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.