Preparation and separation of d(pT)_10.noligonucleotides

Nucleic Acids Research Volume 2 number 3 March 1975 Preparation and separation of d(pT)IO.n oligonuclectides A. J. Raae, J. R. Lillehaug, R. K. Kleppe and K. Kleppe Department of Biochemistry, University of Bergen, Bergen, Norway Received 13 February 1975 ABSTRACT A series of oligomers having the general formula d(pT)10*n n varying from 2 to 20, has been prepared by enzymatic joining of d(pT)lO, annealed on poly dA, employing T4 polynucleotide ligase. The oligomers could be separated on 8 or 12% polyacrylamide gels. Such oligomers may prove useful as molecular weight markers and as initiators for various polymerases. INTRODUCTION Separation of nucleic acids on polyacylamide gels, both for preparative and analytical purposes, has proven to be an exceedingly useful technique 1-3 . This laboratory has for some time employed polyacrylamide gel electrophoresis in studying the mechanism of action of various enzymes involved in the nucleic acid metabolism, in particular TW polynucleotide kinase and ligase. The present work describes one aspect of this study, namely, the nature and separation of the products of the T4 polynucleotide ligase catalyzed joining of d(pT)lO annealed on poly dA. The method presented here may prove useful in other areas of nucleic acids research as well. MATERIALS polynucleotide kinase and ligase were prepared as previously described . Bacterial alkaline phosphatase was a product of Worthington Biochemical Corporation. Acrylamide and T4 N, Nmethylenebisacrylamide were products of Eastman Kodak 423 Nucleic Acids Research Company. [Y-32P] ATP with a specific activity of approximately 50-100 Ci/mmole was prepared according to a modified procedure of Glynn and Chappell'. Poly dA with average molecular weight of approximately lx105 and d(pT)10 were obtained from P-L Bio- d(pT)lO was dephosphorylated by treatalkaline phosphatase6 the reaction being chemicals. Prior to use ment with bacterial carried out at 670 to inactivate possible nucleases. The dT(pT)9 was then phosphorylated at the 5'hydroxyl terminus by employing T4 polynucleotide kinase7 and the phosphorylated oligonucleotide separated from excess ATP by gelfiltration on a column of Sephadex G-50 equilibrated with 0.05M tri- ethylammonium bicarbonate (TEAB). After separation the buffer was removed by lyophilization and the phosphorylated oligo- nucleotide dissolved in a small volume of 10 mM Tris/HCl pH 8.0, containing 0.1 mM EDTA. METHODS .:Conditions for .ioining of d(nT)10. for the T4 A typical reaction mixture polynucleotide ligase catalyzed joining of [5'32P] d(pT)10 contained the following components: 50 mM Tris pH 8.0, 6 mM MgCl2, 10 mM dithiothreitol (DTT), 0.6 mM ATP, 0.14 mM poly dA (phosphate), 0.14 mM [5t_32p] d(pT)10 (phosphate) 400 units of T4 polynucleotide ligase/ml. Poly dA and and [51_32p] d(pT)10 were annealed prior to addition of ATP, DTT and enzyme by heating the reaction mixture at 60°C for 5 minutes then letting it slowly cool to room temperature. The reaction temperature was 20°C and aliquots were removed at various times and assayed for resistance to bacterial alkaline phosphatase as described elsewhere 4. After approximately 1 hour the reaction had usually reached a plateau. The reaction was then 424 Nucleic Acids Research stopped by making the reaction mixture 50% with respect to formic acid and the mixture was then kept for 16 hours at 300C 8. The latter treatment causes complete depurination of poly dA and thus serves to denature the poly dA-d(pT)10.n duplexes. Upon completion of this step the reaction mixture was lyophilized, then dissolved in 100 WI. of 50% glycerol containing 7M urea and the marker dyes bromphenol blue and xylene cyanole. Aliquots of this mixture were subjected to gel electrophoresis. Gel electrophoresis. Vertical gels (18x18x0.2 cm) of 8 or 12% polyacrylamide containing 7M urea were employed. The gels (95% acrylamide and 5% N,N'methylenebisacrylamide) were used with a running buffer of 90 mM Tris, 90 mM boric acid and 0.4 mM EDTA, pH 8.3. The electrophoresis was carried out for 12-18 hours at room temperature and usually at 150 V. Upon completion of electrophoresis the gels were subjected to autoradiography. RESULTS AND DISCUSSION The [5t-32p] d(pT)1O oligonucleotide annealed on poly dA was joined to larger oligonucleotides by T4 polynucleotide ligase and the oligonucleotides thus formed were then sub- jected to gel electrophoresis. Prior to electrophoresis the duplexes were denatured by depurination of poly dA. Heat denaturation alone, with or without formamide, proved unsatisfacto-ry for this purpose as the strands rapidly reannealed. A separation pattern on a 12% polyacrylamide gel of the oligonucleotides formed is shown in Figure 1 A. It is clear that the action of T4 polynucleotide ligase on the duplex poly dA-[51-32P] d(pT)lO resulted in formation of a series of new 425 Nucleic Acids Research d(pT)10 n. oligomers having the general formula tides with such on a varying from 1 to 10 could-clearly be separated n gel. The separation pattern Figure 1 B. In this were run mers case on a 8% gel is given in the lower molecular weight oligomers out of the gel to allow better separation of oligo- of higher molecular weight. Oligomers from d(pT)50 and to d(pT)200 up Oligonucleo- be well separated by this technique. For can both types of gels plot of log (molecular weight) a log or (oligomer size) vs relative mobility produced similar curves shown in Figure 2. In the lower molecular weight regions as these plots were linear. The various oligomers could be eluted from the gel by cutting out the appropriate regions, crushing the gel to smaller pieces with a glassrod in a 15 ml centrifuge tube, then adding 1 M TEAB equal to twice the volume of the gel. This suspension was left at room temperature for approxima- tely 15 hours, after which time the supernatant was off and the gel particles washed 3 times with 1 volume of 1 M TEAB. The TEAB was remove times the then evaporated off by lyophilization and the sample dissolved in water. In order to pipetted a small volume of the urea and the buffer from the gel the sample was subjected to gelfiltration on a small column of Sephadex G-50 (lx15 cm). Usually 70-80% recovery was achieved. The mobility on the gel of single stranded nucleic acids will undoubtedly be influenced by such factors as stacking interactions and any tendency to form secondary and tertiary structures. Therefore, the mobility of various single stranded nucleic acids may be slightly different than that of the 426 Nucleic Acids Research Figure 1. A. Separation of d(pT)10.n oligomers on a 12% polyacrylamide gel. Autoradiogram of the gel. B. Separation of d(pT)10.n oligomers on a 8% polyacrylamide gel. The lower molecular weight oligomers were run out of the gel. Marker oligonucleotide not shown. I I 1.0 ,12 0/0 geL ,8 0/0 gel .0 E 0) 0.5 ._ 0) 50 100 200 10 Size of oligo d(pT)lO,n (nucleotides) I I I. as 4,5 4.0 35 Log moleculor weight Figure 2. Semi-logarithmic plots of the separation data shown in Figure 1. I 427 Nucleic Acids Research d(pT)10*n of the same size. This was clearly revealed by comparing the mobility of d(pT)10 and d(pA) ratio where the of their relative mobilities was 1:1.1. Bearing these restrictions in mind the above oligomers may be useful as molecular weight markers. Furthermore, they are also well suited as initiators for various polymerases9-11 A CKN OWLEDGEMENTS This study was supported in parts by a grant from the Norwegian Research Council for Science and Humanities. We are also grateful to Dr. C.T. Korch for criticism of the manuscript. Mailing address: Dr. Kjell Kileppe, Department of Biochemistry, University of Bergen, Bergen, Norway REFERENCES 1. De Wachter and Fiers, W. (1971) Methods in Enzvmologv, XXI D, 167-178. 2. Jovin, T.M. (1971) Methods in Enzymolory, XI D, 179-187. 3. Burd, J.F. and Wells, R.D. (1974) J. Biol. Chem., 249, 7094-7101. 4. 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