A simple and optimized method of producing silanized surfaces for FISH and replication mapping on combed DNA fibers.
Molecular combing of DNA is an extremely powerful DNA fiber-stretching technique that is often used in DNA replication and genome stability studies. Optimal DNA combing results mainly depend on the quality of the silanized surfaces onto which fibers are stretched. Here we describe an improved method of liquid-phase silanization using trimethoxy-octenylsilane/n-heptane as novel silane/solvent combination. Our simple method produces homogenously modified coverslips in a reproducible manner but does not require any sophisticated or expensive equipment in comparison to other known silanization protocols. However DNA fibers were combed onto these coverslips with very good high-density alignment and stayed irreversibly bound onto the surfaces after various denaturing treatments, as required for different immunofluorescent detection of DNA with incorporated modified nucleotides or FISH.
Labit
Hélène
H
Laboratory of Molecular Genetics, Centre national de la recherche scientifique (CNRS), UMR 8541, Biology Department, Ecole Normale Supérieure, Paris, France.
Goldar
Arach
A
Guilbaud
Guillaume
G
Douarche
Carine
C
Hyrien
Olivier
O
Marheineke
Kathrin
K
D000818:Animals; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D015342:DNA Probes / Q000378:metabolism; D004261:DNA Replication; D004275:DNA, Ribosomal; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D018625:Microscopy, Atomic Force; D009690:Nucleic Acid Conformation; D012821:Silanes / Q000737:chemistry*; D013499:Surface Properties; D014981:Xenopus
D015342:DNA Probes; D004275:DNA, Ribosomal; D012821:Silanes; D004247:DNA