To construct a detailed cytogenetic map of human chromosome region 3p23→p21.3, we determined the order of 26 cosmid markers (cCI 3 series) previously localized within this region by fluorescence in situ hybridization (FISH). Two-color pairwise FISH analysis of prophase chromosomes provided the order of these markers as follows: pter – 245 (D3S647) – 872 (D3S1018) – 818 (D3S996) – 905 (D3S1022) -515 (D3S685) – 1195 (D3S1125) – 718 (D3S935) – 911 (D3S1025) – 878 (D3S1020) – 717 (D3S934) – 401 (D3S664) -[708 (D3S926)/524 (D3S686)] – 848 (D3S1011) – 771 (D3S966) – 917 (D3S1029) – 533 (D3S688) – 470 (D3S676) 940 (D3S1037) – 785 (D3S974) – 810 (D3S988) – 9 (D3S643) -382 (D3S660) – 769 (D3S965) – 792 (D3S978) – 604 (D3S705) – cen. The two-color signals of 524 (D3S686) and 708 (D3S926) were visualized as an overlapping pattern on prophase chromosomes, and, further, the string signals also overlapped on stretched DNAs, allowing us to determine their precise order as pter – D3S926 – D3S686 – cen. The precise order of 26 DNA markers on 3p23→p21.3 can provide useful information for the positional cloning of tumor suppressor gene(s) and cancer breakpoint(s) encompassed in this region.

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