Sep 1, 1980

Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose

Proceedings of the National Academy of Sciences of the United States of America
P S Thomas

Abstract

A simple and rapid method for transferring RNA from agarose gels to nitrocellulose paper for blot hybridization has been developed. Poly(A)+ and ribosomal RNAs transfer efficiently to nitrocellulose paper in high salt (3 M NaCl/0.3 M trisodium citrate) after denaturation with glyoxal and 50% (vol/vol) dimethyl sulfoxide. RNA also binds to nitrocellulose after treatment with methylmercuric hydroxide. The method is sensitive: about 50 pg of specific mRNA per band is readily detectable after hybridization with high specific activity probes (10(8) cpm/microgram). The RNA is stably bound to the nitrocellulose paper by this procedure, allowing removal of the hybridized probes and rehybridization of the RNA blots without loss of sensitivity. The use of nitrocellulose paper for the analysis of RNA by blot hybridization has several advantages over the use of activated paper (diazobenzyloxymethyl-paper). The method is simple, inexpensive, reproducible, and sensitive. In addition, denaturation of DNA with glyoxal and dimethyl sulfoxide promotes transfer and retention of small DNAs (100 nucleotides and larger) to nitrocellulose paper. A related method is also described for dotting RNA and DNA directly onto nitrocellulose paper treated with...Continue Reading

  • References13
  • Citations4855

References

Mentioned in this Paper

Dimethyl Sulfoxide
Electrophoresis, Agar Gel
RNA Denaturation
Nucleic Acid Hybridization Procedure
Methylmercury hydroxide
Rapid Technique
Celloidin
Ribosomes
Binding (Molecular Function)
Chick Embryo

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