8055 - Laserfiche WebLink

Home Browse Search

NEST-SPECIRC BASS DNA FINGERPRINTS 451 Jones Bay Lake Opeongo, Ontario . 1 2 , ~ t N O.5km FIGURE I. - Map of Lake Opeongo with the shoreline of Jones Bay highlighted to show the location of nests (e) from which fry were collected for DNA fingerprinting, Circles denote nests spaced closely together in clusters, for whose fry fingerprints were run on the same gel. ted in a solution of 200 mM Na2HP04 (pH 7,2) and then prehybridized for I h in a solution of 500 mM Na2HP04 (pH 7,2) and 10% SDS, The mem- branes were hybridized overnight in a solution con- taining 200 mM Na2HP04 (pH 7,2), 1% SDS, 1% bovine serum albumin (fraction V), 6% polyethylene glycol 8000, and 32P-labeled probe (Amersham pro- tocol), Probes and hybridization temperatures tested with each enzyme listed above were human mini- satellites 33,15 and 33,6 (Zeneca Ltd,) at 62"C; oli- gonucleotides (CACh and (GACGCrGGAGGT- TCf)4 at 37"C; and (GACA)4, (GATAh, and (GGA T)4 at 42"C, Excess probe was removed by washing the membranes twice for 20 min at their hybridization temperatures in 200 mM Na2HP04 (pH 7,2) and 0,1 % SDS. Membranes were exposed for approximately 3 d to Kodak XAR-5 film at -7(J>C with intensifying screens, Fingerprint scheme and evaluation,-A test gel containing DNA from several smallmouth bass was run for each of the six restriction enzymes, Test gel membranes for each enzyme were hy- bridized with the different probes to compare the fingerprint patterns that were generated, The com- bination of enzyme and probe that produced the best overall banding pattern for an individual, as well as having a high level of polymorphisms be- tween individuals, was selected to test for nest- specific fingerprints. Fish from 15 randomly distributed nests were fingerprinted with the same enzyme-probe com- bination (Figure I), Nest fingerprint gels consisted