Separation of the complementary strands of DNA fragments on polyacrylamide gels

TY - JOUR

T1 - Separation of the complementary strands of DNA fragments on polyacrylamide gels

AU - Szalay, A. A.

AU - Grohmann, K.

AU - Sinsheimer, R. L.

N1 - Funding Information: be resolved on polyacrylamide gels. This separation does not require electrophoresis in denaturing conditions, it only requires denaturation of DNA prior to electrophoresis. Treatment of the DNA fragments with 5 mM Me-HgOH or heating in boiling water for 2-3 min gave very good separation of the complementary strands. The separation is independent of temperature (4°-22°) and pH (7.0-9.0). The ratio of acrylamide to N,N'-bis-acrylamide with gel was found to play a very important role in the strand separation. Best results were achieved when the ratio was 60:1. By choosing the appropriate concentration of acrylamide it is possible to separate complementary strands of fragments containing 75-5500 base pairs. Full length double stranded 0X174 RFIII DNA (5500 b.p.) obtained by cleaving RFI with Providencia stuartii endonuclease could be resolved as two sharp bands on 2% polyacrylamide gel. For preparative isolation of strands containing 600-2000 nucleotide 3% acrylamide gels were most suitable. For analytical purpose gradient gels have good resolution in the range of 75 to 2000 nucleotide long fragments. The basis of strand separation is not at present clear but it appears that the base composition plays an important role. Our results show that the slow moving strand of the doublets is the positive (+) strand in most of the 0X DNA fragments. Preliminary experiments on base composition of separated strands suggest that the slow moving strands are rich in T (30-40%). Available nucleoside sequence analysis on both strands of fragment numbers 9 and 10 generated by Hinc II14 show that the T rich strands are the + strands which migrate on our gels slower than the iri vitro labeled minus (-) strands. In the fragment number 5 produced by Hinc II the negative strand migrates slower. This is not an artifact of Hinc II digestion. Fragment number 3 produced by Hae III from the same region of the 0X genome also shows the same results. Fragments of HeLa mitochondrial DNA and of SV40 DNA obtained by Hpa II and Hind III restriction endonucleases could also be resolved into complementary strands suggesting the general applicability of the method. A combination of this technique with the transfer-method developed by Southern could be useful for identification of the DNA strand with its transcription product. ACKNOWLEDGEMENTS This work was supported by U.S. Public Health Service Grant No. GM 13554. We thank Professors N. Davidson and S. K. Dube for their critical discussions. REFERENCES * Dedicated to Jerome Vinograd 1 Szybalski, W., Kubinski, H., Hradecna, Z. and Summers, W. C. (1971) Methods Enzymol. 21, 388-413 Hayward, G. S. (1972) Virology 49, 342-344 Tibbetts, C, Petersson, U., Johansson, K. and Philipson, L. (1974) J. Virology 13, 370-377

PY - 1977/6

Y1 - 1977/6

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U2 - 10.1093/nar/4.5.1569

DO - 10.1093/nar/4.5.1569

M3 - Article

C2 - 331258

AN - SCOPUS:0017412330

SN - 0305-1048

VL - 4

SP - 1569

EP - 1578

JO - Nucleic Acids Research

JF - Nucleic Acids Research

IS - 5

ER -