Screening for polymorphisms in peas using chemiluminescent detection
Authors
Date
1993
Type
Dissertation
Abstract
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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