Hybridization of N-acetoxy-N-acetyl-2-aminofluorene-labelled RNA to Q-banded metaphase chromosomes.
This paper describes improvements of a recently developed immunocytochemical method for the detection of specific polynucleotide sequences within chromosomes, as well as conditions by which this method can be combined with chromosome banding. The immunocytochemical method involves modification of polynucleotide probes with N-acetoxy-N-acetyl-2-aminofluorene (AAAF)2), and hybridization of the modified probes with metaphase chromosomes; the hybrids are made visible immunocytochemically by means of an antiserum which recognizes AAAF-induced polynucleotide modifications. We have sorted out conditions which allow a high sensitivity of hybrid detection by the above procedure, in combination with chromosome banding. The best results are obtained if the ABC-technique is used for the visualization of the hybrids; the lower limit of detection is estimated to be a sequence of about 7000 nucleotides. The method can be combined with Q-banding of chromosomes, if this is performed not more than 1 day prior to hybridization, and if excitation of the Q-banded chromosomes is kept to a minimum.
D015073:2-Acetylaminofluorene; D001483:Base Sequence; D002478:Cells, Cultured; D002871:Chromosome Banding; D002877:Chromosomes, Human / Q000032:analysis*; D006801:Humans; D007124:Immunoenzyme Techniques; D008214:Lymphocytes / Q000032:analysis; D008677:Metaphase; D009693:Nucleic Acid Hybridization; D011489:Protein Denaturation; D012335:RNA, Ribosomal / Q000378:metabolism*
D012335:RNA, Ribosomal; D015073:2-Acetylaminofluorene