Non-radioactive detection of single copy sequences in maize
--Dan Maillet, Kelly Jo Bates and Alex Richman

Recent advances in non-radioactive nucleic acid labelling and detection techniques have made it possible to detect single copy sequences on Southern blots of maize genomic DNA. We have adapted the digoxigenin based protocol, provided by Boehringer Mannheim (BM), for the detection of maize RFLPs. This contribution identifies changes or clarifications we have made to the steps in the BM protocols. (The steps identified below correspond to those presented in the BM protocols.)

DNA labelling (see DIG DNA labelling and detection kit protocol). We label (step 4) 200ng of DNA for 20 hours to ensure a high ratio of labelled to non-labelled fragments. The probe is precipitated (step 7) overnight at -70 C.

Southern Transfer. We load 0.8% agarose gels , 150ml, 11 by 14cm with 15-20µg of digested genomic DNA per lane and separate by electrophoresis at 20-40 volts in a model H5 gel tank (BRL). DNA is transferred under neutral conditions, as specified in Molecular Cloning (Sambrook et al., 1989), to BM positively charged nylon membranes and cross linked for 3 min in a UV Stratalinker 1800 (Stratagene).

Hybridization and stringency washes (see Lumigen PPD protocol). Wet blot in 2 X SSC. Prehybridize blots in plastic bag with 20ml of hybridization solution for 2 hours at 42 C with mild agitation. Hybridize (step 2) overnight (approx. 16 hours) with 5 to 6ml of hybridization solution containing 10ng per ml of a denatured DIG labelled probe. The probe is denatured by boiling in a water bath for 10 min and quickly cooled in salt and ice.

Chemiluminescent detection. Incubate (step 5) for 2 hours in 100ml of buffer 2. Wash (step 8) 6 X 5 min with 100ml of washing buffer. Drain substrate solution (step 12) and store at 4 C in dark for reuse (up to 5 times), then reseal the bag (step 13).

Comments. The protocols provided by Boehringer Mannheim resulted in luminographs that could be scored; however, background levels were often unacceptably high. Increased washes after incubation with the antibody was the most important change with respect to lowering background. A longer block in buffer 2 also decreased background. Plastic forceps should be used when handling the membrane. For all steps, use a clean plastic box (21 x 14 x 6cm) unless the volume required is lower than 20ml; in which case a sealed plastic bag may be used. If blots are to be reprobed, they must not dry out at any time. Following stripping, blots can be stored in redistilled sterile water at 4 C.

We have reprobed blots 5 times and have not noticed a decrease in signal strength or an increase in background. Using the Boehringer Mannheim protocols with the modifications outlined in this article we have detected single copy sequences (see Figure 1) in order to determine the strain distribution pattern for a 2kb genomic probe (see the following article). We are interested in correspondence with others using the DIG system or variations of this method.

Figure 1. Southern blot analysis using the DIG detection system reveals the strain distribution pattern for a 2Kb genomic probe in the Co x Tx recombinant inbred family. Standard markers of 23130, 9416, and 6557bp are indicated by arrow heads. 


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