Efficient Cloning of DAF Polymorphic Markers from Silver-Stained Polyacrylamide Gels

DNA amplification fingerprinting (DAF) (1) gives about 40–60 bands per polyacrylamide gel lane, which makes the polymerase chain reaction (PCR) pattern rather complex for band isolation from a gel. The presence of minor amplified bands having only a few base pairs difference from major bands interferes with the product cloning because of co-isolation (3). This fact becomes obvious if a multiply re-amplified band is to be cloned. For cloning of a soybean polymorphic DAF marker (Figure 1A), we used the standard cloning procedure, in which the candidate DAF band is re-amplified 3–5 times to enrich the amount of PCR product (6). Instead of the expected unique 450-bp marker, we repeatedly obtained a set of cloned inserts with sizes varying from 423 to 456 bp (Figure 1B). DNA sequencing of six candidate clones showed that only two had the same sequence. These two clones were chosen for Southern hybridization but did not reveal the initial polymorphism (data not shown). Use of a whole DAF PCR mixture as a probe for hybridization with immobilized candidate clones (3) does not solve the problem if non-polymorphic products with homology to the desired DAF marker band are presented in the mixture. One can expect this if a repeated DNA was