Nonradioactive Detection of Nucleic Acids with Biotinylated Probes

Summary

Methods for nonradioactive detection of nucleic acids have replaced ³²P-based techniques in many laboratories. Improvements in sensitivity allow the detection of single-copy genes in Southern blots and low abundance mRNAs in Northern blots. Labelling of (c)DNA probes involves replacement of dTTP by either digoxigenin-dUTP or biotinylated dUTP. Here we describe experimental protocols for Northern blot analysis and for genomic Southern blot using a rapid alkaline transfer technique, followed by procedures for hybridization and blot development with biotinylated probes. A protocol for synthesis of biotin-labeled probes by polymerase chain reaction (PCR) is included. The technical difficulties encountered with densitometric signal quantitation of nonradioactive blots are discussed, and procedures for relative and absolute quantitation of bound probe are presented.