: The direct DNA sequencing procedure
can be automated by the use of fluorescent DNA sequencing primers
(see also Chapter 4) and a commercially available fluorescent gel reader
(l5,17,18). The manipulations for the automated DNA-sequence analy-
sis are essentially the same as those for manual DNA sequencing. If nec-
essary, the products of two SSPRs can be pooled before distribution of
aliquots to be annealed to each of the custom-produced primers. Auto-
mated DNA sequencing of the PCR products routinely yields 275550
bases of sequence, and it is likely that this can be extended by further
“fine tuning” of the reaction conditions.
Acknowledgments
We thank Grant MacGregor for reviewing this manuscript. R. A. G. is a
recipient of the Muscular Dystrophy Association’s Robert G. Sampson Distin-
guished Research Fellowship, and C. T. C. is an investigator of the Howard
Hughes Medical Institute. Supported by DHS grant #DK31428 and Welch
Foundation grant # Q533.
Direct Sequencing of DNA
19

Fig. 3. Direct DNA sequencing with T7 DNA polymerase at 37 or 50%. Two other-
wise identical DNA sequencing reactions were performed at 37 or 5O”C, according to
the procedure described here.
References
1.
2.
3.
4.
5.
Mullis, K and Faloona, F. A. (1987) Specific synthesis of DNA in vitrovia a polymer-ax
catalyzed chain reaction. Methods Enzymol155,335-350.
Saiki, R. K., Scharf, F., Faloona, F., Mullis, R B., Horn, G., Erlich, H. A., and Amheim,
N. (1985) Enzymatic amplification of Bglobin genomic sequences and restriction
sire analysis for diagnosis of sickle cell anemia. Science230, 1350-1354.
Wrischnik, L. A., Higuchi, R. G., Stoneking, M., Erlich, H. A., Arnheim, N., and Wil-
son, A. C. (1987) Length mutations in human mitochondrial DNA: Direct sequenc-
ing of enzymatically amplified DNA. Nucleic Acids Res. 15,529-542.
McMahon, G., Davis, E., and Wogan, G. N. (1988) Characterization oft-ki-ras oncogene
alleles by direct sequencing of enzymatically amplified DNA from carcinogen-induced
tumors. Pm. Nat1 Acad. Sci. USA 84,49’74-49’78.
Wong, C., Dowling, C. E., Saiki, R. K, Higuchi, R. G., Erlich, H. A., and Kazazian, H.
H. Jr. (1987) Characterization of beta-thalassemia mutations using direct genomic
sequencing of amplified single copy DNA. Nature 30,384-386.
Gibbs, Nguyen, and Caskey
6.
10.
11
12.
13
14.

15.
16
17
18.
Yandell, D. W. (1989) Direct genomic sequencing of alleles at the retmo-
blastoma locust Applications to carrier diagnosis and genetic counsellmg, m
Cancer Cells: Molecular Daagnosfws of Human Cancer, vol 7, Cold Spring
Harbor Laboratory, Cold Spring Harbor, NY, pp 223-227.
Gyllensten, U. B. and Erlich, H. (1988) Cenerauon of smgle stranded DNA by the
polymerase chain reacuon and its application to dtrect sequencmg of the HLA-DQA
locus Proc NatL Acad Sn. US4 85,7652-‘7656.
Winship, P. R. (1989) An improved method for directly sequencing PCR amphfied
material using dimethyl sulphoxide. Nucleic Acxis Res. 17,1266
Carothers, A. M , Urlaub, G , Mucha, J., Grunberger, D , and Chasm, L. A
(1989)
Pomt mutauon analysts m a human gene: Rapid preparation of total RNA, PCR amph-
ficauon of cDNA, and Taq sequencing by a novel method Bio.?&nzquts 7,494-499
Kogan, S. C., Doherty, M., and Gitschier, J. (1987) An improved method for prenatal
dtagnosls of generic diseases by analysts of amphfied DNA sequences Apphcauon to
hemophilia A. N. En&J, Med. 317,98.5-990
Gibbs, R. A, Chamberlam, J. S , and Caskey C. T. (1989) Dlagnosts of new mutauon
diseases using the polymerase chain reaction, m The Polymerase Churn Reactron Pnn-
n/&s and Applacatrons (Erbch, H , ed.), Stockton, New York, pp. 171-191
Kawasaki, E (1989) Detecuon of gene expression, m The Polymerase Charn Reactron
Pnnnples and Appkcattons (Erbch, H., ed.), Stockton, New York, pp. 89-97
Chugwm, J. M., Przybyla, A. E., McDonald, R J , and Rutter, W J (1979) Isolauon of
biologically active nbonucleic acid from sources enriched m nbonuclease. B:ochemrs-
try
18,52945299.
Satkt, R. K., Celfand, D. H., Stoffel, S., Scharf, S J , Hlgucht, R , Horn, G T., and

Mulhs, R. B. (1988) Pnmerdlrected enzymatic ampllficauon of DNA with a therm*
stable DNA polymerase. Scaenu 239,48’7-491.
Gibbs, R. A., Nguyen, P. N., McBride, L J , Koepf, S. M , and Caskey, C T (1989)
Identificauon of mutations leading to the Lesch-Nyhan syndrome by automated
direct DNA sequencing of an vrtro amplified cDNA Proc NatL Acad Sn USA 89,
1919-1923.
Inms, M A , Myambo, K. B , Gelfand, D. H., and Brow, M A (1988) DNA sequencmg
wuh Thus acquahcm DNA polymerase and direct sequencing of polymerase cham
reaction amplified DNA Proc. NatL Acad. Ser. USA 85,94369440
McBride, L J., Koepf, S. M , Gibbs, R A, Nguyen, P N , Salser, W , Mayrand, P E ,
Hunkaplller, M. W., and Kromck, M. N. (1989) Automated DNA sequencing meth-
ods using polymerase chain reactton Clm. Chem ,35,21962201
Smith, L. M., Sanders, J. Z., Raiser, R. J., Hughes, P., Dodd, C , Connell, C. R., Hemer,
C., Kent, S. B H., and Hood, L. E. (1986) Fluorescence detecnon m automated DNA
sequence analysts Nature 321, 674
CHAPTER 3
Direct Sequencing
of PCR-AmpMed DNA
Peter M. Green and Francesco Giannelli
1. Introduction
The polymerase chain reaction (seechapter 1) allows the rapid isolation
of specific DNA targets that may be used as sequencing templates either
directly or after cloning into M13. The latter procedure allows single-strand
sequencing, but is otherwise undesirable not only because it is slow, but also
because a significant proportion of the amplified DNA molecules contain
replication errors. These are expected to occur at a frequency of 1 in 10,000
bases incorporated (I), and will also be amplified during subsequent cycles.
This means that at least three clones from independent amplification experi-
ments must be sequenced in order to identify these replication errors and
determine the final consensus sequence. Direct sequencing of the PCR prod-

uct bypasses this problem since it produces an “average sequence” of all the
copies of the target, and any miscopied molecule is bound to represent only
a very small proportion of the total (unless one starts PCR with very few
molecules of the target DNA),
The technique described here for the direct sequencing of PCR prod-
ucts is based on the “traditional” dideoxynucleotide (ddNTP) sequencing
method developed by Sanger, Nicklen, and Coulson in 1977 (2). The proce-
dure uses a modified T7 DNA polymerase, SequenaseTM (USB) , in place of
the Klenow enzyme, and ddNTPs to terminate specifically DNA synthesis at
either A, C, G, or T in such a way as to produce a population of molecules
where every possible length is represented in sufficient amounts to be
detected by autoradiography after fractionation on a denaturing polyacryl-
amide gel. This results in a “ladder” of bands across four tracks that are read
From:
Methods in Molecular Biology, Vol 9. Protocols m Human Molecular Genetics
Edlted by- C Mathew Copyright Q 1991 The Humana Press Inc , Clifton, NJ
21
22
Green and Giannelli
upward to give the sequence of a particular template DNA When sequenc-
ing large numbers of products with the same primer, it is more useful to load
all the “A” tracks adjacent, then the “Cs,” and so on, and look for pattern
changes that would indicate a mutation (3).
However, direct sequencing of PCR products requires first of all the elimi-
nation of primers, dNTPs, PCR buffer, and Tuq polymerase, since these would
interfere with sequencing. This is done by binding the DNA to a glass bead
suspension (GeneClean from Bio lOl), washing it, and then eluting it in a
small vol. The DNA binds to the “Glassmilk” suspension while other ingredi-
ents are washed away. The oligonucleotides appear to bind too tightly to be
efficiently eluted.

2. Materials
1. GeneClean kit (Bio 101). This contains all the ingredients needed to
purify the PCR products prior to sequencing, i.e., sodium iodide (satu-
rated solution), “Glassmilk” suspension, and “NEW” wash buffer.
2. TE: 10 mMTris-HCI, pH 8,0.1 mMEDTA.
3. SequenaseTM (USB). This is the trade name for a modified T7 DNA
polymerase. It should be noted, however, that the sequencing strategy
of the Sequenase kit differs from that described here and is not very
useful for sequencing PCR products.
4. dNTP/ddNTP mixes:
Mix. UL
A” C G” To
Stock solutions
0.5 mMdCTP
0.5 mMdGTP
0.5 mMdITP
0.05 mM ddATP
0.05 mM ddCTP
0.05 mM ddGTP
0.05 mM ddTIP
TE
80 80 80 80
80
80 I
80
80
80 80 80 80
0.8
80
80

80
260 180 180 180
Final vol = 500 500 500 500
5. 5x Sequenase TM buffer: 200 mM Tris-HCl, pH 7.5, 100 mM MgCl,,
250 mM NaCl.
6. Dimethyl sulfoxide (DMSO): Freeze in lOO-uL aliquots.
7. a[S”]dATP (600 Ci/mmol). Store in 4u.L aliquots at -70°C.
8. 100 mMDithiothreito1 (DTT): Freeze in 106uL aliquots.
Direct DNA Sequencing
23
Notched
Plate
Backplale
(40 Y 20cm)
Spacers
(0 4mm thick)
Ploles ioped-
together
Gel stand
Lower buffer
Fig. 1. Diagram of apparatus for polyacrylamide gels. The equipment should in-
clude a safety lid that completely covers the gel stand to protect against shocks, and
an aluminum
plate to clamp on front of the gel plates as a heatsink.
9. Oligonucleotide primers at 100 ng&L: Either the primers used for PCR
or primers internal to the product can be used.
10. Microtiter plates: U-shaped wells are best and they must be resistant to
boiling (e.g., Nunc).
11. Chase solution: 0.25 mMdATP, 0.25 mMdCTP, 0.25 mM dGTP, 0.25
mMdTIP, 10% DMSO.

12. Gel loading dyes: 10 mg Bromophenol blue; 10 mg xylene cyanol; 3@
mL deionized formamide.
13. Polyacrylamide gel electrophoresis. The basic apparatus for running
sequencing gels is shown in Fig. 1. It can either be bought from various
suppliers or constructed in a university or hospital workshop.
14. “Repelcoten (BDH) or similar siliconizing solution.
24
15.
16.
17.
18.
19.
20.
21.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Green and Giannelli
40% Acrylamide stock solution: 76 g acrylamide, 4 g N,N-methylene-
bisacrylamide; made up to 200 mL with distilled water. Deionize for 30
min by stirring with -10 g “Amberlite” mixed bed resin (BDH). Filter
the solution into darkened bottles and store at 4OC. Acrylamide is highly
toxic and should be prepared in a fume hood with gloves, goggles, and a

face maskwhen weighing out solid. It is possible to buy ready-topour gel
mixes from several companies. This is safer and convenient but expensive!
10% Ammonium persulfate (AI’S). Make up 10 mL at a time; keep at
4°C for up to 1 mo.
N,N,N,N-Tetramethylethylenediamine (TEMED).
10x TBE buffer: 089MTris-base, 0.89Mboric acid, O.OZMEDTA.
Gel fixing solution: 10% Acetic acid; 10% methanol. Make up 1L that
can be reused a number of times.
X-ray film (e.g., Kodak XS-1) and cassettes.
Along with standard laboratory equipment, the following are useful: gel
drier, salad spinner, and Hamilton repeater syringe (for dispensing 2 PL
repeatedly with a yellow tip).
3. Method
3.1. Gene Cleaning of PCR Product
After removing the paraffin oil, add 2.5 vol of saturated sodium iodide
(supplied with kit) to l-2 Itg of PCR product in a 0.5mL Eppendorf
tube.
Add 5 I.~L of the “Glassmilk” suspension, vortex, and leave for 5 min at
room temperature.
Spin tubes for 15 s at full speed (-14,000 r-pm) in a microcentrifuge tube.
Remove and discard the supernatant with a yellow tip.
Add 200 PL of “NEW” wash buffer (supplied with the kit and kept at
-2OOC).
Vortex and spin for 15 s. Repeat steps 4-6 twice.
After removing the supernatant, respin the pellet for 15 s and remove
residual liquid (including any remaining paraffin) with a drawn-out
Pasteur pipet.
Add 5 ltL of TE to the pellet and resuspend with the automatic plpet.
Incubate for 5 min at 55OC (either in a water bath or a PCR machine).
Spin for 30 s. Transfer the supernatan t to a fresh 0.5mL Eppendorf tube.

Repeat Steps 8 and 9. Combine the supernatants to give 10 PL of puri-
fied PCR product. If desired, run 1 PL on a gel to check recovery (should
be 80-90% or hieher).
Direct DNA Sequencing
25
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
1.
2.
3.
4.
3.2. Sequencing Reaction
In order to sequence eight templates simultaneously with the same
primer, make up the following primer premix: 5 l,tL Primer (at 100
ng/pL), 18 ltL 5x Sequenase buffer, 25 ltL TE, and 6 l.tL DMSO (see
Note 3).
Aliquot 6 yL of this primer premix to eight 0.5mL Eppendorftubes and
add 1 l.tL of each purified PCR product (“template”) per tube, mixing
with a yellow tip each time. Put these tubes to one side for a few minutes.

Dispense 2 ltL of each dNTP/ddNTP mix into four microtiter wells, for
each template wrth the Hamilton repeater. Mark the plate by template
number (1-8) and nucleotide mix (A’, Co, Go, and TO).
Make up the enzyme/label premix: 4 PL a[Ss5]dATP (600 Ci/mmol), 8
ltL 0 IMD’IT, 19 j.tL TE, 3 I,~L DMSO, and 1 uL Sequenase (12.5 U).
Heat the primer/template mixes (from Step 2) to 95°C for 5 min
(either in a boiling water bath or a PCRmachine). Snap-cool on an ice/
water bath.
Add 4 FL of the enzyme/label mix (from Step 4) to the side of the tubes
containing the primer/template mixes. Flick-spin to mix.
Use the Hamilton repeater to dispense 2 p.L from tube 1 to each of the
four microtiter wells labeled l-A, l-C, l-G, and 1-T. Repeat for tem-
plate/primer mixes 2-8.
Use the salad spinner to spin down the drops in a microtiter plate. Al-
ternatively, tap on the bench to knock the droplets down to the bottom.
Put tape around the edge of plate and float on 37°C water bath for 5 min.
Add 2 l.tL of chase solution to every well, spin to mix, and again incubate
at 37OC for 5 min.
Add 2 yL of running dyes to each well, and spin to mix.
When the gel is ready for loading (see below), heat to 95OC for 3 min by
floating on a “simmeringn water bath (fast boiling will flood the plate).
Snap-chill on ice/water. Samples are now ready for loading on the gel.
3.3. Polyacrylamide Gel Electrophoresis
The glass plates (Fig. 1) must be cleaned thoroughly in soap and water,
and then dried with paper towels and ethanol.
Use “Repelcote” to siliconize the notched plate only. Clean with etha-
nol. Repeat if this is the first use of the plate.
Tape the plates together with the 0.4mm spacers down each side, ensur-
ing that the tape sticks firmly all round, especially at the bottom.
Make up the gel mix: ‘75 mL 40% Acrylamide, 25 mL 10x TBE, and

230 g urea; make up to 500 mL with distilled water.