Fluorescent In Situ Hybridization Using Oligonucleotide-Based Probes

Efficient and consistent chromosome identification is the foundation for successful cytogenetic studies. Fluorescent in situ hybridization (FISH) has been the most popular technique for chromosome identification in plants. Large insert genomic DNA clones, such as bacterial artificial chromosome (BAC) clones, and repetitive DNA sequences have been the most commonly used DNA probes for FISH. However, most of such traditional probes can only be used to identify a single chromosome or are too polymorphic to consistently identify the same chromosome in the target species. In contrast, FISH using oligonucleotide (oligo)-based probes is highly versatile. In this procedure, a large number of oligos specific to a chromosomal region, to an entire chromosome, or to multiple chromosomes are computationally identified, synthesized in parallel, and labeled as probes. In addition, each oligo probe can be used for thousands of FISH experiments and represents an infinite resource. In this chapter we describe a detailed protocol for amplification and labeling of oligo-based probes, relevant chromosome preparation, and FISH procedures.