Subtractive hybridization is a valuable, relatively inexpensive molecular tool, but it can require many complex steps to achieve adequate enrichment for target-specific DNA. The use of microplate wells to immobilize subtracter fragments eliminated the need to separate target from subtracter following hybridization. Subtracted target DNA was PCR amplified directly from the supernatant, and one round of hybridization provided sufficient enrichment for target-specific fragments. Sequencing of only 10 clones from each experiment provided enough information for primer design. Had it not done so, additional clones could have been sequenced.
Maxisorp microplates (Nalge Nunc) contain a polystyrene surface that binds DNA noncovalently. Since immobilized DNA is stable for at least 6 to 12 h at 68°C (27
), we expected that the 70°C, 2-h hybridization would occur without significant loss of subtracter DNA to the solution. A large excess of subtracter over target DNA was used to allow for any loss of subtracter due to reannealing.
Three formamide concentrations were chosen based on Na2+
concentration, GC content, and fragment sizes. Formamide is a denaturant and has the effect of decreasing the melting temperature of the DNA hybrid (23
). Overly stringent conditions result in hybridization of perfect matches only, and subtraction products could lack the variability necessary for primer design. If too relaxed, the conditions result in no product at all. Since all three formamide concentrations resulted in similar bands, products of the 35% concentration were chosen for sequencing because this concentration produced a sufficient amount of unhybridized DNA for cloning. Ultimately, the design of primers that distinguish target from subtracter host Bacteroidales
indicated that an appropriate stringency range was attained. Future experiments might include a greater range of stringencies or sequencing of products from more than one stringency for comparison, but because our primer design objectives were met, we did not pursue this further.
No fragments of less than 300 bp were recovered in either of the experiments, for which there are several possible explanations. Some of the restriction fragments ligated to each other, forming chimeric sequences; some small fragments may have been lost in this way. PCR bias may have resulted in preferential amplification of the most-abundant larger restriction fragments. Alternatively, the inverted terminal repeats created by ligation of identical linkers to either end of the fragments may have resulted in the formation of panhandle structures that prevented primer binding and amplification (1
). This would be more kinetically favorable with smaller fragment sizes. Inouye and Hondo found that hybridization efficiency declined as fragment size fell below 227 bp (17
). These observations suggest the use of a restriction enzyme that produces fragments larger than 200 bp.
The intent in using the entire 16S rRNA gene, internal transcribed spacer, and part of the 23S rRNA gene was to obtain as much sequence information as possible for primer design. Three of 20 total subtracted fragments aligned with the 23S rRNA gene, but the small number of database sequences available for comparison made it difficult to use these fragments for primer design. Fourteen of the 20 subtraction products were from the 16S rRNA gene, and all 10 of the elk feces-specific clones were from a region known to be hypervariable (E. coli
positions 940 to 1370) (6
Primer DF475F was the first and only dog-specific primer designed and tested, and it did not require stringent optimization to exclude amplification of other host fecal DNAs. This illustrates the ability of this method to enrich for regions of variability. Primer EF990R was the third elk-specific primer tested and the first to successfully distinguish cow and elk fecal Bacteroidales sequences. It did not distinguish elk and sheep Bacteroidales sequences, which was not the intent of this experiment, but that may be accomplished with another subtractive hybridization. Alternatively, it may be possible to design a primer based on the 5′ and 3′ ends of a target fragment. Any cross-reacting DNA could be sequenced, allowing more directed primer design.
Primer EF990R amplified elk fecal DNAs and did not amplify cow fecal DNAs, even when four times more cow fecal DNA was added. A previous study using blind samples (12
) demonstrated that Bacteroidales
source-specific primers identified fecal sources correctly when the sources comprised as little as 1% of the total fecal contamination in the sample.
Previous studies reported the immobilization of up to seven genomic subtracters in one well (26
). An initial attempt to use seven subtracters with an elk-specific target resulted in subtraction products without enough variability for primer design. Future experiments may determine what limitations exist and what adjustments can be made in the use of multiple subtracters.
Microplate subtractive hybridization was successfully employed to generate a unique source-specific marker for dog fecal pollution and a marker that differentiates elk and cow fecal pollution. The results demonstrate the method's ability to enrich for variable regions of the 16S rRNA gene. In addition, the method could readily be adapted for other gene targets. The capacity to characterize markers that distinguish sources of fecal pollution without obtaining large numbers of clones for each new host will expedite the addition of new source markers for fecal pollution.