An Efficient Method of Cotton DNA Fibers Preparation for FISH

Abstract

Fluorescence in situ hybridization(FISH) has become the most important technique applied in molecular cytogenetics,especially in developing physical maps in plants.As a key technique,FISH on cotton DNA fibers stretched has not been reported yet,possibly owning to the difficulty in their DNA fiber preparation as well as the existence of thick cytoplasm and hard cell walls.Here we present a method of highly efficient preparation of stretched DNA fibers in cotton.Cotton cotyledons germinated in dark moisture chamber for one week were chopped with a sharp sterile scalpel in a Petri dish that contained ice-cold nucleus isolation buffer(MgSO4 10 mmol L?1,KCl 5 mmol L?1,HEPES 0.5 mmol L?1,DTT 1 mg mL?1,Triton X-100 0.25%,PVP40 2%) followed by sequential filtration through 100,50,and 30 μm nylon meshes.Nuclei were obtained by centrifuging the filtrates at 16 000× g for 1 min.Mixture of nucleus lysis buffer(0.5% SDS,5 mmol L-1 EDTA,100 mmol L?1 Tris,pH 7.0) and nuclei was incubated on the slide for 9 min,DNA fibers obtained by dragging and stretching with a clean slide edge from the end to another end on the liquid surface.After incubated at 60℃ overnight,the slides were pretreated with DNase-free RNase and then rinsed in 2×SSC.The probes and DNA fibers were denatured separately,and hybridization mixture was incubated on the slide overnight,followed by post-hybridization rinses in 2×SSC,1×4T.The slides were blocked with 5% BSA and covered with antibody for 1h.After rinsed with 1×TNT the slides were counterstained with 4',6-diamidino-2-phenylin-dole(DAPI) and followed by rinsing in 1×PBS.After mounting the slides with Vectashield mounting medium the hybridization signals were observed under a fluorescence microscope.Images were captured by a charge-coupled device(CCD) system and brought together to make the plate using Adobe Photoshop 7.0 software.The results indicated that it was easier to release nuclei from cells in nucleus isolation buffer by chopping cotyledon,and slowly and smoothly dragging the nuclei solution with a slide edge from the end to another end on slide treated with poly-L-lysine.Highly stretched and intact DNA fibers were obtained.This method is very simple and rapid,which takes only 30 min to finish the entire process,and it is also safe because poisonous mercaptoethanol is replaced by dithiothreitol.The linear or near-linear stretches of beads on-a-string signals with cotton genomic DNA and 45S rDNA as probes showed that the DNA fibers were suitable for FISH.