Abstract
We have developed a system for the enzymatic in vitro synthesis of non-radioactively labeled RNA which is derivatized with the hapten digoxigenin (DIG). The labeling reaction as well as the conditions for hybridization and detection of hybrids by an antibody-conjugate and a coupled colour reaction were analyzed and adapted for high sensitivity and low background. In addition, data on the performance and sensitivity of digoxigenin-labeled RNA probes in Southern and Northern blots are presented.
Full text
PDF![5843](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0634/332324/00b95c308328/nar00203-0230.png)
![5844](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0634/332324/6040206b7036/nar00203-0231.png)
![5845](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0634/332324/9f4bfff6c2be/nar00203-0232.png)
![5846](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0634/332324/ba71cfa6d34e/nar00203-0233.png)
![5847](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0634/332324/0b2669bc8904/nar00203-0234.png)
![5848](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0634/332324/f42a71983a13/nar00203-0235.png)
![5849](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0634/332324/3a681505ad74/nar00203-0236.png)
![5850](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0634/332324/951e103abe62/nar00203-0237.png)
![5851](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0634/332324/56012aa808c2/nar00203-0238.png)
Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Beck S., O'Keeffe T., Coull J. M., Köster H. Chemiluminescent detection of DNA: application for DNA sequencing and hybridization. Nucleic Acids Res. 1989 Jul 11;17(13):5115–5123. doi: 10.1093/nar/17.13.5115. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Behar K. L., den Hollander J. A., Stromski M. E., Ogino T., Shulman R. G., Petroff O. A., Prichard J. W. High-resolution 1H nuclear magnetic resonance study of cerebral hypoxia in vivo. Proc Natl Acad Sci U S A. 1983 Aug;80(16):4945–4948. doi: 10.1073/pnas.80.16.4945. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bronstein I., Edwards B., Voyta J. C. 1,2-dioxetanes: novel chemiluminescent enzyme substrates. Applications to immunoassays. J Biolumin Chemilumin. 1989 Jul;4(1):99–111. doi: 10.1002/bio.1170040116. [DOI] [PubMed] [Google Scholar]
- Brown J. E., Klement J. F., McAllister W. T. Sequences of three promoters for the bacteriophage SP6 RNA polymerase. Nucleic Acids Res. 1986 Apr 25;14(8):3521–3526. doi: 10.1093/nar/14.8.3521. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Butler E. T., Chamberlin M. J. Bacteriophage SP6-specific RNA polymerase. I. Isolation and characterization of the enzyme. J Biol Chem. 1982 May 25;257(10):5772–5778. [PubMed] [Google Scholar]
- Coutlee F., Viscidi R. P., Yolken R. H. Comparison of colorimetric, fluorescent, and enzymatic amplification substrate systems in an enzyme immunoassay for detection of DNA-RNA hybrids. J Clin Microbiol. 1989 May;27(5):1002–1007. doi: 10.1128/jcm.27.5.1002-1007.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cox K. H., DeLeon D. V., Angerer L. M., Angerer R. C. Detection of mrnas in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Dev Biol. 1984 Feb;101(2):485–502. doi: 10.1016/0012-1606(84)90162-3. [DOI] [PubMed] [Google Scholar]
- Forster A. C., McInnes J. L., Skingle D. C., Symons R. H. Non-radioactive hybridization probes prepared by the chemical labelling of DNA and RNA with a novel reagent, photobiotin. Nucleic Acids Res. 1985 Feb 11;13(3):745–761. doi: 10.1093/nar/13.3.745. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Heiles H. B., Genersch E., Kessler C., Neumann R., Eggers H. J. In situ hybridization with digoxigenin-labeled DNA of human papillomaviruses (HPV 16/18) in HeLa and SiHa cells. Biotechniques. 1988 Nov-Dec;6(10):978–981. [PubMed] [Google Scholar]
- Henikoff S., Keene M. A., Fechtel K., Fristrom J. W. Gene within a gene: nested Drosophila genes encode unrelated proteins on opposite DNA strands. Cell. 1986 Jan 17;44(1):33–42. doi: 10.1016/0092-8674(86)90482-4. [DOI] [PubMed] [Google Scholar]
- Ishikawa E., Imagawa M., Hashida S., Yoshitake S., Hamaguchi Y., Ueno T. Enzyme-labeling of antibodies and their fragments for enzyme immunoassay and immunohistochemical staining. J Immunoassay. 1983;4(3):209–327. doi: 10.1080/15321818308057011. [DOI] [PubMed] [Google Scholar]
- Ishikawa E., Imagawa M., Hashida S., Yoshitake S., Hamaguchi Y., Ueno T. Enzyme-labeling of antibodies and their fragments for enzyme immunoassay and immunohistochemical staining. J Immunoassay. 1983;4(3):209–327. doi: 10.1080/15321818308057011. [DOI] [PubMed] [Google Scholar]
- LARDY H. A., PEANASKY R. Metabolic functions of biotin. Physiol Rev. 1953 Oct;33(4):560–565. doi: 10.1152/physrev.1953.33.4.560. [DOI] [PubMed] [Google Scholar]
- Langer P. R., Waldrop A. A., Ward D. C. Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6633–6637. doi: 10.1073/pnas.78.11.6633. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lichter P., Tang C. J., Call K., Hermanson G., Evans G. A., Housman D., Ward D. C. High-resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones. Science. 1990 Jan 5;247(4938):64–69. doi: 10.1126/science.2294592. [DOI] [PubMed] [Google Scholar]
- Melton D. A. Injected anti-sense RNAs specifically block messenger RNA translation in vivo. Proc Natl Acad Sci U S A. 1985 Jan;82(1):144–148. doi: 10.1073/pnas.82.1.144. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Milligan J. F., Groebe D. R., Witherell G. W., Uhlenbeck O. C. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 1987 Nov 11;15(21):8783–8798. doi: 10.1093/nar/15.21.8783. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Morris C. E., Klement J. F., McAllister W. T. Cloning and expression of the bacteriophage T3 RNA polymerase gene. Gene. 1986;41(2-3):193–200. doi: 10.1016/0378-1119(86)90098-3. [DOI] [PubMed] [Google Scholar]
- Mössner E., Boll M., Pfleiderer G. Purification of human and bovine alkaline phosphatases by affinity chromatography. Hoppe Seylers Z Physiol Chem. 1980 Apr;361(4):543–549. doi: 10.1515/bchm2.1980.361.1.543. [DOI] [PubMed] [Google Scholar]
- Nielsen D. A., Shapiro D. J. Preparation of capped RNA transcripts using T7 RNA polymerase. Nucleic Acids Res. 1986 Jul 25;14(14):5936–5936. doi: 10.1093/nar/14.14.5936. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pfeiffer F., Gilbert W. A. VecBase, a cloning vector sequence data base. Protein Seq Data Anal. 1988;1(4):269–280. [PubMed] [Google Scholar]
- Ponte P., Ng S. Y., Engel J., Gunning P., Kedes L. Evolutionary conservation in the untranslated regions of actin mRNAs: DNA sequence of a human beta-actin cDNA. Nucleic Acids Res. 1984 Feb 10;12(3):1687–1696. doi: 10.1093/nar/12.3.1687. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Riley L. K., Marshall M. E., Coleman M. S. A method for biotinylating oligonucleotide probes for use in molecular hybridizations. DNA. 1986 Aug;5(4):333–337. doi: 10.1089/dna.1986.5.333. [DOI] [PubMed] [Google Scholar]
- Schray K. J., Artz P. G., Hevey R. C. Determination of avidin and biotin by fluorescence polarization. Anal Chem. 1988 May 1;60(9):853–855. doi: 10.1021/ac00160a006. [DOI] [PubMed] [Google Scholar]
- Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
- Sutton R. E., Boothroyd J. C. Evidence for trans splicing in trypanosomes. Cell. 1986 Nov 21;47(4):527–535. doi: 10.1016/0092-8674(86)90617-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Theissen G., Richter A., Lukacs N. Degree of biotinylation in nucleic acids estimated by a gel retardation assay. Anal Biochem. 1989 May 15;179(1):98–105. doi: 10.1016/0003-2697(89)90207-8. [DOI] [PubMed] [Google Scholar]
- Wilchek M., Bayer E. A. The avidin-biotin complex in bioanalytical applications. Anal Biochem. 1988 May 15;171(1):1–32. doi: 10.1016/0003-2697(88)90120-0. [DOI] [PubMed] [Google Scholar]