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`ILMN EXHIBIT 1024
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`Volume 17 Number 18 1939
`
`Nucleic Acids Research
`
`A new and versatile reagent for incorporating multiple primary aliphatic amines into synthetic
`oltgonucleotides
`
`
`Paul S.Nelson. Rivka Shcmtan—Gold‘ and Roy Leon
`
`
`Organic Chemistry Division. Clontech Laboratories. lnc.. 3040 Fabian Way. Palo Alto, CA 94303 and
`'Biogrowth Inc.. 3065 Atlas Road. Richmond. CA 94306. USA
`
`
`Received August I. 1989: Accepted August
`
`I I. 1989
`
`ABSTRACT
`A novel and versatile phosphoramidite, N-Fmoc-0'-DMT-03-cyanoethoxydiisopropylamino-
`phosphinyl—3—amino—l .2—propanediol (1. Fig. 1). has been synthesized and used to incorporate primary
`aliphatic amines into synthetic oligonucleotides. lts convenient preparation and use in solid phase
`oligonucleotide synthesis is described. Using phosphoramidite I, an amincrmodified oligonucleotide
`probe complementary to Ml3mp1S DNA was constructed with five primary amines attached to the
`5’—tern1inus. The amino—modified oligonucleotide was subsequently labeled with biotin and employed
`in a dot—b|ot hybridization assay. As little as 0.5 ng of M I3mp|8 target DNA was colorimctrically
`‘detected.
`
`INTRODUCTION
`
`in non—radioactively labeled oligonuclcotides has prompted the
`Increased interest
`development of chemical methods to modify oligonucleotides. Recent methods which utilize
`phosphoramidite chemistry to introduce modifications into oligonucleotides during solid
`phase synthesis have proven to be the most convenient‘ "7. Various phosphoramidites
`containing masked primary aliphatic amines have been employed to introduce amino groups
`into oligonucleotides'"1“‘. The primary aliphatic amine group is the chemical modification
`most widely used since it reacts with many commercially available labeling reagents.
`Moreover, since the chemistry for conjugating amine groups is well established, virtually
`any reporter molecule can be covalently attached to an oligonucleotide through this linkage.
`Smith at at‘ and Sproat er al3 designed 5‘-amino-5'-deoxyribonucleoside
`phosphoramidites which incorporate primary aliphatic amines at the 5’cnds of synthetic
`oligonucleotides. Amino-modified oligonucieotides prepared from these reagents were
`successfully labeled with fluorophores and biotin. The use of 5’-amino~5’~deoxyribo-
`nucleoside phosphoramidites, however, is limited because their preparation requires many
`synthetic steps, they allow the incorporation of only one primary amine which can be added
`only at the 5’-terminus. and the resulting oligonucleotide has an additional base which
`might change its annealing properties.
`Agrawal er (:23 was the first to report a conveniently prepared phosphoramidite amino-
`modifying reagent constructed from an aminoethanol backbone
`rather
`than a
`deoxyribonucleoside. This greatly simplified the chemistry for incorporating primary
`aliphatic amines into synthetic oligonucleotides. Subsequently, Connolly‘ and Sinha and
`Cook5 prepared various phosphoramidite analogs from NI-I2-(CH3),,-OH backbones for the
`same purpose. A similar cyciic phosphoramidite reagent has also been reported by Connell
`at at“. Although the synthetic preparation of these compounds is convenient, they are
`
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`HEN
`
`OH
`
`-T)
`
`FrnocNH
`
`OH
`2
`
`/\r\o~
`
`OH
`3
`
`FrnocNH
`
`com
`
`T
`
`FrnocNH /\]/\ cow
`
`OH
`4
`
`%
`
`P—"’OCH2CH2CN
`
`°\
`N/
`f
`
`1
`
`Figure 1. Synthetic scheme for N-Fmot:-0'-DMT-O2-cyantrctliortydiisupropylaminophosphinyl-3-amintr1.2-
`propanediol I 1}.
`
`limited in utility because only a single prirrtary aliphatic amine‘can be incorporated and
`at only the 5’—terrninus.
`Nucleoside phosphorarnidite reagents containing a masked primary amine group on
`the base heterocycle“ '3 offer the advantage of allowing both the 3'-phosphoramidite and
`5’-hydroxyl to participate in solid phase oligonucleotide synthesis. Therefore, an amino-
`modified phosphoramidite can be incorporated internally at any position in the synthetic
`oligonucleotide. Another advantage is that multiple amines can be added through repetitive
`coupling cycles. Ruth and co-workers7“° have reported phospltoramidites prepared from
`C-5 substituted deoxyuridine compounds that can incorporate in this manner. Other
`researchers have reported similar compounds"“3‘ However, the preparation of such
`reagents requires many synthetic steps and undesired modified nucleotide bases are added
`which might change the hybridization properties of the oligonucleotide.
`We have designed a new and improved phosphoramidite reagent. N-Fmoc-O‘-DMT-
`O5’--cyanoethoxydiisopropylaminophosphinyl—3~amino-1,2-propanediol (1, Fig. 1),
`that
`incorporates primary aliphatic amines during solid phase oligonucleotide synthesis. Two
`important criteria have been addressed in our design of this reagent. First, the reagent
`is versatile, i.e., it can be incorporated at any position of the oligonucleotidc during the
`course of solid phase synthesis. Thus, the 5'-terminus as well as any internal position can
`be modified. Multiple modifications can also be achieved by repetitive coupling cycles.
`Secondly, the preparation of reagent 1 is convenient; the entire synthetic strategy involves
`only three steps and all reagents are readily available. Herein is described the synthesis,
`use. and application of phosphoramidite l.
`
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`MATERIALS AND METHODS
`
`(:|:)3-Amino—1.2—propanedioI, 4,4’-dimethoxytrityl chloride. and chloro-N.N~diisopropyl-
`beta-cyanoethoxyphosphine were purchased from Aldrich Chemical Company.
`9-Fluorenylmethyl chloroformate was obtained from Chemical Dynamics Corporation. Both
`dichloromethane and N .N-diisopropylethylamine were dried by distilling from calcium
`hydride. Analtech Silica Gel GF (0.25 mm} plates were used for thin layer chromatography
`. and developed with concentrated sulfuric acid or visualized by UV light. M l3mpl8 single
`stranded DNA was purchased from New England Biolabs. HPLC analysis was done on
`a Rainin Rabbit HPX system using a C18 Microsorb (4.6 mm><25 cm. 5 am) reverse
`phase column. Microanalyses were determined by Chemical Analytical Services at
`University of California, Berkeley. A Perkin-Elmer 1310 Infrared Spectrometer was used
`for infrared measurements and ‘H and “P NMR spectra were run on a Bruker AM-400
`instrument using tetramethylsilane as an internal standard ('H) or trimethyl phosphate as
`_-an external standard {-"P). Oligonucleotide synthesis was performed on a Biosearch
`Cyclone DNA synthesizer according to the manufacturer‘s protocol.
`( :1: ) N-Fmoc-3-amino-I .2-propanedio! G)
`9-Fluorenylmethyl chloroforrnate (10.0 g, 33 mmol) was added to a stirring solution of
`I~,(:|:) 3-amino-1,2-propanediol (2, 2.9 g. 32 mmol) and N.N-diisopropylethylarnine (6.7
`ml. 38 mmol) in dry dimethylformamide (85 ml). The mixture was stirred 30 min. at
`_room temperature and then poured into cold saturated sodium bicarbonate solution (500
`ml) to precipitate the product. The white solid was collected by filtration, washed several
`times with water, and dried in a vacuum oven at 50°C. Recrystallization was accomplished
`"by dissolving the solid in a minimum amount of hot ethyl acetate and adding hexane until
`the mixture became cloudy. Two crops yielded 7.7 g (77%) of white crystals. TLC showed
`one spot at R; 0.3 (methattol—dichloromethane, 1:19). mp.
`131 — 133°C;
`‘H NMR
`(CDC13): 3.33 (m, 211), 3.58 (m, 2H), 3.77 (m, 1H), 4.21 (t, 1H), 4.46 (d, 21-1). 7.32
`(1, 2H), 7.41 (t. 2H), 7.59 (d, 2H), 7.77 (cl, 2H) ppm; IR (nujol) 3320, 1685. 1530, 1270,
`1160 cm‘ ‘.
`
`Anal. Calc. for C,8H,9O4N: C, 68.98; H, 6.11; N, 4.47.
`Found: C, 68.89; H, 6.16; N, 4.39.
`(:t;) N-Fmoc-0’-DMT-3-amino-I ,2—propanedt'o.’ (4)
`(:2) N-Fmoc-3-amino-1,2-propanediol (3, 7.7 g, 24.5 mmol) was dissolved in anhydrous
`; pyridine (90 ml) and 4,4‘-dimethoxytrityl chloride (10.0 g, 29.5 mmol) was added. The
`mixture was stirred for 4.5 hours at ambient temperature. Methanol (10 ml) was added
`to the reaction mixture which was stirred for another 10 min. The solvent was removed
`
`in vacuo and the residual pyridine was removed by coevaporation with toluene. The product
`was purified on a short column (5><20 cm) of silica gel, eluting successively with
`dichloromethane (700 ml), methanol-dichloromethane (l:200, 1500 ml) and methanol-
`dichloromethane (1:50, 1500 ml). Fractions showing a single spot on TLC at Rf 0.3
`(methanol-dichloromethane, 1:50) were pooled and the solvent was removed by rotary
`evaporation to give 9.33 g (62% yield) of a yellow amorphous solid. Decomp. 74°C;
`‘H NMR (CDC13): 3.18 (m.2H), 3.40 (m. 2H), 3.77 (5, 6H. —OMe), 3.88 (m. 1H),
`4.19 (t, 1H), 4.37 (d, 2H), 6.83 (m, 4H), 7.25 (m, 11H), 7.40 (t, 2H), 7.56 (d, 2H),
`1 7.76 (d. 2H) 131:-m; IR [nujol) 3420. 1710. 1610. 1510, 1160 cm'''.
`i
`Calc for C39H3jO{,N: C,
`H,
`N.
`1 Found: C, 75.68; H. 6.08; N, 2.35.
`
`
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`mt--1
`"'1:r_
`q.
`
`aéo
`
`260
`
`see
`
`also
`
`use
`
`1;‘.-0
`
`160
`PPM
`
`30
`
`6'0
`
`40
`
`
`
`Figure 2. “P NMR spectrum of N-Fmoc-0'-DMT-O2-cyanoethoxydiisopropylaminophosphinyl-3-amino-I.2-
`propandiol (1) using trimethyl phosphate as external control.
`
`N-Fmoc-0’-DMT-03-cjvanoerhoxydiisopropylttminophosphinyl-3-amino-I .2-propanediolfl)
`(:t:) N-Fmoc-0‘-DMT-3-amino-1.2-propanediol (4, 4.0 g, 6.7 mmol, dried overnight at
`35°C under high vacuum) and N,N-diisopropylethylamine (5.3 ml, 30.4 mmol) were
`dissolved in dry dichloromethane (45 ml) under argon. Chloro-N,N—diisopropyl beta-
`cyanoethoxyphosphine (3.2 g, 13.4 mmol) was slowly added over a period of 5 min. and
`the solution was stirred for 30 min at room temperature. Methanol (1 ml) was added and
`the solution was stirred an additional 10 min. The reaction mixture was partitioned --
`-
`-
`ethyl acetate (170 ml) and 10% cold aqueous sodium bicarbonate (275 ml). The organic;
`phase was washed with 10% aq. sodium bicarbonate (275 ml) and dried over anhy -
`sodium sulfate. After removing the solvent in vacuo, the gummy residue was purified em.
`a silica gel column (5><20 cm). The desired product eluted isocratically u-=
`dichloromethane—hexane—triethylamine (252752). The fractions displaying overlaying s o --~.:
`(diastereomers) on TLC at R; 0.2—0.25 (same solvent system) were pooled and I:
`solvent was removed in vacuo. The residue was dissolved in ethyl acetate (15 ml) =
`‘I
`precipitated in cold petroleum ether (500 ml). Collection by filtration yielded 3.5 g -
`?
`a fluffy pale yellow solid. HPLC analysis: retention times of the diastereomers were 7.
`
`
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`Nucleic Acids Research
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`and 8.0 min (90% acetonitrile-10% 0.1 M triethylammonium acetate. pH 7. 1.5 ml/min).
`‘-"P NMR (CDCI3): 147.0 and 147.3 ppm (Fig. 2).
`Anal. Calc. for C4gH54N307P: C, 70.66; H, 6.67; N. 5.15.
`Found: C, 70.39; H, 6.89; N, 5.29.
`
`Oligottncieoride Synthesis
`[X=
`The arnintrmodified oligonucleotide 5’—XXXXXaCACAATTCCACACAAC—3'
`primary amine modifications
`introduced by couplings with N-Fmoc-O'-DMT-
`Olcyanoethoxydiisopropylaminophosphinyl—3—arnino-1,2—propanediol (l)] was synthesized
`on a 1.0 micromolc scale. N-Fmoc-0'-DMT-O3-cyanocthoxydiisopropylamino—
`phosphinyl—3-amino-l .2-propanediol (1) was consecutively coupled in the last 5 cycles using
`a concentration of 0.3 M. The coupling efficiency of I was determined by measuring the
`deprotectecl dimethoxytrityl cation concentration. The average coupling efficiency of 1 was
`95%. Deprotection was accomplished with concentrated ammonium hydroxide according
`to manufacturer’s recommendations. The oligomer CACAATTCCACACAAC was
`synthesized to be used as a control.
`Biorinyiarion of Amino—modi}‘ied 0h'g(mude0tide
`' The total crude amino—modified oligonucleotide product was dissolved in 0.8 ml of 0.1
`M NaHC'O3/Na2CO3 (pH 9). Biotir1—X—X—NI-IS ester. manufactured and supplied by
`Clontech Laboratories, Inc., in dimethylformamide (0.25 ml, 100 mg/ml) was added, the
`mixture briefly vortexed, and allowed to incubate at ambient temperature for 16 hrs. After
`purification on a Sephadex G-25 column. the extent of biotinylation was quantitated by
`employing a photometric ninhydrin analysis”, measuring UV absorption (570 nm) before
`and after biotinylation. The biotinylation reaction derivatized 65 % of the primary aliphatic
`amines. The biotinylated oligonucleotide was purified by electrophoresis on a 20%
`denaturing polyacrylamide gel using published procedures”. The appropriate band was
`excised from the gel, crushed, eluted with 0.1 M ammonium bicarbonate. and desalted
`on a Sephadex G—25 column. Both the purified biotinylated probe and the control
`(CACAATTCCACACAAC) were labeled with 331’ at
`the 3‘-ends using tertninal
`deoxynucleotidyi transferase and [alpha—33P] cordycepin triphosphatem and were analyzed
`by gel electrophoresis (Fig. 3).
`Hybria'i'zan'on Procedures
`The hybridization procedure was a modification of the procedure of Jablonski er al'’.
`Varying amounts of Ml3mpl8 single stranded DNA and lambda phage DNA (control),
`50 ng, 10 ng,
`1 ng, 0.5 ng, and 0.1 ng, were diluted in 0.1 ml TE buffer (10 mM Tris-Cl.
`1 mM EDTA. pH 8) and denatured with 0.] volume of 3 N sodium hydroxide for 15
`min at room temperature. The samples were neutralized with 1 volume of 2 M ammonium
`acetate, and immobilized on nitrocellulose filters (prewetted with distilled water and 2 M
`ammonium acetate) using a dot-blot apparatus (BRL). The nitrocellulose filters were baked
`in a vacuum oven at 30°C for 2 hrs.
`
`44~444-——————~4~———____————————______————4—~'r——_———~r4—————————~
`
`The blots were prehybridized for 1 hr at 35°C in 10 ml 5:-<SSC, 0.5% BSA, 0.5%
`polyvinylpyrrolidone, 1% SDS. containing 50 pg/ml sheared and denatured salmon testes
`DNA and 50 pg/ml IRNA. The biotinylated oligonucleotide probe or the control probe
`(5’--"ZP labeled by polynucleotide kinase“) were added to the hybridization solution to give
`a final concentration of 25 ng/ml. and incubated overnight at 35°C. The nitrocellulose
`filters were washed once in 2 XSSC, 0.5% SDS at room temperature and twice at 37°C
`(15 min each wash). Lastly, the filters were washed twice with 2 XSSC at 37°C (15 min
`each wash).
`
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`Figure 3. Autoradiogr-am oi" “P-labeled oiigonucleoticles resolved by a 20% denaturing polyacrylamide gel. Lane
`I: 3'—"2P labeled control probe CACAATTCCACACAAC: Lane 2: 3‘-‘DP labeled hiotinylated probe BBBBB-
`CACAATTCCACACAAC. (B = covalently attached hiolins); Lane 3: 35-”? labeled 20mcr marker.
`
`Colorimetric detection of the hybridized biotinylated probe was carried out using the
`BlueGene (BRL) nonradioactive detection system according to the manufacturer’s suggested
`procedure. Autoradiography of the filters hybridized with the HP-labeled probe was done
`overnight at —30°C with an intensifying screen.
`
`RESULTS AND DISCUSSION
`The synthetic scheme for preparing N-Fmoc-O’-DMT-O3-cyanoethoxydiisopropylarnino-
`phosphinyl—3—arnino-l.2-propanediol (1) is shown in Figure I. (:|;)3-Amino—l,2—propanecliol
`(2) was reacted with 9—f1uorenyltnethyl chloroformate in dry dimethylformamide at room
`temperature to give N-Fmoc-3-amino-1.2-propanediol (3) in 77% recrystallized yield. The
`primary alcohol of 3 was selectively protected by treatment with 4,4’-dimethoxytrityl
`chloride in anhydrous pyridine to give N-Fmoc-O‘-DMT~3-amino—l.2-propanediol (4).
`Compound 4 was phosphitylated with chloro-N,N-diisopropyl—beta-cyanoethoxyphosphine
`to yield the desired phosphoramidite 1 as a diastereomeric mixture. The diastereomers
`of 1 were isolated by column chromatography on silica gel and subsequently characterized
`by 3‘P NMR (Fig. 2). Two peaks were observed at 147.0 and 147.3 ppm using trimethyl
`phosphate as an extemal standard. Analytical HPLC on a RP-C18 column also exhibited
`two peaks at retention times of 7.3 and 8.0 minutes (isocratic: 90% acetonitrile, 10% 0.!
`M triethylammonium acetate, pH 7; How = [.5 rnl/min}.
`We employed phosphoramidite 1
`in automated solid phase DNA synthesis using
`conventional cyanoethyl phosphoramjdite chemistry. Both internal and external couplings
`of l were readily achieved and could be quantitated by measuring the deblocked
`dimethoxytrityl cation concentration. An oligonucieotidc sequence complementary to
`Ml3mpl8 DNA (5'—XXXXX—CACAATTCCACACAAC-3', X=primary amine
`modifications introduced by couplings with 1) was synthesized having the 5'-terminus
`modified with five consecutive couplings of l. The average coupling efficiency of
`phosphoramidite 1 was determined to be 95%. The Fmoc protecting groups were
`
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`Nucleic Acids Research
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`
`
`l I
`
`0.1
`
`A
`
`B
`
`Figmt 4. Dot-blot hybridization of labeled oligonucleotide probes to nitrocellulose bound DNA. The immobilized
`DNAs were Ml3rrrpl8 ss DNA (lane a. target) and lambda phage DNA (lane b. negative control). Blot A employed
`the biotinylated probe with streptavidin-alkaline phosphatase colorirnctricdetection. Blot B employed the 5'-‘ 2P
`labeled control probe and is an autoradiogram of an overnight exposure.
`
`‘
`
`conveniently removed with ammonium hydroxide in the final deprotection step, and the
`-"amino-modified oligonucleotide was ready for labeling.
`The amino-modified oligonucleotide containing a tail of five primary aliphatic amines
`‘ gilt its 5'-terminus was biotinylated for hybridization testing. Biotins were attached through
`;a long linking arm using biotin-X-X-NI-IS ester, a reagent which contains a I4-atom spacer
`consisting of two e—aminocaproic acid moieties linked in series. The long linking arm was
`used to extend the biotins out from the oligonucleotide, making them more available for
`detection. Purification of the multi—biotinylated probe was accomplished by electrophoresis
`ll on a 20% denaturing polyacrylamide gel. Since the 5’-tenninus of the purified biotinylated
`was modified, it was necessary to label on the 3'-end using [alpha—-“PI cordycepin
`ztriphosphate and terminal trarisferasem for analysis by gel electrophoresis. Figure 3 shows
`. -a
`sizable mobility difference between the biotinylated probe and the control
`‘ (CACAATTCCACACAAC).
`The biotinylated probe hybridized specifically to complementary M13mpl8 single
`. stranded DNA (target) immobilized on nitrocellulose filters. Corresponding amounts of
`| “Jatnbda phage DNA were used in parallel as negative controls. Hybridization was effectively
`jjierformed overnight at 37°C in the presence of 25 ngfml probe. Colorimetric detection,
`employing an alkaline phosphatase-streptavidin conjugate, demonstrated sensitivity at 0.5
`Eng (Fig. 4A). No significant nonspecific background was observed with the lambda phage
`§'DNA negative control. The control probe (CACAATTCCACACAAC) was 33P—labeled
`at its 5'—terrninus3' and employed in an identical hybridization experiment. A similar
`"
`itivity of 0.5 ng was obtained following overnight autoradiography (Fig. 4B).
`
`
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`Nucleic Acids Research
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`CONCLUSIONS
`
`N—Fm0c—O 1 —DMT—03«cyanoethoxydi isopropylaminophosphinyl-3-amino-1 , 2-propanediol
`(1), is a new and versatile phosphoramidite which introduces a primary aliphatic amine
`during solid phase oligonucleotide synthesis. The reagent can be incorporated at any position
`during solid phase oligonucleotide synthesis and multiple amine groups can be added by-
`repetitive coupling cycles. We demonstrated its use by synthesizing an oligonucleotide
`probe with a tail of five primary amine groups at its 5'—termjnus. The primary amine groups
`were labeled with biotin and the probe successfully hybridized to target DNA immobilized
`on nitrocellulose. Colorimetric detection showed specific hybridization with a sensitivity
`of 0.5 ng. This reagent is especially useful for applications in non-radioactive hybridization
`probe diagnostics. Other potential applications include automated DNA sequencing”-'5-”,
`electron microscopyz, X—ray Crystallography”, and site specific cleavage of DNA”.
`
`ACKNOWLEDGEMENTS
`
`We are thankful to Drs. Ken S. Fong, Paul Siebert, and Carol Talkington-Vcrscr for their
`helpful suggestions.
`
`*.’3‘!":'**!-"’!"'
`
`REFERENCES
`1. Smith. L.M.. Fung. S.. Hunkapillcr, M.W.. Hunkapillcr. T.J.. and Hood. L.E. (1985) Nucl. Acids Res.
`13, 2399-2412.
`Sproat. B.S.. Beijer. B.. and Rider. P. (1987) Nucl. Acids Res. 15. 6131-6196.
`Agrawal, S.. Christodoulou. C., and Gait. M.J. (I986) Nuci. Acids Res. 14. 6227-6245.
`Connolly. BA. (1987) Nucl. Acids Res. I5. 3131-3139.
`Sinha. N.D. and Cook. R.M. (I983) Nuci. Acids Res. 16. 2659-2669.
`Connell, C., Fung, S.. Heiner. C., Bridgham. J.. Chakerian. V1. Heron. E,. Jones, B,, Menchen, 8,, Mnrdan.
`W.. Raff. M.. Recknor. M. Smith. L.. Springer. J.. Won. S.. and Hunl<api|ler.M. (1987) BinTechniqt:es
`5. 342-345.
`7. Ruth. J.L. (1984) DNA 3. 123.
`8. Ruth. J.L., Morgan. (2.. and Pasko. A. (I985) DNA 4. 93.
`9. Jablonski. 13.. Mo-ornaw. E.W.. Tullis. R.H., and Ruth.
`.l.L. (I986) Nucl. Acids Res. 14. 6115-6128.
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