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Fig. 1. Whole-genome subtraction between ntn mutant embryos and wild-type siblings by RDA. (A) Analysis of RDA products by agarose gel electrophoresis. Driver and tester amplicons were prepared by digestion of genomic DNA from wild-type and ntn mutant embryos using BglII, EcoRI, HindIII, SpeI and XbaI restriction endonucleases, respectively. Lane M, 100 bp DNA ladder as size markers; lane D, driver amplicons; lane T, tester amplicons; lanes 1-4, RDA products of the first, second, third and fourth rounds of subtraction, respectively. (B) Southern blot hybridization analysis of genomic DNA using RDA product E340 as a probe. Genomic DNAs from a wild-type fish of AB strain (lane 1), wild-type siblings (lane 2) and ntn mutant embryos (lane 3) were digested with EcoRI and hybridized to E340 probe. The sizes of markers in kb are indicated on the left. Black and white arrowheads on the right indicate the 3.1 kb and 0.3 kb EcoRI-digested DNA fragments representing polymorphic alleles, respectively. (C) An example of amplicon Southern blot hybridization analysis. Amplicons were prepared from BglII-digested genomic DNA from pools of five wild-type (lane 1) or ntn mutant (lanes 2-10) embryos. Blots were hybridized to B460 (upper) or B470 (lower) probe. Black and white arrowheads indicate fragments in the amplicons hybridizing to B460 and B470 probes, respectively. (D) Amplicon Southern hybridization mapping of the ntn locus using polymorphic RDA products. Amplicons prepared from 93 pools of five ntn mutant embryos were hybridized to respective RDA products. Black and white boxes indicate the presence and absence of amplicons hybridizing to RDA products, respectively. The numbers of pools containing recombinant(s) are given below. (E) Genetic and physical maps of the zebrafish ntn region on the linkage group 16. Numbers in the genetic map (upper) indicate genetic distances in cM between the markers. Physical map around the ntn locus is shown below with markers. Distances in the map are approximately to scale. Short horizontal lines below the physical map indicate YAC, PAC and BAC clones. BAC clones, 82P21, 72A23 and 105K9, were isolated by screening with C18orf1-like cDNAs as probes. B18S is a marker derived from the Sp6 end of BAC 18M9.





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