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Screening for polymorphisms in peas using chemiluminescent detection

Bolitho, K. M.
Fields of Research
ANZSRC::0604 Genetics , ANZSRC::070305 Crop and Pasture Improvement (Selection and Breeding)
A library constructed of Eco RI random (0.5 to 2 kb) pea DNA fragments derived from the breeding line OSU 442-15 and cloned into a Bluescript vector was constructed to produce non-radioactive probes to be used for the screening of RFLPs (restriction length fragment polymorphisms) by chemiluminescent detection. The label DIG (digoxigenin) was incorporated into probe DNA as the inserts of selected recombinant plasmids were amplified by peR (polymerase chain reaction). The efficiency of this operation was evaluated according to fragment size by electrophoresis and DIG concentration using dot blot analysis. Selected probes were then hybridized to Southern blots of genomic DNA from both OSU 442-15 and the cultivar Primo, each separately digested by one of eight restriction endonucleases. DNA specific binding was than detected by the emission of luminescence as a chemiluminescent substrate was dephosphorylated by alkaline phosphatase attached to the DIG label via an anti-DIG antibody. Although no polymorphisms were detected, distinct bands were revealed suggesting that the nonradioactive method was successful.
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