Nonradioactive labeling of large DNA fragments for genome walking, RFLP and northern blot analysis

Adhami F, Müller S, Hauser MT (1999)

Publication Type: Journal article

Publication year: 1999

Journal

Biotechniques Eaton Publishing

Book Volume: 27

Pages Range: 314-320

Journal Issue: 2

DOI: 10.2144/99272st02

Abstract

In this report, we present a simple nonradioactive labeling procedure for DNA fragments of high specific labeling density that can be used for a variety of applications. The protocol is based on the universal mono- functional platinium reagent for chemical digoxigenin (DIG) labeling of nucleic acids. The labeling protocol was optimized for large DNA templates as complete bacterial artificial chromosomes (BAC). Variations of incubation time and temperature improved the labeling density such that about 30% of the nucleotides were DIG-modified within 30 min. Furthermore, the refined procedure generates in a single-tube reaction and without prior digestion- labeled DNA fragments of 0.5-4.0 kb from a 130-kb template. Hybridization experiments were performed on Southern and northern blots and allowed the detection of single copy genes in 2.5 μg genomic DNA from Arabidopsis thaliana, which has a haploid genome size of 0.13 pg (ca. 120 Mb) and medium expressed transcripts from 0.8 μg poly(A)+ RNA, respectively. The extremely high specific labeling density, the stability and the universal application of the probe generated with the platinium reagent makes this method a useful alternative to classical radioactive nuclei acids labeling techniques.

Authors with CRIS profile

Sabine Müller

Involved external institutions

Universität für Bodenkultur Wien (BOKU) / University of Natural Resources and Life Sciences, Vienna AT Austria (AT)

How to cite

APA:

Adhami, F., Müller, S., & Hauser, M.-T. (1999). Nonradioactive labeling of large DNA fragments for genome walking, RFLP and northern blot analysis. Biotechniques, 27(2), 314-320. https://doi.org/10.2144/99272st02

MLA:

Adhami, Farzaneh, Sabine Müller, and Marie-Theres Hauser. "Nonradioactive labeling of large DNA fragments for genome walking, RFLP and northern blot analysis." Biotechniques 27.2 (1999): 314-320.

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