Volume 16 Number 21 1988
Nucleic Acids Research
Direct sequencing of polymerase chain reaction amplified DNA fragments through the
incorporation of deoxynudeoside a-thiotriphosphates
Kay L.Nakamaye + , Gerald Gish, Fritz Eckstein* and Hans-Peter Vosberg1
Max-Planck-Institut fur Expenmentelle Medizin, Abteilung Chemie, Hermann-Rein-Strasse 3, D-3400
Gottingen and 'Max-Planck-Institut fur Medizinische Forschung, Jahnstrasse 29, D-6900 Heidelberg,
FRG
Received September 5, 1988, Accepted October 7, 1988
ABSTRACT
The direct sequencing of DNA generated by the polynucleotide chain reaction, via the incorporation of phosphorothioate nucleotides and followed by
treatment with an alkylating reagent that cleaves specifically at the
phosphorothioate positions, is described. The Tag polymerase used in the
amplification reaction incorporates the Sp-diastereomer of the deoxynucleoside 5'-O-(l-thiotriphosphates) as efficiently as the natural nucleotides. Chemical degradation of the phosphorothioate-containing DNA fragment can be performed with either 2-iodoethanol or 2,3-epoxy-l-propanol. The higher reactivity of 2,3-epoxy-l-propanol allows less reagent to
be used to obtain the same amount of degradation as with 2-iodoethanol.
INTRODUCTION
Analysis of the molecular basis of genetic disease, examining DNA for
phenotypical mutations, studying polymorphisms, and typing of DNA are a
few among many problems that require detailed and accurate nucleotide
sequence information of DNA or RNA. These studies, which involve the
analysis of small quantities of DNA or RNA, have benefited greatly from
the large degree of amplification that is possible using the recently developed polynucleotide chain reaction (PCR) technique (1,2). The PCR technique has now a variety of applications from forensic analysis (analyzing
biological evidence from crimes), to the identification of low levels of certain DNA fragments such as HIV (3) and neoplastic cells (which typically
are at a level of 1 cell to 1 x 10^ normal cells) (4), and as well, the
preparation of sufficient DNA for cloning (5). A useful complement to the
PCR procedure is the ability to sequence the amplified products and to this
end both the Sanger and Maxam-Gilbert methods have been used (5-7).
Recently, we reported an alternative method for DNA sequencing involving the incorporation of phosphorothioate groups into the nucleic acid
£> IRL Press Limited, Oxford, England.
9947
Nucleic Acids Research
and selective degradation of the strands at the position of the phosphorothioate (8). Conceptually, this method has the advantage that the DNA
fragments generated from a PCR could be sequenced directly and operationally it is not complicated to perform. We report here our studies using
Taq polymerase to incorporate phosphorothioates into DNA using PCR and
further investigations into the chemistry of the degradation reaction. The
result is a PCR sequencing technique where four separate polymerization
reactions are conducted in the presence of three natural deoxynucleoside
triphosphates and one deoxynucleoside a-thiotriphosphate (each reaction
using a different dNTPaS). After purification, the sequence of the reaction
products is revealed by treatment with an alkylating reagent that causes
partial random degradation at the phosphorothioate positions, followed by
polyacrylamide gel electrophoresis. Conditions are reported allowing
either one or both strands of the PCR fragments to be sequenced.
EXPERIMENTAL
Materials
DNA was either closed circular double stranded (ceds, RFIV)
M13mp2TAA (9), Bgl I-linearized double stranded (ds, RFIII)
M13mp2TAA or Bgl I-linearized ds M13mpl8. In addition, a ds DNA
restriction fragment from the human myosin heavy chain was used. Taq
Polymerase (5 U/(a.l) was supplied by Perkin Elmer Cetus. T4 polynucleotide kinase (30 U/p.1) was purchased from US Biochemicals. The Spdeoxynucleoside 5'-O-(l-thiotriphosphates) were prepared as in (10). The
enyzme Bgl I and the nucleoside triphosphates were purchased from
Boehringer Mannheim. [/y-32p]ATP (>5000 Ci/mmol) was purchased from
Amersham.
Both 2-iodoethanol and 2,3-epoxy-l-propanol were purchased from Sigma. Synthesized using an Applied Biosystems 380B DNA
Synthesizer were the oligonucleotides:
M13SEQ
M13PCR
MHC1
MHC2
5'-d[AGGGTTTTCCCAGTCACG]-3'
S'-dtCACCCTGGCGCCCAATAq-S1
5'-d[GATGGATGCTGACCTGTq-3'
S'-dfGAGGTGGCAATGGTCTGGJ-S1
The PCR amplifications were conducted using Techne Dri-Block DB-1 adjustable heating blocks with glycerol in the heating wells for efficient heat
transfer to the reactions.
9948
Nucleic Acids Research
Methods
A. Sequencing of DNA Labelled After PCR Amplification
1. Phosphorylation of Oligonucleotide Primer
One oligonucleotide primer was phosphorylated prior to PCR amplification as described (9). Briefly, 2.0 A260~units (40 u\g) of oligonucleotide was treated with 30 units polynucleotide kinase in a reaction mixture
(30 |il) composed of 100 mM Tris-HCl, pH 8.0, 10 mM 2-mercaptoethanol,
10 mM MgCl2, and 1 mM ATP. After 2 hr at 37°C the reaction mixture was
heated at 70°C for 15 min and the product purified using a SEP-PAK Ci 8
cartridge (Waters Associates) (11). The phosphorylated oligonucleotide
was stored at -20°C as an aqueous solution at a concentration of 10 A260"
units/ml.
2. Polymerase Chain Reaction
The PCR amplifications were performed manually. Linear ds DNA,
from 1 ng to 1 mg, was dissolved in 320 |xl of a solution containing 63 mM
KC1, 12.5 mM Tris-HCl, pH 8.4, 0.016 % (w/v) glycerin, 3.8 mM MgCl2, 0.8
(J.M (0.07 A260-u n its) of 5'-phosphorylated oligonucleotide primer and
0.8 |0.M (0.07 A260-units) of a non-phosphorylated oligonucleotide primer.
Annealing of the oligonucleotides was performed by heating at 96 - 97°C
for 5 min to denature the DNA then cooling at 37°C for 5 min. The solution
was partitioned (80 p.1) i n t 0 f ° u r 750 \il Eppendorf tubes, diluted to
100 (0.1 with a mixture of three dNTPs and one dNTPaS (10) to give a final
concentration of 250 (i.M in each nucleotide, and 4 U of Tag DNA
polymerase was added. Paraffin oil (100 jxl) was layered over each
reaction mixture. The reactions were subjected to the temperature cycle
of 71°C for 3 min, 96 - 97°C for 1 min, and 37°C for 2 min for 12 to 30
cycles depending upon the amount of starting DNA. For the last cycle the
reaction time at 71°C was extended to 7 min then the samples were
allowed to cool to room temperature. The paraffin oil was pipetted away
from the reaction solution and completely removed by two ethyl ether
washes (200 \±\ each). The PCR product could be easily visualized on
ethidium bromide 1.5 % agarose gel (9), using about 8 u.1 of the reaction
mixture.
3. Labelling of PCR Fragments
Each PCR product was passed through a Sephadex G50 spun column
(ca. 1 ml) equilibrated with H2O to remove the unreacted nucleotides and
salt (12). The flow-through from the column was precipitated by adding
9949
Nucleic Acids Research
10 \i\ 3 M sodium acetate pH 6.0 and 500 u.1 absolute ethanol. After chilling for 15 min at -78°C followed by centrifugation for 15 min the supernatant liquid was decanted and the pellet washed with 750 (il 70 % ethanol then dried for 2 min in a Speed Vac Concentrator.
To each pellet was added 30 mCi of [Y-32p]ATP and the solutions
again dried. Each pellet was taken up in 4.5 |il of 20 mM Tris-HCl, pH 8.0,
20 mM MgCl2, and 2.4 mM 2-mercaptoethanol. T4 polynucleotide kinase
(0.5 |il, 15 U) was added to the solution and the reaction allowed to proceed for 45 min at room temperature. The reaction mixture was mixed
with 2 |il of stop mix (96 % formamide, 10 mM EDTA, 0.1 % (w/v) bromophenol blue and 0.1 % (w/v) xylene cyanol ff) and applied to an 8 % polyacrylamide sequencing gel. After electrophoresis the PCR fragment was visualized by 5 min exposure of Kodak X-Omat XAR-5 film. Often the PCR
fragment was observed as 2 bands probably due to incomplete denaturation. Both bands of the PCR fragment were excised and extracted from the
polyacrylamide using the method of Rubin (13).
4. Sequencing Reactions
The radioactively labelled PCR fragments were dissolved in enough
water to give approximately 500 cps/|il solutions (as determined with a
Geiger counter).
For each sequencing reaction 4 ^.1 of DNA was mixed
well with 2 ^1 of stop mix (96 % formamide, 10 mM EDTA, 0.1 % (w/v)
bromophenol blue and 0.1 % (w/v) xylene cyanol ff) containing 7 % (v/v)
iodoethanol or 0.5 % (v/v) 2,3-epoxy-l-propanol. The stop mix solution
was prepared fresh prior to each experiment to avoid any possibility of
hydrolysis of the alkylating reagents. The samples were heated at 95°C for
3 min and then cooled on ice before being applied to an 8 % polyacrylamide sequencing gel. After electrophoresis the gel was dried and exposed
on Kodak X-Omat XAR-5 film, usually for about 48 hr without an
enhancing screen or 12 hours with a screen.
B. Sequencing of DNA Labelled Prior to PCR Amplification
1. Radioactive Labelling of Oligonucleotide Primer for PCR
Oligonucleotide primer (0.2 A260-units, 4 |ig) was phosphorylated in
a 30 (J.1 reaction solution composed of 100 mM Tris-HCl, pH 8.0, 10 mM 2mercaptoethanol, 10 mM MgCl2, 60 |iCi [y-32p]ATP and 60 units T4 polynucleotide kinase. After 45 min at 37°C the reaction mixture was heated
at 70°C for 15 min and the product purified using a SEP-PAK Cl8 cartridge
(Waters Associates) (11). The phosphorylated oligonucleotide was stored
9950
Nucleic Acids Research
as a 3.6 A260-units/ml stock aqueous solution and had a specific activity
of 3 x 1()9 cpm/A260-unit2. PCR Amplification and Sequencing
The PCR was performed as described above using the radioactively
labelled primer. After 15 cycles the paraffin oil was removed and the DNA
precipitated through the addition of 10 |il 3M sodium acetate, pH 6.0 and
250 u.1 absolute ethanol, chilling at -80°C for 30 min followed by a
20 min centrifugation. Excess ethanol was removed by drying the pellets
for 2 min and the DNA was dissolved in 20 u.1 of 10 mM Tris-HCl, 0.1 mM
EDTA, pH 8.0 and 2 u.1 stop mix. The PCR fragments were purified on an
8 % polyacrylamide sequencing gel as described above. Isolation of the
DNA from the gel and the sequencing reaction were performed as described above.
RESULTS
Incorporation of Phosphorothioate Nucleotides into PCR Fragments
The critical factor in combining PCR with phosphorothioate sequencing is the ability of Taq polymerase to use deoxynucleoside 5'-O-(l-thiotriphosphates) as substrates. To test this we used ds M13 DNA to prepare
PCR fragments of known length. As one oligonucleotide we used the M13
general sequencing primer (M13 SEQ) which hybridizes in M13mp2TAA to
base positions 6258 -6275. A second primer (M13PCR) was prepared
which had the same sequence as bases 5994 - 6011 in M13mp2TAA. The
PCR fragment should thus be 281 bases long. The same primers were also
used with M13mpl8 which yields a PCR fragment 335 bases long.
As can be seen in Figure 1, the PCR amplification can be performed
using three normal nucleotides and one phosphorothioate-containing
nucleotide. The yields are similar to that obtained using all natural
nucleotides. By varying the MgCl2 concentration we found 3 mM to give
an optimial yield of DNA. Slight differences in yield were observed (Figure
2) with the different dNTPaS mixes, with dGTPaS = dCTPaS > dATPaS =
dTTPaS, but a significant amount of each PCR fragment could be prepared
and, most importantly, no increase in the number of PCR cycles was
required to obtain enough DNA from the four reactions for sequencing. A
comparison of Figures 1 and 2 indicates that the DNA used for
amplification can be either linear ds or ccds (RFIV) with no reduction in
the yield of PCR fragment obtained. We have also found that the
9951
Nucleic Acids Research
A
B C
D
Fig. 1 Dependence of the PCR yield on Mg2+ concentration
Experiments were carried out as described in Materials and Methods with
350 ng linear ds M13mpl8 DNA, lu.M each of M13 Seq and 5'phosphorylated M13PCR oligonucleotides, 250 mM each of dATP, dCTP,
dTTP and dGTPaS, the indicated MgCl2 concentration and 4 units of Taq
polymerase for 20 cycles. Analysis was carried out by gel electrophoresis
using an ethidium bromide 1.5 % agarose gel. A, length standard d>X174
DNA-Hae III digest (the arrow indicates a length of 310 bp); B, lmM
MgCl2; C, 2.0 mM MgCl2; D, 3.5 mM MgCl2-
conditions described here can be used with ss DNA to give good yield of
PCR product (data not shown).
Several parameters appear to be important in maximizing the effectiveness of the PCR amplifications. Foremost is the quality of the Taq
polymerase. Our best results were obtained with the Perkin Elmer/Cetus
enzyme. Two factors relating to the thermal cycle must be controlled to
achieve good amplification. First, care must be taken when denaturing the
DNA. Poor denaturation hinders the annealing of new primers. We found
that temperatures less than 95 - 96°C gave poor results. Second, thermal
transfer is very important. We obtained good results by using glycerol in
a well-controlled, variable temperature thermal block to obtain efficient
heat transfer to the (0.75 ml) Eppendorf tubes. The other temperatures
appear to be less important, although the polymerizations conducted at
70°C appear to produce fewer side products than at lower temperatures.
The temperature of polymerization probably aids the purity of the PCR
product by increasing the specificity of the oligomer annealing and is thus
9952
Nucleic Acids Research
A
B
C
D E
F G
Fig. 2 Suitability of dNTPaS as substrates for PCR amplification
Experiments were carried out as described in Materials and Methods using
Taq polymerase to amplify 250 ng of ccds M13mp2TAA DNA (lane B, 15
cycles) or 200 ng of linear ds M13mpl8 DNA (lane C-F, 20 cycles) in the
presence of three normal dNTP and one dNTPaS analog. Analysis was carried out by gel electrophoresis using an ethidium bromide 1.5 % agarose
gel. A and G, length standard <DX174 DNA-Hae III digest (the arrows indicate a length of 310 bp); B, dATP replaced by dATPaS ; C, dATP replaced
by dATPaS ; D, dCTP replaced by dCTPaS; E, dGTP replaced by dGTPaS; F,
dTTP replaced by dTTPaS.
related to the length of the oligomers. The 18mer oligonucleotides used in
our studies gave relatively clean PCR production at 70°C. The size of the
PCR fragment will dictate the length of time for the polymerization, but we
have found no difficulties with a polymerization time of 3 min to prepare
DNA as large as 600 bases.
Sequencing of Phosphorothioate PCR Fragments
In order to obtain a DNA sequence from the PCR fragments we
developed two efficient methods of labelling only one of the strands. First,
in order to reduce the exposure to radioactivity, we tested labelling the
DNA after the PCR (Method A). The amplifications were conducted with
one phosphorylated oligonucleotide primer and a second that was not
phosphorylated. This kept the 5'-end hydroxyl of one strand free for
radioactive labelling after the PCR using polynucleotide kinase and
[y-32p]ATP. To sequence the other strand a second set of PCR amplification
was performed in which the phosphorylation of the oligonucleotide
9953
Nucleic Acids Research
9954
Nucleic Acids Research
primers was interchanged. In the second method a radioactively labelled
oligonucleotide primer was used in the PCR thereby giving labelled DNA
directly (Method B). Both methods of labelling the fragments worked well.
Before performing the sequencing reactions we routinely tested the
purity of the PCR fragments by performing an 8 % polyacrylamide sequencing gel. This showed that although agarose gel analysis indicated that the
PCR fragments were quite pure, several smaller pieces were also present.
For the best results it was necessary to gel-purify the PCR fragments prior
to performing the sequencing reaction. Often during the gel purification
we observed two bands with rather similar mobility in the region of the
PCR fragment, the lower band often being somewhat dispersed. Both
bands have been isolated individually and found to give identical sequencing patterns.
In this study both 2-iodoethanol and 2,3-epoxy-l-propanol were
tested as reagents to degrade the phosphorothioate-containing DNA (8).
The reagent was added to the stop mix and the concentration adjusted
until a proper level of cleavage was obtained. It is important to note that
at the concentration of reagent used here the heating of the DNA at 95°C,
that is normally carried out to denature the strands prior to electrophoresis, acts to accelerate the cleavage reaction. Samples applied to the gel
without this heating step showed marked reductions in the level of cleavage. Both reagents worked well but we found that the higher reactivity
of 2,3-epoxy-l-propanol required that a concentration 10 times lower
than that with 2-iodoethanol be used to achieve an equal level of cleavage.
High concentrations of 2,3-epoxy-l-propanol tended to interfere with the
resolution of the gel electrophoresis. For example, when a stop mix containing 5 % (v/v) of 2,3-epoxy-l-propanol was used the DNA sequence was
still visible but very blurred and difficult to read. Shown in Figure 3 is a
sequencing result using 2,3-epoxy-l-propanol to cleave the phosphorothioate-containing PCR fragments amplified from M13mpl8. The sequen-
Fig. 3 DNA seqencing of isolated PCR fragments amplified from M13mpl8.
Radioactively-labelled phosphorothioate-containing DNA prepared from
15 PCR amplification cycles of 200 ng linear ds M13mpl8 was degraded
using 2,3-epoxy-l-propanol as described in the Experimental section
(Method A). Polyacrylamide gel electrophoresis using an 8 % sequencing
gel was used to analyse the cleavage products.
9955
Nucleic Acids Research
cing pattern, produced starting with ng quantities of M13mpl8, is identical
to the published and accepted known sequence.
We also carried out experiments on a 510 bp restriction fragment
from the human genome containing a part of the beta-myosin heavy chain
whose sequence was not completely known. Portions from both ends of
the sequence were known and allowed us to prepare oligonucleotide primers HPVI and HPV2. Using these primers a PCR fragment of 192 bases
was produced that span the unknown region of the DNA and the sequence
was determined. To confirm the sequence the PCR amplification was
performed twice and both stands were analysed. There were no
ambiguities in the observed sequence.
DISCUSSION
The PCR methodology is ideally suited for many applications in rapidly producing sufficient amounts of DNA for analysis, but most methods
reported to date on sequencing the DNA produced utilize conventional
strategies and therefore require that additional steps be carried out. For
example, in one very useful procedure the PCR fragment is made to allow
direct cloning for further amplification and is sequenced after cloning (5).
Other reported methods use a third oligonucleotide (6) to hybridize to a
sequence within the PCR fragment which is then used as a site for polymerization and subsequent Sanger (14) or Maxam-Gilbert (15) sequencing.
We have investigated a possible improvement on these techniques which
involves the incorporation of dNTPaS into the PCR products and therefore
allows the DNA to be sequenced directly using the phosphorothioate approach (8).
Although many polymerases, including the Klenow fragment of DNA
pol I (1), can be used in PCR amplification the use of Taq polymerase
greatly simplifies the method (16). The Taq polymerase efficiently incorporates the Sp-diastereomers of dNTPaS into the growing DNA polymer.
The configuration of the incorporated phosphorothioate was not studied
but is probably Rp as has been found previously for all other DNA polymerases (17). These results also show that the presence of phosphorothioate
groups in the template strand does not inhibit polymerization.
Our attempts to sequence the phosphorothioate-containing DNA directly after the PCR amplification were only moderately successful due to
the presence of a high background of smaller fragments. This was probably caused by our inability to reliably estimate the number of PCR cyc9956
Nucleic Acids Research
les required to obtain essentially pure DNA. To obtain consistent results
we found it best to gel purify the product before performing the sequencing reactions. During this step we often observed two dominate bands.
Isolation of each band and sequencing showed that both were identical in
base composition. We conclude that this effect is due to incomplete denaturation of the DNA and that the upper band is still the double stranded
PCR product while the lower band is single stranded or a mixture of single
stranded and partially annealed single stranded PCR product.
J-CH^OH
0
•
o4-s-
C
* 0
: S—CH'I
OH
OH
i
2v-
| HS-CH2
S'-s"N
I
S-CH :
OH
•
OH
HO-
O-P-OI
CH,
T
OH
Scheme 1. Mechanism of phosphorothioate alkylation and cleavage.
9957
Nucleic Acids Research
Both 2-iodoethanol and 2,3-epoxy-l-propanol were found to be effective in degrading phosphorothioate-containing DNA. The difference in
reactivity suggests that 2,3-epoxy-l-propanol is the more electrophilic
alkylating reagent. This is in accord with studies carried out with the
dinucleoside monophosphate d[Cp(S)T]. The predominate reaction with
both alkylating reagents is the conversion of d[Cp(S)T] to d[CpT] following
route a of Scheme 1. Under comparable conditions this conversion is more
rapid with 2,3-epoxy-l-propanol. From our data it appears that the ratedetermining step is alkylation of the phosphorothioate sulfur and not the
events that occur later leading to d[CpT] or products resulting from P-0
bond breakage. Paradoxically, the reaction pathways that provide the
cleavage giving the sequence data, routes b and c in Scheme 1, are minor.
From the sequencing data it appears that with DNA one of these two routes
must dominate as the products, bearing either a free 3'-hydroxyl or 3'phosphate, should be observed as two bands in the sequencing gel.
Indeed, some doubling of bands was observed with the smaller sequence
fragments but the difference in relative intensity was sufficient to
distinguish the sequence and thereby resolve any ambiguity. The
tendency to observe doubling was reduced when using 2,3-epoxy-lpropanol and as is observed in Figure 3, the sequence can be read without
ambiguity starting from the first base added to the primer. In these
studies, we observed no incorrect bases in the sequence. This indicates
that the Taq polymerase has sufficient fidelity of replication using dNTPaS
compounds.
It was particularly satisfying to confirm the method on a human
genome fragment whose sequence was only partially known. The
simplicity, speed, and convenience of the method suggests that it can
prove very useful in aiding studies that require accurate sequencing of
small quantities of DNA. For maximal application it might be desirable to
have the PCR fragments fluorescently-labelled. Indeed, our initial
experiments show that the procedures described here can be used to
prepare such DNA through amplification with an oligonucleotide primer
bearing a fluorescent marker at the 5'-end.
ACKNOWLEDGEMENTS
Acknowledgement is made to the National Science Foundation International Programs Division Grant No. INT-8722658, and the National
9958
Nucleic Acids Research
Institute of Health - AREA Program for their support to K. L. N.
technical assistance of A. Fahrenholz is greatly appreciated.
The
"•"Present address: Department of Chemistry, Gonzaga University, Spokane, WA 99203, USA
•To whom requests for reprints should be sent
REFERENCES
1. Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, H.A. and
Arnheim, N. (1985) Science 230, 1350-1354.
2. Marx, J.L. (1988) Science 240, 1408-1409.
3. Murakawa, G.J., Zaia, J.A., Spallone, P.A., Stephens, D.A., Kaplan, B.E.,
Wallace, R.B. and Rossi, J.J. (1988) DNA 7, 287-295.
4. Crescenzi, M., Seto, M., Herzig, G.P., Weiss, P.D., Griffith, R.C. and
Korsmeyer, S.J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 4869-4873.
5. Scharf, S.J., Horn, G.T. and Erlich, H.A. (1986) Science 233, 1076-1078.
6. Wrischnik, L.A., Higuchi, R.G., Stoneking, M., Erlich, H.A., Arnheim, N. and
Wilson, A.C. (1987) Nucleic Acids Res. 15, 529-542.
7. Stoflet, E. S., Koeberl, D. D., Sarkar, G. and Sommer, S.S. (1988) Science
239, 491-194.
8. Gish, G. and Eckstein, F. (1988) Science 240, 1520-1522.
9. Taylor, J.W., Ott, J. and Eckstein, F. (1985) Nucleic Acids Res. 13, 87658785.
10. Taylor, J.W., Schmidt, W., Cosstick, R., Okruszek, A. and Eckstein, F.
(1985) Nucleic Acids Res. 13, 8749-8764.
11. Ott, J. and Eckstein, F. (1987) Biochemistry 26, 8237-8241.
12. Maniatis, T., Fritisch, E.F. and Sambrook, J. (1982) Molecular Cloning: A
Laboratory Manual, pp. 466-467, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y.
13. Rubin, G. M., (1975) Methods in Cell Biology, Vol. Xn Yeast Cells, Ed. by
D. M. Prescott, pp. 45-65, Academic Press, N.Y.
14. Sanger, F., Nicklen, S., Coulson, A.R. (1977) Proc. Natl. Acad. Sci. U.S.A.
74, 5463-5467.
15. Maxam, A. M. and Gilbert, W., ibid., 560-564.
16. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, SJ., Higuchi, R., Horn, G.T.,
Mullis, K.B. and Erlick, H.A. (1988) Science 239, 487-491.
17. Eckstein, F. (1985) Ann. Rev. Biochem. 54, 367-402.
9959