Chemical Synthesis of a Primer and Its Use in the Sequence Analysis of the Lysozyme Gene of Bacteriophage T4* (DNA Sequencing/Repair Synthesis/Hybridization) R

Proc. Nat. Acad. Sci. USA Vol. 71, No. 6, pp. 2510-2514, June 1974

Chemical Synthesis of a Primer and Its Use in the Sequence Analysis of the Lysozyme Gene of Bacteriophage T4* (DNA sequencing/repair synthesis/hybridization) R. PADMANABHANt, ERNEST JAY, AND RAY WU Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14850 Communicated by Leon A. Heppel, February 8, 1974

ABSTRACT We have developed a general approach for the mRNA sequence shown in Fig. 1, have been reported determining the nucleotide sequence of a gene, with the (12, 13). aid of a deoxyribonucleotide primer of defined sequence. The selection of the primer sequence was based on a short MATERIALS AND METHODS segment of mRNA sequence of T4 phage lysozyme. A tetradecadeoxyribonucleotide primer was chemically syn- T4 DNA. T4 DNA was prepared according to the pre- thesized and its sequence verified by sequence analysis. viously procedure (14, 15). For our purpose, T4 This primer was found to bind to the single-stranded published region of the exonuclease Ill-treated T4 DNA, and specific DNA obtained by phenol extraction of the T4 phage was nucleotides were incorporated to its 3' end. The result found to be sufficiently pure for sequence analysis. Therefore, indicated that this primer was bound to the expected the sucrose density gradient purification step (15) was location on the T4 DNA. Therefore, long sequences of the omitted. T4 lysozyme gene can now be determined from this specific was a gift of starting point. The Enzymes. The spleen phosphodiesterase Dr. G. Bernardi. Escherichia coli exonuclease III and DNA One of the new approaches proposed for the sequence deter- polymerase I were purified according to Jovin et al. (16). mination of specific internal regions of DNA is the use of Deoxynucleotidyl terminal transferase was a gift of Drs. oligonucleotides of defined sequences as primers (1, 2) for the Roychoudhury and Kossel. DNA polymerase I-catalyzed repair synthesis to provide Methods. Chemical synthesis of the tetradecadeoxynucleo- labeled oligonucleotides which can be sequenced by established tide (14-mer) was according to the general methods developed methods (3). The feasibility of this approach has been ex- by Khorana et al. (17-19). A summary of conditions for the perimentally verified by using an octanucleotide, which is synthesis of the tetradecanucleotide and the yield of the oligo- complementary to the left-hand cohesive end of 186 DNA, nucleotides is given in Table 1. as primer for the repair synthesis. A sequence of eight nucleo- Exonuclease III Treatment of T4 DNA. The procedure is tides added to the C' end of the primer has been determined as described previously (7), except that the extent of digestion (4). Subsequently, several sequences from internal regions of was 26%. DNA have been determined with the aid of primers (5-7). Hybridization of the Primer to DNA. An incubation mixture In this communication, this method is used to determine the containing 0.4-0.6 pmoles of exonuclease III-treated T4 DNA sequence of a specific segment of T4 DNA coding for the ly- in potassium phosphate buffer (70 mM, pH 6.9) and 5 pmoles sozyme. The reason for choosing this system is that the com- of the primer was heated to 750 for 15 min and then cooled plete amino-acid sequence of this enzyme is known (8); thus, slowly to 45°. The reaction mixture was kept at 450 for 12 DNA sequence information will be valuable for understand- hr and then chilled to 5°. The solution was supplemented ing the degeneracy in the genetic code. Moreover, Streisinger with 10 mM Mg++, 15 mM dithiothreitol, 60 mM NaCl, and et al. (9) were able to derive an unique 17-nucleotide-long 2-3,M labeled deoxynucleoside triphosphates. The repair sequence of the mRNA coding for the T4 lysozyme by analy- synthesis was started by the addition of 10 units of DNA sis of double-frameshift mutants and with the use of the polymerase I in a final volume of 0.44 ml. The unutilized genetic code (10, 11). We report here the chemical synthesis primer and labeled deoxynucleoside triphosphates were sep- of a tetradecadeoxynucleotide, d(A-G-T-C-C-A-T-C-A-C- arated from the DNA- oligonucleotide duplex by using a T-T-A-A), which corresponds to part of the mRNA sequence column (0.7 cm X 46 cm) of agarose (1.5 M, saturated with as shown in Fig. 1, except that dpT was substituted for pU. The exact sequence of this tetradecadeoxynucleotide was verified by direct sequence analysis and it was used as a specific 36 40 primer in the sequence determination of the lysozyme gene Amino acid - Ser - Pro - Ser - Leu - Asn - Ala - beyond the 3' end of the primer. mRNA The chemical syntheses of several shorter segments of -A-G-U-C-C1A-U-CA-C-U-U-A-A-U-G-N- sequence 5 7 9 12 14 16 oligodeoxynucleotides, which are complementary to part of defined (9) 1 3 DNA primer Abbreviation: TEAB, triethylammonium bicarbonate. synthesized d(A-G-T-C-C-A-T-C-A-C-T-T-A-A) * This is paper XV in a series on "Nucleotide Sequence Analysis of DNA." Paper XIV is by R. Hamilton and R. Wu, J. Biol. FIG. 1. Sequence of a specific segment of mRNA coding for Chem., in press. the T4 lysozyme. The mRNA sequence was derived from analysis t Present address: Institute for Molecular Virology, St. Louis of the amino acid sequences of wild-type and double-frameshift University, School of Medicine, St. Louis, Mo. 63110. lysozyme mutants (9). 2510 Downloaded by guest on September 24, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Sequence Analysis of Lysozyme Gene of T4 2511 TABLE 1. Summary of conditions and yields of condensation reactions* (A) 5'-phosphate- (B) Pyri- amount containing amount TPS dine Time Yieldt Size of 3'-OH-containing component (,umoles) component (umoles) (Jmoles) (ml) (hr) % product: d(mmt-bzA-OH) 2000 d(pmbG-OAc) 3140 6, 000 25 5 68 dimer d(mmt-bzA-mbG-OH) 1360 d(pT-OAc) 4000 10,000 20 5 67 trimer d(mmt-bzA-mbG-T-OH) 930 d(panC-anC-OAc) 2400 5,400 15 7 43 pentamer d(mmt-bzA-mbG-T-anC-anC-OH) 400 d(pbzA-T-OAc) 1500 4,500 10 7 10§ heptamer d(mmt-bzA-mbG-T-anC-anC-bzA-T-OH) 41 d(panC-bzA-OAc) 600 1,440 5 6.5 32 nonamer d(mmt-bzA-mbG-T-anC-anC-bzA-T- 8 d(panC-T-T-bza- 100 600 2 6 31 14-mer anC-bzA-OH) bzA-OAc)

* Abbreviations and symbols used in this table: anC, N-anisoyldeoxycytidine; bzA, AN-benzoyldeoxyadenosine; mbG, N-a-methyl- butyryldeoxyguanosine; mmt, monomethoxytrityl-; TPS, tri-isopropylbenzene sulfonyl chloride. t The percent yield of all products is given as yield determined spectroscopically after complete purification by partition extraction or DEAE-cellulose column purification followed by precipitation. T At the end of each step of the condensation reaction, a small amount of the protected compound was treated with NH40H followed by acetic acid to remove the protecting groups. The unprotected compounds were characterized after venom or spleen phosphodiesterase digestion and fractionation on paper chromatography (23). In every case, the expected ratios of each nucleotide and nucleoside were found. § The isolated yield was poor because a large amount of this protected heptamer was lost on the DEAE-cellulose column during the column purification step. This was due to the fact that the DEAE-cellulose used for this column had never been used before and probably a large amount of the heptamer was more or less irreversibly bound to the DEAE-cellulose. It was found that the loss of the precious long oligonucleotides can be minimized if protected mononucleotides or dinucleotides are first passed through the DEAEcellulose column to saturate these irreversible binding sites. DEAE-cellulose which had been repeatedly used for the purification of protected oligonucleotides was found to give good recovery of protected long oligomers. calf thymus DNA, 0.1 mg/ml, and washed free of the DNA). was carried out at 370 for 15 hr. The labeled mononucleotides NH4HCO3 (0.05 M) containing 0.1 mM ethylenediamine- and nucleosides were then separated and analyzed as de- tetraacetate (EDTA) was used as the elution buffer (1, 4). scribed earlier (23). Fractions of 1.5 ml were collected and counted for 32p by RESULTS Cerenkov radiation in the liquid scintillation counter. It has been shown that the genes in T4 phage are circularly Dissociation of DNA Oligonucleotide Duplex. After repeated permuted (24) and that the lysozyme gene is transcribed from evaporation to remove NH4HCO3, the DNA- oligonucleotide the L-strand of T4 DNA (25). This means that, statis- duplex was heated in 50 yl of water in a boiling water bath tically, the lysozyme gene is distributed along the entire for 10 min. It was then fractionated as a line (1 cm) on one- k-strand length, in different DNA molecules. If 26% of the dimensional homochromatography on DEAE-cellulose using nucleotides are removed from the 3' end of the H-strand, partially hydrolyzed yeast RNA (Homo III). Alternatively, it means that 26% of the total ly)sozyme gene has been dissociation was achieved on a Sephadex G-100 column (0.5 rendered single-stranded, on a statistical basis. Thus, the cm X 63 cm) at 650 using 0.5 mM Tris HCl buffer (pH 8.0 maximum extent of hybridization of the synthetic tetra- at 650) as elution buffer. decanucleotide primer to the single-stranded template regions 3'-End-Group Analysis of Oligonucleotides. In the case when on the Ikstrand will be only 26%. one-dimensional homochromatography was used for the 5'-End-Group Labeling of the Tetradecanucleotide for Its dissociation of the DNA oligonucleotide hybrid, the samples Sequence Analysis from Its 3' End and for Its Use as a Primer. were scraped out of the DEAE-cellulose thin-layer plate using The oligonucleotide was labeled at the 5' end with 32p by the Eppendorf plastic tips (W. Sarstedt, Inc.) with a cotton polynucleotide kinase (26) in an incubation volume of 10 plug at the narrow end. A glass scraper (22) was attached at gl containing 200 pmoles of 14-mer, 66 mMI Tris- HCl (pH the wider end and the sample was collected in the tip by using 7.8), 6.6 mM MgCl2, 15 mM dithiothreitol, 0.066 mM [,y_32P]_ a mild vacuum. Urea was removed from the sample by wash- ATP, and 5 units of the enzyme. The incubation was carried ing with approximately 2 ml each of 95% and 50% ethanol out for 2 hr at 37°. The labeled 14-mm was purified on a and then with water. The sample was eluted with 50-100 Sephadex G-50 column to remove the excess [32P]ATP. Mul of 2 M triethylammonium bicarbonate (TEAB). After For sequence analysis, the labeled primer was digested removal of the TEAB by evaporation, the dry sample was partially with venom phosphodiesterase and fractionated by taken up in 20 ,ul of 0.05 M NH4 HCO3 (pH 8.0), and the RNA two-dimensional homochromatography (20-22). As shown in carrrier from the Homo-mix in the sample was digested with Fig. 2a, 14 spots were well resolved. From the characteristic 4 gg of pancreatic RNAse (Worthington) and 5 units of mobility shifts of the oligonucleotides in our improved two- RNAse T, for 8 hr at 37°. The sample was further digested dimensional homochromatography system (7, 21, 32) and for 1 hr at 370 with 0.04 lug of micrococcal nuclease in the the knowledge that Spot 1 is [32P]dpA by cochromatography presence of CaC12 (0.8 mM) and a mixture of four 3'-deoxy- and coelectrophoresis with dpA, the complete sequence of mononucleotides and nucleosides (0.1 MAmol each). Finally, the tetradecamer was deduced to be [5'-32P]d(pA-G-T-C-C- spleen phosphodiesterase digestion was carried out in a vol- A-T-C-A-C-T-T-A-A). ume of 0.05 ml which contained potassium phosphate buffer For its use as a primer, the 32P-labeled tetradecanucleotide (25 mMI, pH 6.0), ethylenediaminetetraacetate (1.4 mMI), was purified by one-dimensional chromatography on poly- Tween 80 (0.04%), and the enzyme (0.25 lg). The incubation ethyleneimine-cellulose thin-layer chromatography plate Downloaded by guest on September 24, 2021 2512 Biochemistry: Padmanabhan et al. Proc. Nat. Acad. Sci. USA 71 (1974)

(cellulose impregnated with polyethyleneimine) using 1 M Sequence Analysis from Its 5' End and for Its Use as a Primer. LiCI (27) and 7 M urea. This removed any nonphosphorylated The 14-mer was labeled at the 3' end with [32P]UTP and starting material which would otherwise compete with the deoxynucleotidyl terminal transferase (28) in an incubation labeled 14-mer in the hybridization experiments with the T4 volume of 5,ul containing [32P]UTP (150 ,uM), Co++ ions DNA template. (1.6 mM), dithiothreitol (16 mM), K-cacodylate (240 mM, 3'-End-Group Labeling of the Tetradecanucleotide for Its pH 7.6), and 30 units of the enzyme. The incubation was I a 14 0.9 A q13 1.3 A * 12 1.0 tT * 11 1.1 @ 10 1.3 C @ 9 2.5 IA @ 8 C/ 1.8 .7 1.8 0KT 0 6

3.1 y * 5 2.2 C>s/C 1.8 O 3 1.9

3.0 i 1 a [cml 0 pA B

I I b 14 1.3 A 13 1.8 O *12 1.1 * 11 1.5 0 10 * 9 3.1 IA

1.7 0N 1.8 aR 0 @ 6 - 0 y 5

* 4

3.1 A I II 8Icml Q Ap

B FIG. 2. (Legend appears at bottom of next page.) Downloaded by guest on September 24, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Sequence Analysis of Lysozyme Gene of T4 2513

TABLE 2. Binding of the synthetic 14-mer to exonuclease seemed to be almost quantitative, the purification step by III-treated T4 DNA polyethyleneimine-cellulose chromatography to remove the small amounts of unutilized 14-mer was omitted. Experi- % Binding of [32PJ144mer to Exonuclease III-Treated T4 DNA. ment Components of binding reaction Binding The hybridization of [32P]14-mer or 14-mer[32P]pU to exo- A T4 DNA + 14-mer 9 nuclease III-treated T4 DNA followed by repair synthesis B T4 DNA + 14-mer + polymerase 2 was carried out as described in Materials and Methods. Table C T4 DNA + 14-mer + polymerase 14 2 shows that the binding of 14-mer to T4 DNA is increased + dtkTP in the presence of DNA polymerase I, which stabilizes the D T4 DNA + 14-mer + polymerase + 16 duplex formation (Experiment B). This stabilizing effect of dtTP + d4TP DNA polymerase in the duplex formation has already been E T4 DNA + 14-mer + polymerase 25 shown in the binding of a nonanucleotide to the left-hand + dtTP + d6TP + d(.TP cohesive end of P2 DNA. (R. Padmanabhan, unpublished results). The extent of binding of the [ 42P11-mer to T4 The primer used was [6'-terminal-'2P] d(pA-G-T-C-C-A-T-C-A- DNA was slightly increased in the presence of the enzyme C-T-T-A-A). Exonuclease III-treated T4 DNA (0.43 pmole) was hybridized with 5.2 pmole of [32P)114-mer as described under and one (Experiment C) or two deoxynucleoside triphosphates (Experiment D). But in the presence of three deoxyribonucleo- Materials and Methods. The dot - over the nucleoside part of the labeled triphosphate denotes 3H-labeled compounds. The side triphosphates (Experiment E), the binding almost maximum possible extent of binding was 26% with this DNA. reached the maximum possible value of 26%. Sequence analysis of the [82P]14-mer extended in the presence of DNA poly- carried out for 3 hr at 37°. The "diaddition" product was merase and dTTP showed that only one dT was added to the converted to a "monoaddition" product by treatment with 3' end of the primer (Table 3). This result was in agreement alkali followed by bacterial alkaline phosphatase (28). The with the mRNA sequence predicted by Streisinger et al. (9) product was purified on a Sephadex G-50 column. (see Fig. 1). When the 15-mer synthesized enzymatically For sequence analysis by partial spleen phosphodiesterase by the addition of pU (by terminal deoxynucleotidyl trans- digestion, the [12P]14-mer was prepared by oxidation of the ferase) to the 3' end of the 14-mer was tested for binding to 14-mer-['2P]pU with periodate followed by elimination (29). T4 DNA, binding occurred but to a lesser extent (Table Fig. 2b shows the fractionation of a partial spleen phospho- 4) than the 14-mer to T4 DNA. This could be due to (a) diesterase digest of the [32P]14-mer by two-dimensional homo- the fact that the 15-mer was used in much lower concentra- chromatography. Spot 1 was shown to be [32P]dAp and, from tion than the 14-mer; (b) the decreased stability of the hy- a series of successive additions of nucleotides causing char- drogen bond between U in the primer with dA in the tem- acteristic shifts in mobility of spots 2 to 14, the complete plate; or (c) the competition for binding of some 14-mer sequence was deduced to be [3'-terminal-'2P~d(A-G-T-C-C- present as a contaminant in the 15-mer. Although the pres- A-T-C-A-C-T-T-A-Ap). ence of polymerase still increased the duplex formation, The nucleotide next to the 14-mer at the 3' terminus was addition of one or two deoxynucleoside triphosphates did not shown to be dpT (Tables 2 and 3). Thus the transferase affect the extent of binding. product of the 14-mer, 14-mer-[32P]pU (a i5-mer), was also Results in Table 3 indicate that a d(pT) was added to the used as a primer for hybridization to T4 DNA and repair 3'. end of the 14-mer (Experiment 1) in the presence of [3H1- synthesis. Since the conversion of the 14-mer to 14-mer[32PJpU dTTP and DNA polymerase I. This is consistent with the TABLE 3. Nearest-neighbor and 3' end group analyses of oligonucleotides after repair synthesis

Products after enzyme digestion Experiment Primer used for repair synthesis Nucleoside Nucleotide Sequence present 1 [32P14-mer + dtTP + Polymerase dT - 14-mer-dT-OH 2 14-mer-[32P]pU-OH dA$ 13-mer-dA[32PlpU-OH 3 14-mer-['2P]pU-OH + dGTP dG dAf 13-mer-dA-[32PJpU-dGMOH Oligonucleotides after repair synthesis by DNA polymerase I were isolated by one-dimensional homochromatography and enzymatically digested as described under Materials and Methods. The nucleotides and nucleosides were separated and analyzed as described earlier (23).

FIG. 2 (on preceding page). Two-dimensional homochromatography of the products resulting from partial enzyme digestion of labeled 14-mer. Dimension I, electrophoresis on cellulose acetate strip in pyridine acetate at pH 3.5; Dimension II, homochromatography on DEAE-cellulose plate in 2% partially hydrolyzed yeast RNA containing 7 M urea (7, 20). Origin of electrophoresis is at origin of arrows; Y = position of yellow dye marker; B = position of blue dye marker. The distances between spots along Dimension II, in cm, are indicated by numbers on left. (a) Map of ['2P]oligonucleotides resulting from partial venom phosphodiesterase digestion (2jsg, 5-60 min) of [2P 14- mer (20 pmole in 5 IAI containing 15 ,ug of RNA carriers). Spot 1 is [32P]dpA, spot 2 is [5'-terminal-32PJd(pA-G), spot 3 is [5'-32Pfd(pA- G-T), etc. and spot 14 is [5'-'2Pld(pA-G-T-C-C-A-T-C-A-C-T-T-A-A). (b) Map of oligonucleotides resulting from partial spleen phosphodiesterase digestion of [3'-terminal-"P] 14-mer. The [3'-terminal-12P14-mer was prepared as described in Results and partially digested with 0.25 uig of spleen phosphodiesterase [0.1 unit according to Bernardi et al. (31), in the presence of 30 jug of partially hydrolyzed RNA carrier] in a final volume of 10 jMl for 5-60 min and fractionated. Spot 1 is [P32PdAp, Spot 2 is P [3'-terminal-32P]d(A-A-p), etc. and Spot 14 is [.3'-terminal-32Pld(A-G-T-C-C-A-T-C-A-CT-T-A-Ap). Downloaded by guest on September 24, 2021 2514 Biochemistry: Padmanabhan et al. Proc. Nat. Acad. Sci. USA 71 (1974) TABLE 4. Binding of the 15-mer to exonuclease The chemical synthesis of the tetradecanucleotide was carried III-treated T4 DNA out in the laboratory of Dr. H. G. Khorana between March and July of 1972. We are indebted to Dr. H. G. Khorana for his helpful advice and generous support; we also thank Dr. K. Experi- % Agarwal for expert guidance. This work has been supported by ment Components of binding reaction binding grants GM-18887 from the National Institutes of Health and A T4DNA+i5-mer 2.6 GB-40036X from the National Science Foundation. B T4 DNA + 15-mer + polymerase 4.5 C T4 DNA + 15-mer + polymerase 4.6 1. Wu, R. (1972) Nature New Biol. 236, 198-200. 2. Wu, R., Donelson, J., Padmanabhan, R. & Hamilton, R. + d4T? (1972) Bull. Inst. Pasteur Paris 70, 203-233. D T4 DNA + 15-iner + polymerase 4.7 3. Wu, R. & Taylor, E. (1971) J. Mol. Biol. 57, 491-511. + dGTP + dOTP 4. Padmanabhan, R., Padmanabhan, R. & Wu, R. (1972) Biochem. Biophys. Res. Commun. 48, 1295-1302. The primer used was 14-mer-[P2PjpU-OH (the 15-mer). 5. Sanger, F., Donelson, J. E., Coulson, A. R., Kossel, H.. & Exonuclease III-treated T4 DNA (0.57 pmole.) was'.hybridized Fisher, D. (1973) Proc. Nat. Acad. Sci. USA 70, 1209-1213. with the primer (0.78 pmole) as described under Materials and 6. Loewen, P. C. & Khorana, H. G. (1973) J. Biol. Chem. 248, 3489-3499. Methods. The dot (-) over the nucleoside part of the labeled 7. Wu, R., Tu, C. D. & Padmanabhan, R. (1973) Biochem. triphosphate denotes 'H-labeled compounds. The maximum Biophys. Res. Commun. 55, 1092-1099. possible binding was 26% with this DNA. 8. Tsugita; A. & Inouye, M. (1968) J. Mol. Biol. 37, 201-212. 9. Streisinger, G., Okada, Y., Emrich, J., Newton, J., Tsugita, fact that the 15-mer (14-mer-["2P]pU) is also bound to T4 A., Terzahgi, E. & Inouye, M. (1966) Cold Spring Harbor Symp. Quant. Biol. 31, 77-84. DNA and serves as a primer see Table 4). Nearest neighbor 10. Nirenberg, M., Leder, P., Bernfield, M., Brimacombe, R., analysis of the 14-mer-{"2PlpU showed the transfer of "2P Trupin, J., Rottman, F. & O'Neal, C. (1965) Proc. Nat. from~['2P]pU to dpk, which was at the 3' end of the 14-mner Acad. Sci. USA 53, 1161-1168. (Table 3, Experimeht 2), as expected. In Experiment S of 11. Khbrana, H. G., Buchi, H., Ghosh, H., Gupta, N., Jacob, T. fM., Kossel, H., Morgan, R., Narang, S. A., Ohtsuka, Table 3, the 14-mer-["2P]pU was extended to a 16-mer in the E. & Wells, R. D. (i966) Cold Spring Harbor Symp. Quant. presence of ['H]dGTP and DNA polymerase I. The 16-mer Biol. 31, 39-49. showed a dG at the 3' end and no dpG was found. Therefore, 12. Narang, S. A., Itakura, K., Bahl, C. P. & Wigfield, Y. Y. the sequence of nucleotides beyond the 3' end of the chemi- (1972) Biochem. Biophys. Res. Commun. 49, 445-452. cally synthesized 14-mer is d(pT-G), which is in agreement 13. Doel, M. T. & Smith, M. (1973) FEBS Lett. 34, 99-102. 14. Thomas, C. A., Jr. & Abelson, J. (1966) in Procedures in with the RNA sequence as shown in Fig. 1. Nucleic Acid Research, ed. Cantoni, G. L. & Davies, D. R. (Academic Press, New York), Vol. 1, pp. 553-561. DISCUSSION 15. Wu, R., Padmanabhan, R. & Bambara, R. (1974) in The use of this general approach for DNA sequence deter- Methods in Enzymology, eds., Grossman, L. & Moldave, K. mination is limited to the proper selection of the primer se- (Academic Press, New York), Vol. 29, part E, p. 231. 16. Jovin, T. M., Englundj P. T. & Bertsch, L. L. (1969) J. quence which will hybridize to the single-stranded region of Biol. Chem. 244, 2996-3008. the template DNA of interest. Current methods in selecting 17. Khorana, H. G. et al. (1972) J. Mol. Biol. 72, 209-426. the primer sequence include (a) the method based on knowl- 18. Agarwal, K. L., Yamasaki, A. & Khorana, H. G. (1971) edge of the RNA sequencesuch as tRNA (6) or mRNA (this J. Amer. Chem. Soc. 93, 2754-2762. 19. Jay, E., Agarwal, K. L., Cashion, P. J., Fridkin, M. & communication) and (b) the method based on knowledge Khorana, H. G. (1972) Amer. Chem. Soc., Abstract. of the protein sequence coded by the gerionie of interest, such 20. Brownlee, G. G. & Sanger, F. (1969) Eur. J. Biochem. 11, as the endolysin gene from X phage, and the correct choice 395-399. of the triplet in cases where the amino-acid code is degenerate 21. Ghangas, G. S., Jay, E., Bambara, R. & Wu, R. (1973) (1; 7). Biochem. Biophys. Res. Commun. 54, 998-1007. 22. Barrell, B. G. (1971) in Procedures in Nucleic Acid Research, The method used in this communication for choosing the eds. Cantoni, G. L. & Davies, D. R. (Academic Press, primer sequence has the potential advantage that the DNA New York), Vol. 2, pp. 751-796. sequence information obtained beyond the primer would 23. Wu, R. (1970) J. Mol. Biol. 51, 501-521. reveal the frequency of the actual use of the triplets among 24. Streisinger, G., Edgar, R. S. & Denhardt, G. H. (1964) Proc. Nat. Acad. Sci. USA 51, 775-779; and Thomas, the degenerate codewords. This is important in understanding C. A., Jr. & MacHattie, L. A. (1964) Proc. Nat. Acad. Sci. the degeneracy in the genetic code. The knowledge of the USA 52, 1297-1301. exact DNA seqcencob which codes for a protein is also helpful 25. Kasai, T., Bautz, E. K. F., Guha, A. & Szybalski, W. (1968) in the elucidation of the tertiary structure of its mRNA J. Mol. Biol. 34, 709-711. cannot be obtained in 26. Richardson, C. C. (1965) Proc. Nat. Acad. Sci. USA 54, transcript. Such valuable information 158-165. cases where the primer is bound to the DNA at an unknown 27. Southern, E. M. & Mitchell, A. R. (1971) Biochem. J. 123, location (5). 613-617. Once the- primer is synthesized, a specific ribonucleotide 28. Roychoudhury, R., Fisher, D. & Kossel, H. (1971) Bio- can be added to its 3' end by the deoxynucleotidyl terminal chem. Biophys. Res. Commun. 45, 430-435. 29. Neu, H. C. & Heppel, L. A. (1964) J. Biol. Chem. 239, transferase for use as a more versatile primer. Such a primer, 2927-2934. the 14-mer-['*P]pU for example, has an added advantage 3d. Thomas, C. A., Jr. (1966) in Progress in Nucleic Acid in the determination of longer sequences beyond its 3' end Research and Molecular Biology, eds. Davidson, J. N. & because the oligonucleotides incorporated beyond the 3' end Cohn, W. E. (Academic Press, New York), Vol. 5, pp. can be 315-337. of pU (in polymerase I-catalyzed repair synthesis) 31. Bernardi, A. & Bernardi, G. (1968) Biochim. Biophys. Acta cleaved off from the primer with pancreatic ribonuclease, 155, 360-370. thus making sequence analysis of the newly synthesized seg- 32. Jay, E., Bambara, R., Padmanabhan, R. & Wu, R. (1974) ment easier. Nucleic Acids Research 1, 331-354. Downloaded by guest on September 24, 2021