Fiber-FISH: experiences and a refined protocol.
One of the most time-consuming steps in positional cloning is the physical mapping of probes from the critical chromosomal region and the assembly of a genomic contig of large insert probes. New high-resolution Fiber-FISH techniques have significantly facilitated this tedious task by enabling rapid direct visualization of the order, degree of overlap and gap sizes of adjacent large insert clones. We have developed a method, where agarose-embedded DNA (PFGE block) is used as a source for preparing linearized DNA targets on microscope slides. This modification of the fiber-FISH technique has been successfully used in physical mapping in the 1-300 kb range as well as for detecting genomic rearrangements. Here, we present a refined protocol of our original technique. The application of this technique to agarose embedded yeast cells is also demonstrated. Finally, critical steps and trouble shooting of the method are addressed.
Heiskanen
M
M
Laboratory Department of Helsinki, University Central Hospital, Finland.
Kallioniemi
O
O
Palotie
A
A
D002874:Chromosome Mapping; D018244:Chromosomes, Artificial, Yeast; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D016521:Electrophoresis, Gel, Pulsed-Field; D005069:Evaluation Studies as Topic; D015321:Gene Rearrangement; D006801:Humans; D007091:Image Processing, Computer-Assisted; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D015335:Molecular Probes; D012441:Saccharomyces cerevisiae / Q000235:genetics
D015335:Molecular Probes; D004247:DNA