BACKGROUND
single nucleotide polymorphisms  play a significant role in the genetic basis for many acquired and inherited genetic disorders.
this has driven the development of protocols to characterise snps, to both detect novel polymorphisms and discriminate between allelic states, including snps implicated in disease.
a number of these approaches employ restriction endonucleases to discriminate between sequences.
although naturally occurring restriction endonucleases are highly efficient at cleaving dsdna at specific locations and have been used successfully to discriminate polymorphisms such as restriction fragment length polymorphisms , many snps are not associated with a natural restriction site.
for other applications such as cloning, restriction endonucleases are used to great effect, however, a restriction site may not be conveniently located, or, too many restriction sites may exist within a defined sequence.
to overcome these limitations, a number of efforts have been made to develop endonucleases that do not recognise a single specific dna motif, but are sequence-adaptable.
the first generation of these sequence-adaptable endonucleases involved the use of the dna strand-invading peptide nucleic acids  with the ssdna-specific s <dig> nuclease  <cit> , a combination which has since been adapted in colorimetric  <cit>  and fluorometric  <cit>  assays for snps detection and cloning  <cit> , including the isolation of large dna fragments from entire e. coli genomic templates  <cit> .
the cleavage mechanism typically employs two pnas to mediate the induction of sequence-specific stretches of ssdna in a dsdna template that can form a substrate for a ssdna-specific endonuclease.
the orientation of the two strand-invading pnas relative to each other determines the location and size of the ssdna region, with the majority of reports employing two trans-orientated pnas that partially overlap and therefore form single-stranded regions on both strands of dna.
this strategy is suitable for a range of restriction-based applications where complete cleavage of the sample is desired.
however, when this approach is applied as a means to discriminate between two sequences that differ by a snp, the ability to distinguish relies solely on the pna binding to one sequence variant but not the other , rather than specific recognition of the snp itself.
this approach therefore becomes problematic when non-specific binding occurs either at the site of interest in the non-targeted sequence or at any other nonspecific site, as the binding event produces nuclease susceptible ssdna regions regardless of the sequence that it binds to.
consequently, to improve the specificity of this approach, the discrimination mechanism needs to rely not on the presence or absence of the dna binding molecule, but rather the presence or absence of the snp itself.
in this investigation, the development of a novel adaptation of an s <dig> nuclease-mediated cleavage mechanism is described whereby dna cleavage is not mediated by the presence or absence of the binding molecule, but by the induction of small ssdna 'bubbles' surrounding a snp.
in this model, inducible mismatches surrounding the mutation site  generated a mismatch 'bubble' that was exaggerated in the mutant sequence relative to the wild-type sequence, which thereby increased the susceptibility of the mutant sequence to dna strand cleavage by the ssdna-specific s <dig> nuclease.
increasing the 'bubble' size resulted in an increase in the cleavage of the mutant sequence relative to the wild-type.
demonstration of this mechanism was made possible by the development of a novel melting curve assay to empirically calculate tm and annealing rates of complimentary and mismatched oligonucleotides.
this in vitro 'cleavage bubble' model can be easily modified to target any site by changing the sequence of the binding oligonucleotide and hence, has the potential to be tailored to discriminate between any two or more snps.
RESULTS
this investigation presents a new in vitro snp discrimination mechanism, demonstrated by discriminating between the wild-type and mutant sequence of the mitochondrial a8344g merrf mutation.
this mutation was chosen as a target as it is one of the most common pathological mtdna heteroplasmic variants affecting mt-trnalys synthesis  <cit>  and that pna sequence discrimination of this snp has been previously demonstrated  <cit> .
the discrimination mechanism presented was based on s <dig> nuclease-mediated cleavage of single stranded 'bubbles' created by induced mismatches between an annealed oligonucleotide and target mtdna sequence.
however, this mechanism differs from previous s <dig> nuclease/pna cleavage models, as cis- rather than trans-orientated regions of single stranded dna were generated and cleaved  and that both wild-type and mutant templates are targeted by the bubble-forming oligonucleotides, rather than exclusively targeting one sequence and not the other, a common approach in many pna-based snp discrimination strategies.
therefore, it is the bubble size difference between the mutant and wild-type sequence, rather that the presence or absence of the binding molecule, that dictates cleavage.
design and generation of nuclease-sensitive 'bubbles'
to assess the efficacy of s <dig> nuclease-mediated sequence discrimination between the wild-type and mutant templates, a series of gradually increasing mismatches were generated to determine both the minimal 'bubble' size that would confer nuclease sensitivity on the mutant template while leaving the wild-type template uncleaved, and, the maximum size of the mismatch 'bubble' that would render both templates sensitive to the nuclease treatment.
furthermore, since any mismatch configurations that existed within the minimum and maximum defined 'bubbles' would create different sized regions of ssdna, this analysis would provide a means to observe variation in the cleavage rate due to differential substrate recognition by the nuclease.
to create oligonucleotides that bind both templates but induce a differential cleavage capacity between wild-type and mutant sequences, mismatches were created in the immediate adjacent nucleotides surrounding the mutation site of the binding oligonucleotide .
as the two template sequences  were identical except for the mutation site , all oligonucleotides were complimentary to the wild-type sequence at the mutation site but mismatched relative to the mutant template .
for all mismatched oligonucleotide combinations, the mismatch bubbles are larger by one base pair when the oligonucleotides anneal to the mutant sequence, as compared to wild-type sequence, and hence the mutant sequence is rendered more susceptible to cleavage by s <dig> nuclease than the wild-type sequence.
an oligonucleotide designated ' <dig> × 1' produced a single base mismatch when bound to the wild-type sequence, but produced a two base mismatch when bound to the mutant template .
similarly, the oligonucleotide ' <dig> × 1' produced a single base mismatch on either side of the mutation site in the wild-type, but produced a three base mismatch  when bound to the mutant template.
finally, the ' <dig> × 2' oligonucleotide produced a two base mismatch on either side of the mutation site in the wild-type sequence, but produced a five base mismatch  in the mutant template.
this therefore produced a series of mismatch 'bubbles' that spanned  <dig>   <dig>   <dig>   <dig>   <dig>  and 5-bp which could be individually analysed for their sensitivity to s <dig> nuclease.
a wild-type and mutant template sequences differ by a single nucleotide change, indicated in bold.
note in the  <dig> ×  <dig>   <dig> ×  <dig> and  <dig> ×  <dig> sequences, the nucleotide at the mutation site is complementary to that of the wild-type sequence.
b induced nucleotide mismatches to generate mismatch "bubbles" in  <dig> ×  <dig>   <dig> ×  <dig>  and  <dig> ×  <dig> oligonucleotides that differ from the template sequence are indicated by being underlined.
differential cleavage capacity produced by induced mismatches demonstrates selective degradation of the mutant template
an initial s <dig> nuclease cleavage assay to determine the effects of 'bubble' size on cleavage sensitivity demonstrated that differential cleavage between the wild-type and mutant templates was observed for some, but not all, annealed oligonucleotides .
both self- and  <dig> ×  <dig> complementary oligonucleotides showed no significant difference between wild-type and mutant sequences, and both the 1-bp  and 2-bp mismatch  generated by the  <dig> ×  <dig> oligonucleotide remained resistant to the nuclease .
however, the  <dig> ×  <dig> oligonucleotide induced a significant difference in cleavage between wild-type and mutant templates , while maintaining no significant difference between the wild-type/ <dig> ×  <dig> and the wild-type complementary bound templates .
the  <dig> ×  <dig> bound oligonucleotide produced a greater difference in nuclease sensitivity between the wild-type and mutant template sequences  and almost complete degradation of mutant template was achieved, however, the wild-type/ <dig> ×  <dig> was also sensitive to the nuclease .
considering one of the primary aims was to minimise wild-type sequence degradation, and that wild-type/ <dig> ×  <dig> was sensitive to nuclease digestion, the  <dig> ×  <dig> oligonucleotide was discontinued throughout the rest of the study.
theoretically, the wild-type/ <dig> ×  <dig> complex should consist of two mismatched base pairs followed by a matched base pair and then two more mismatches.
given that the dissociation and annealing of dsdna is a cooperative process, it is likely that the annealed middle position of the wild-type/ <dig> ×  <dig> complex is destabilised by its mismatched neighbours and is, therefore, as susceptible to s <dig> nuclease digestion as the mutant/ <dig> ×  <dig> complex.
considering that the  <dig> ×  <dig> oligonucleotide showed a promising degree of selectivity between wild-type and mutant sequences, it was analysed further over 5-hours in the presence of s <dig> nuclease .
wild-type/wild-type, and wild-type/mutant templates were also included in this assay, so that stepwise  <dig> ,  <dig> ,  <dig>  and  <dig>  base mismatches could be analysed simultaneously.
although an increase in 'bubble' size resulted in a stepwise increase in nuclease sensitivity at each time point, the rate of degradation over time was virtually identical for each 'bubble' size, suggesting that the bubble size difference was not influencing the degradation rate as was initially predicted.
furthermore, when the incubation time was extended, both wild-type and mutant sequences were equally sensitive to nuclease, and hence the sequence specificity of the nuclease degradation was lost relative to the 90-minute incubation .
novel melting curve assay facilitates determination of tm and optimal annealing conditions in the presence of increasing mismatched bases
a number of factors, such as dna concentration, salt concentration, and sequence composition will affect the melting and reassociation kinetics of double-stranded dna.
these characteristics have been used to develop a range of algorithms to estimate the melting temperature  of perfectly complimentary oligonucleotides ; however, there are few algorithms that describe the change of tm generated by the presence of a single mismatch, let alone multiple mismatches.
furthermore, these algorithms often predict quite different tm's for a single oligonucleotide pair and thus a calculated tm is at best an estimate of unknown accuracy.
therefore, a plausible hypothesis for the s <dig> nuclease degradation of perfectly matched oligonucleotides  may be imperfect annealing due to uncertainty concerning the real tm.
to explore this hypothesis, a novel fluorescence-based high resolution melting curve protocol was developed to optimise the annealing conditions empirically by careful measurement of observed tm's over a range of conditions for the mismatched oligonucleotide sets.
this protocol, modified from a conventional melting curve assay, therefore allowed us to determine the optimal annealing temperature  for each oligonucleotide set.
the dsdna-specific dye, lc green plus, which has been optimised for high resolution melting curve analysis, was used to monitor the disassociation of annealed complexes as the dsdna is melted into ssdna.
the melting temperature  of each oligonucleotide mismatch set was determined by calculating the average negative first derivative  across the annealing temperature range.
-df/dt describes the rate of change of the logarithmic normalised fluorescence data from which the tm is interpreted as the point at which -df/dt is at its maximum .
a single base pair increase in mismatch 'bubble' size resulted in an approximate 5°c decrease in tm  which was similar to the predicted tm change , of which variation in tm would be due to differences in mismatch base composition and the relative position of the mismatch to the snp  <cit> .
additionally, these values for tm were on average  <dig> °c higher than predicted , which is consistent with an increase in dsdna template stability in the presence of an intercalating fluorescent dye  <cit> .
a predicted tm was determined using an online tm calculator , which initially calculates a tm value as if the mismatch was not present, and then subsequently corrects this initial value by the addition of a destabilisation factor dependant on the identity of the mismatch being either a dg, dc, or neither a dg/dc  <cit> .
b Δtm-Δtm is used as a relative measure of observed annealed complex stability relative to predicted values.
a value of zero indicates there is no deviation between observed and predicted tm values.
annealed templates are considered less-stable as the value becomes more negative, whereas a more positive value indicates greater template stability than predicted.
c values indicate mean temperature change per single base increase/decrease in "bubble" size.
d average Δtm describes the temperature difference as a result of the lc green dye binding to dsdna.
this value is used to correct the annealing temperature.
e average of difference in observed tm and observed annealing temperature, indicative of the decrease away from the tm needed to achieve optimal annealing conditions.
the optimal annealing temperature of each mismatched oligonucleotide combination was determined by measuring the rate of dsdna disassociation relative to the observed tm, i.e., the slope of the gradient formed between tm ± 1°c in the disassociation profile, across a broad temperature range of 20°c to 80°c in 5°c increments .
the annealing rate was determined to be almost the same for both wild-type and mutant complimentary template sequences and demonstrated a decrease in annealing rate as the mismatch 'bubble' size increased.
the annealing rate was also decreased for the mutant template when compared with the wild-type for both  <dig> ×  <dig> and  <dig> ×  <dig> oligonucleotides, indicative of the lower stability of the mutant bound sequence.
therefore, it is clear that the number and type of mismatch affects the reassociation kinetics of oligonucleotide binding and that an increase in base-pair mismatch reduces its ability to hybridise.
however, although each mismatch bubble size demonstrated different annealing rates, each oligonucleotide set produced a similar annealing curve, such that an optimal peak height for a given oligonucleotide set was observed at approximately tm minus  <dig> °c ±  <dig>  sd .
above this temperature, the annealing rate demonstrated a sharp decrease as the annealing temperature approached the tm, whereas, below this temperature, a comparatively modest decrease in annealing was observed.
therefore, a corrected ta could be calculated for each oligonucleotide annealing set, which was determined by subtracting the dye-induced temperature increase from the optimal annealing temperature ]).
the relative steepness of the gradient used to calculate the optimised annealing temperature is indicative of the thermal stability of the annealed template  <cit> : as demonstrated in figure  <dig>  there is an apparent 'trade-off' as the annealing temperature moves away from the optimal tm.
a steeper gradient implies greater stability due to more accurate hybridisation, whereas a shallower gradient implies less stable binding, whether it be due to the incorporation of 'forced' mismatches due to fast annealing as the temperature decreases away from the tm, or, due to less annealed dsdna product formed during slower annealing conditions as the temperature increases above the tm.
the data presented were generated using a broad range of temperatures with increments of 5°c.
the level of resolution achieved was suitable for the purpose of this investigation and permitted a relatively simple one-step determination of tm.
however, a more finely resolved estimation of tm and annealing parameters might be necessary to discriminate between more subtle nucleotide changes, such as a/t class iv snps, which could be achieved by decreasing the temperature interval and thus permitting the use of smaller temperature increments between data points.
optimised annealing conditions enhances stability of nuclease resistant templates and enables efficient nuclease degradation of targeted mutant sequences
utilising the empirically determined ta from the fluorescence assay and data presented in figure  <dig> and table  <dig>  each oligonucleotide combination was annealed and subjected again to nuclease treatment for up to  <dig> hours .
no significant degradation of complimentary and single base mismatch  sets for wild-type and mutant oligonucleotides was observed over  <dig> hours.
some minimal digestion of wild-type/ <dig> ×  <dig> mismatch was observed at  <dig> and  <dig> hours.
strikingly however, both mutant/ <dig> ×  <dig> and mutant/ <dig> ×  <dig> annealed templates were significantly digested over the time course in a stepwise manner: the  <dig> ×  <dig> oligonucleotide was approximately 80% digested after  <dig> hours by s <dig> nuclease, whereas the  <dig> ×  <dig> annealed template was approximately 90% digested after  <dig> hours and was not detectable after  <dig> hours.
note that under these conditions, the perfectly annealed  combinations were not detectably digested.
a single-base mismatch that was present in both the mixed complimentary and wild-type/ <dig> ×  <dig> complexes was not recognised by s <dig> nuclease under these conditions, but two consecutive base mismatches were recognised as a ssdna substrate by s <dig> nuclease as seen in the mutant/ <dig> ×  <dig> and mutant/ <dig> ×  <dig> templates.
note that two non-consecutive mismatches, as in the wild-type/ <dig> ×  <dig> complex, were not sufficient for s <dig> nuclease cleavage.
the requirement for at least two consecutive mismatches for s <dig> nuclease cleavage contrasts with several reports of successful cleavage with a single mismatch  <cit> .
however, the observation here may be attributed to the significantly lower enzyme concentration used in this study , together with the careful matching of annealing temperature to each oligonucleotide combination allowing for a more stable duplex even in the presence of mismatches.
the dramatic improvement in both the stability of the perfectly annealed templates over a significantly greater time frame  and the stepwise increase in degradation rate across the mutant/ <dig> ×  <dig> and mutant/ <dig> ×  <dig> template oligonucleotide combinations highlight the significant effect that the number and type of mismatches have on duplex stability, and emphasise the importance of empirical optimisation of the annealing conditions in hybridisation experiments.
furthermore, these data demonstrate the effectiveness of the novel fluorometric melting curve assay described here.
CONCLUSIONS
the work presented in this investigation demonstrates a new set of conditions under which s <dig> nuclease can be rendered sequence specific .
as well as meeting the requirement for discrimination of a single base point mutation, the method described provides a simple mechanism to vary the rate of cleavage dependent on the orientation of the mismatch relative to the mutation site and requires only transient binding of the oligonucleotide to the template while the s <dig> nuclease is present to cleave the substrates.
the generation of s <dig> nuclease sensitive mismatch 'bubbles' is likely to have a broader application than mtdna haplotype discrimination alone, including alternative snp detection strategies and other manipulations that require sequence specific cleavage of dna, such as the isolation of specific genomic sequences for cloning in the absence of unique restriction sites.
the fluorometric assay presented was able to both rapidly and simultaneously determine tm and annealing conditions for complimentary and mismatched oligonucleotides.
of broader interest, this new fluorescence assay can be used independently of the s <dig> nuclease assay, providing an efficient hybridisation analysis protocol  that may supersede traditional assays, such as filter-hybridisation-  <cit>  and sequencing-based radiolabelling  <cit> , and temperature-controlled spectrophotometry  <cit> ; all of which are labour intensive and require specialised equipment.
furthermore, this methodology has the potential to be routinely used to determine optimum tm and annealing conditions for complimentary and mismatched oligonucleotides in a range of applications that rely on accurate hybridisation, such as oligonucleotide-  and probe-based assays , or, protocols that assess the level of degenerate hybridisation, such as snp detection/screening or evolutionary divergence in comparative taxonomical studies.

 single nucleotide polymorphisms play a significant role in the genetic basis for many acquired and inherited genetic disorders. this has driven the development of protocols to characterise snps, to both detect novel polymorphisms and discriminate between allelic states, including snps implicated in disease. a number of these approaches employ restriction endonucleases to discriminate between sequences. although naturally occurring restriction endonucleases are highly efficient at cleaving dsdna at specific locations and have been used successfully to discriminate polymorphisms such as restriction fragment length polymorphisms, many snps are not associated with a natural restriction site. for other applications such as cloning, restriction endonucleases are used to great effect, however, a restriction site may not be conveniently located, or, too many restriction sites may exist within a defined sequence. to overcome these limitations, a number of efforts have been made to develop endonucleases that do not recognise a single specific dna motif, but are sequence-adaptable. the first generation of these sequence-adaptable endonucleases involved the use of the dna strand-invading peptide nucleic acids with the ssdna-specific s nuclease, a combination which has since been adapted in colorimetric and fluorometric assays for snps detection and cloning, including the isolation of large dna fragments from entire e. coli genomic templates. the cleavage mechanism typically employs two pnas to mediate the induction of sequence-specific stretches of ssdna in a dsdna template that can form a substrate for a ssdna-specific endonuclease. the orientation of the two strand-invading pnas relative to each other determines the location and size of the ssdna region, with the majority of reports employing two trans-orientated pnas that partially overlap and therefore form single-stranded regions on both strands of dna. this strategy is suitable for a range of restriction-based applications where complete cleavage of the sample is desired. however, when this approach is applied as a means to discriminate between two sequences that differ by a snp, the ability to distinguish relies solely on the pna binding to one sequence variant but not the other, rather than specific recognition of the snp itself. this approach therefore becomes problematic when non-specific binding occurs either at the site of interest in the non-targeted sequence or at any other nonspecific site, as the binding event produces nuclease susceptible ssdna regions regardless of the sequence that it binds to. consequently, to improve the specificity of this approach, the discrimination mechanism needs to rely not on the presence or absence of the dna binding molecule, but rather the presence or absence of the snp itself. in this investigation, the development of a novel adaptation of an s nuclease-mediated cleavage mechanism is described whereby dna cleavage is not mediated by the presence or absence of the binding molecule, but by the induction of small ssdna'bubbles' surrounding a snp. in this model, inducible mismatches surrounding the mutation site generated a mismatch'bubble' that was exaggerated in the mutant sequence relative to the wild-type sequence, which thereby increased the susceptibility of the mutant sequence to dna strand cleavage by the ssdna-specific s nuclease. increasing the'bubble' size resulted in an increase in the cleavage of the mutant sequence relative to the wild-type. demonstration of this mechanism was made possible by the development of a novel melting curve assay to empirically calculate tm and annealing rates of complimentary and mismatched oligonucleotides. this in vitro'cleavage bubble' model can be easily modified to target any site by changing the sequence of the binding oligonucleotide and hence, has the potential to be tailored to discriminate between any two or more snps.  this investigation presents a new in vitro snp discrimination mechanism, demonstrated by discriminating between the wild-type and mutant sequence of the mitochondrial a8344g merrf mutation. this mutation was chosen as a target as it is one of the most common pathological mtdna heteroplasmic variants affecting mt-trnalys synthesis and that pna sequence discrimination of this snp has been previously demonstrated. the discrimination mechanism presented was based on s nuclease-mediated cleavage of single stranded'bubbles' created by induced mismatches between an annealed oligonucleotide and target mtdna sequence. however, this mechanism differs from previous s nuclease/pna cleavage models, as cis- rather than trans-orientated regions of single stranded dna were generated and cleaved and that both wild-type and mutant templates are targeted by the bubble-forming oligonucleotides, rather than exclusively targeting one sequence and not the other, a common approach in many pna-based snp discrimination strategies. therefore, it is the bubble size difference between the mutant and wild-type sequence, rather that the presence or absence of the binding molecule, that dictates cleavage. design and generation of nuclease-sensitive'bubbles' to assess the efficacy of s nuclease-mediated sequence discrimination between the wild-type and mutant templates, a series of gradually increasing mismatches were generated to determine both the minimal'bubble' size that would confer nuclease sensitivity on the mutant template while leaving the wild-type template uncleaved, and, the maximum size of the mismatch'bubble' that would render both templates sensitive to the nuclease treatment. furthermore, since any mismatch configurations that existed within the minimum and maximum defined'bubbles' would create different sized regions of ssdna, this analysis would provide a means to observe variation in the cleavage rate due to differential substrate recognition by the nuclease. to create oligonucleotides that bind both templates but induce a differential cleavage capacity between wild-type and mutant sequences, mismatches were created in the immediate adjacent nucleotides surrounding the mutation site of the binding oligonucleotide. as the two template sequences were identical except for the mutation site, all oligonucleotides were complimentary to the wild-type sequence at the mutation site but mismatched relative to the mutant template. for all mismatched oligonucleotide combinations, the mismatch bubbles are larger by one base pair when the oligonucleotides anneal to the mutant sequence, as compared to wild-type sequence, and hence the mutant sequence is rendered more susceptible to cleavage by s nuclease than the wild-type sequence. an oligonucleotide designated' × 1' produced a single base mismatch when bound to the wild-type sequence, but produced a two base mismatch when bound to the mutant template. similarly, the oligonucleotide' × 1' produced a single base mismatch on either side of the mutation site in the wild-type, but produced a three base mismatch when bound to the mutant template. finally, the' × 2' oligonucleotide produced a two base mismatch on either side of the mutation site in the wild-type sequence, but produced a five base mismatch in the mutant template. this therefore produced a series of mismatch'bubbles' that spanned and 5-bp which could be individually analysed for their sensitivity to s nuclease. a wild-type and mutant template sequences differ by a single nucleotide change, indicated in bold. note in the × × and × sequences, the nucleotide at the mutation site is complementary to that of the wild-type sequence. b induced nucleotide mismatches to generate mismatch "bubbles" in × × and × oligonucleotides that differ from the template sequence are indicated by being underlined. differential cleavage capacity produced by induced mismatches demonstrates selective degradation of the mutant template an initial s nuclease cleavage assay to determine the effects of'bubble' size on cleavage sensitivity demonstrated that differential cleavage between the wild-type and mutant templates was observed for some, but not all, annealed oligonucleotides. both self- and × complementary oligonucleotides showed no significant difference between wild-type and mutant sequences, and both the 1-bp and 2-bp mismatch generated by the × oligonucleotide remained resistant to the nuclease. however, the × oligonucleotide induced a significant difference in cleavage between wild-type and mutant templates, while maintaining no significant difference between the wild-type/ × and the wild-type complementary bound templates. the × bound oligonucleotide produced a greater difference in nuclease sensitivity between the wild-type and mutant template sequences and almost complete degradation of mutant template was achieved, however, the wild-type/ × was also sensitive to the nuclease. considering one of the primary aims was to minimise wild-type sequence degradation, and that wild-type/ × was sensitive to nuclease digestion, the × oligonucleotide was discontinued throughout the rest of the study. theoretically, the wild-type/ × complex should consist of two mismatched base pairs followed by a matched base pair and then two more mismatches. given that the dissociation and annealing of dsdna is a cooperative process, it is likely that the annealed middle position of the wild-type/ × complex is destabilised by its mismatched neighbours and is, therefore, as susceptible to s nuclease digestion as the mutant/ × complex. considering that the × oligonucleotide showed a promising degree of selectivity between wild-type and mutant sequences, it was analysed further over 5-hours in the presence of s nuclease. wild-type/wild-type, and wild-type/mutant templates were also included in this assay, so that stepwise,, and base mismatches could be analysed simultaneously. although an increase in'bubble' size resulted in a stepwise increase in nuclease sensitivity at each time point, the rate of degradation over time was virtually identical for each'bubble' size, suggesting that the bubble size difference was not influencing the degradation rate as was initially predicted. furthermore, when the incubation time was extended, both wild-type and mutant sequences were equally sensitive to nuclease, and hence the sequence specificity of the nuclease degradation was lost relative to the 90-minute incubation. novel melting curve assay facilitates determination of tm and optimal annealing conditions in the presence of increasing mismatched bases a number of factors, such as dna concentration, salt concentration, and sequence composition will affect the melting and reassociation kinetics of double-stranded dna. these characteristics have been used to develop a range of algorithms to estimate the melting temperature of perfectly complimentary oligonucleotides; however, there are few algorithms that describe the change of tm generated by the presence of a single mismatch, let alone multiple mismatches. furthermore, these algorithms often predict quite different tm's for a single oligonucleotide pair and thus a calculated tm is at best an estimate of unknown accuracy. therefore, a plausible hypothesis for the s nuclease degradation of perfectly matched oligonucleotides may be imperfect annealing due to uncertainty concerning the real tm. to explore this hypothesis, a novel fluorescence-based high resolution melting curve protocol was developed to optimise the annealing conditions empirically by careful measurement of observed tm's over a range of conditions for the mismatched oligonucleotide sets. this protocol, modified from a conventional melting curve assay, therefore allowed us to determine the optimal annealing temperature for each oligonucleotide set. the dsdna-specific dye, lc green plus, which has been optimised for high resolution melting curve analysis, was used to monitor the disassociation of annealed complexes as the dsdna is melted into ssdna. the melting temperature of each oligonucleotide mismatch set was determined by calculating the average negative first derivative across the annealing temperature range. -df/dt describes the rate of change of the logarithmic normalised fluorescence data from which the tm is interpreted as the point at which -df/dt is at its maximum. a single base pair increase in mismatch'bubble' size resulted in an approximate 5°c decrease in tm which was similar to the predicted tm change, of which variation in tm would be due to differences in mismatch base composition and the relative position of the mismatch to the snp. additionally, these values for tm were on average °c higher than predicted, which is consistent with an increase in dsdna template stability in the presence of an intercalating fluorescent dye. a predicted tm was determined using an online tm calculator, which initially calculates a tm value as if the mismatch was not present, and then subsequently corrects this initial value by the addition of a destabilisation factor dependant on the identity of the mismatch being either a dg, dc, or neither a dg/dc. b Δtm-Δtm is used as a relative measure of observed annealed complex stability relative to predicted values. a value of zero indicates there is no deviation between observed and predicted tm values. annealed templates are considered less-stable as the value becomes more negative, whereas a more positive value indicates greater template stability than predicted. c values indicate mean temperature change per single base increase/decrease in "bubble" size. d average Δtm describes the temperature difference as a result of the lc green dye binding to dsdna. this value is used to correct the annealing temperature. e average of difference in observed tm and observed annealing temperature, indicative of the decrease away from the tm needed to achieve optimal annealing conditions. the optimal annealing temperature of each mismatched oligonucleotide combination was determined by measuring the rate of dsdna disassociation relative to the observed tm, i.e., the slope of the gradient formed between tm ± 1°c in the disassociation profile, across a broad temperature range of 20°c to 80°c in 5°c increments. the annealing rate was determined to be almost the same for both wild-type and mutant complimentary template sequences and demonstrated a decrease in annealing rate as the mismatch'bubble' size increased. the annealing rate was also decreased for the mutant template when compared with the wild-type for both × and × oligonucleotides, indicative of the lower stability of the mutant bound sequence. therefore, it is clear that the number and type of mismatch affects the reassociation kinetics of oligonucleotide binding and that an increase in base-pair mismatch reduces its ability to hybridise. however, although each mismatch bubble size demonstrated different annealing rates, each oligonucleotide set produced a similar annealing curve, such that an optimal peak height for a given oligonucleotide set was observed at approximately tm minus °c ± sd. above this temperature, the annealing rate demonstrated a sharp decrease as the annealing temperature approached the tm, whereas, below this temperature, a comparatively modest decrease in annealing was observed. therefore, a corrected ta could be calculated for each oligonucleotide annealing set, which was determined by subtracting the dye-induced temperature increase from the optimal annealing temperature ]). the relative steepness of the gradient used to calculate the optimised annealing temperature is indicative of the thermal stability of the annealed template: as demonstrated in figure there is an apparent'trade-off' as the annealing temperature moves away from the optimal tm. a steeper gradient implies greater stability due to more accurate hybridisation, whereas a shallower gradient implies less stable binding, whether it be due to the incorporation of'forced' mismatches due to fast annealing as the temperature decreases away from the tm, or, due to less annealed dsdna product formed during slower annealing conditions as the temperature increases above the tm. the data presented were generated using a broad range of temperatures with increments of 5°c. the level of resolution achieved was suitable for the purpose of this investigation and permitted a relatively simple one-step determination of tm. however, a more finely resolved estimation of tm and annealing parameters might be necessary to discriminate between more subtle nucleotide changes, such as a/t class iv snps, which could be achieved by decreasing the temperature interval and thus permitting the use of smaller temperature increments between data points. optimised annealing conditions enhances stability of nuclease resistant templates and enables efficient nuclease degradation of targeted mutant sequences utilising the empirically determined ta from the fluorescence assay and data presented in figure and table each oligonucleotide combination was annealed and subjected again to nuclease treatment for up to hours. no significant degradation of complimentary and single base mismatch sets for wild-type and mutant oligonucleotides was observed over hours. some minimal digestion of wild-type/ × mismatch was observed at and hours. strikingly however, both mutant/ × and mutant/ × annealed templates were significantly digested over the time course in a stepwise manner: the × oligonucleotide was approximately 80% digested after hours by s nuclease, whereas the × annealed template was approximately 90% digested after hours and was not detectable after hours. note that under these conditions, the perfectly annealed combinations were not detectably digested. a single-base mismatch that was present in both the mixed complimentary and wild-type/ × complexes was not recognised by s nuclease under these conditions, but two consecutive base mismatches were recognised as a ssdna substrate by s nuclease as seen in the mutant/ × and mutant/ × templates. note that two non-consecutive mismatches, as in the wild-type/ × complex, were not sufficient for s nuclease cleavage. the requirement for at least two consecutive mismatches for s nuclease cleavage contrasts with several reports of successful cleavage with a single mismatch. however, the observation here may be attributed to the significantly lower enzyme concentration used in this study, together with the careful matching of annealing temperature to each oligonucleotide combination allowing for a more stable duplex even in the presence of mismatches. the dramatic improvement in both the stability of the perfectly annealed templates over a significantly greater time frame and the stepwise increase in degradation rate across the mutant/ × and mutant/ × template oligonucleotide combinations highlight the significant effect that the number and type of mismatches have on duplex stability, and emphasise the importance of empirical optimisation of the annealing conditions in hybridisation experiments. furthermore, these data demonstrate the effectiveness of the novel fluorometric melting curve assay described here.  the work presented in this investigation demonstrates a new set of conditions under which s nuclease can be rendered sequence specific. as well as meeting the requirement for discrimination of a single base point mutation, the method described provides a simple mechanism to vary the rate of cleavage dependent on the orientation of the mismatch relative to the mutation site and requires only transient binding of the oligonucleotide to the template while the s nuclease is present to cleave the substrates. the generation of s nuclease sensitive mismatch'bubbles' is likely to have a broader application than mtdna haplotype discrimination alone, including alternative snp detection strategies and other manipulations that require sequence specific cleavage of dna, such as the isolation of specific genomic sequences for cloning in the absence of unique restriction sites. the fluorometric assay presented was able to both rapidly and simultaneously determine tm and annealing conditions for complimentary and mismatched oligonucleotides. of broader interest, this new fluorescence assay can be used independently of the s nuclease assay, providing an efficient hybridisation analysis protocol that may supersede traditional assays, such as filter-hybridisation- and sequencing-based radiolabelling, and temperature-controlled spectrophotometry; all of which are labour intensive and require specialised equipment. furthermore, this methodology has the potential to be routinely used to determine optimum tm and annealing conditions for complimentary and mismatched oligonucleotides in a range of applications that rely on accurate hybridisation, such as oligonucleotide- and probe-based assays, or, protocols that assess the level of degenerate hybridisation, such as snp detection/screening or evolutionary divergence in comparative taxonomical studies. 
in this model, generation of oligonucleotide-mediated ssdna mismatch'bubbles' in the presence of s nuclease resulted in the selective degradation of the mutant template while maintaining wild-type template integrity. many snp discrimination strategies employ natural restriction endonucleases to discriminate between allelic states. however, snps are often not associated with a restriction site and therefore, a number of attempts have been made to generate sequence-adaptable restriction endonucleases. furthermore, the demonstrated fluorometric assay has broad application potential, offering a rapid, sensitive and high-throughput means to determine tm and annealing rates as an alternative to conventional hybridisation detection strategies. a series of mismatch'bubbles' that spanned 0-5-bp surrounding a point mutation was generated and analysed for sensitivity to s nuclease. the in vitro'cleavage bubble' model presented is sequence-adaptable as determined by the binding oligonucleotide, and hence, has the potential to be tailored to discriminate between any two or more snps. increasing the size of the mismatch increased the rate of mutant sequence degradation, until a threshold above which discrimination was lost and the wild-type sequence was degraded. this level of fine discrimination was possible due to the development of a novel high-resolution melting curve assay to empirically determine changes in tm and to optimise annealing conditions of the mismatched oligonucleotide sets.
 many snp discrimination strategies employ natural restriction endonucleases to discriminate between allelic states. however, snps are often not associated with a restriction site and therefore, a number of attempts have been made to generate sequence-adaptable restriction endonucleases. in this study, a simple, sequence-adaptable snp discrimination mechanism between a'wild-type' and'mutant' template is demonstrated. this model differs from other artificial restriction endonuclease models as cis- rather than trans-orientated regions of single stranded dna were generated and cleaved, and therefore, overcomes potential issues of either inefficient or non-specific binding when only a single variant is targeted.  a series of mismatch'bubbles' that spanned 0-5-bp surrounding a point mutation was generated and analysed for sensitivity to s nuclease. in this model, generation of oligonucleotide-mediated ssdna mismatch'bubbles' in the presence of s nuclease resulted in the selective degradation of the mutant template while maintaining wild-type template integrity. increasing the size of the mismatch increased the rate of mutant sequence degradation, until a threshold above which discrimination was lost and the wild-type sequence was degraded. this level of fine discrimination was possible due to the development of a novel high-resolution melting curve assay to empirically determine changes in tm and to optimise annealing conditions of the mismatched oligonucleotide sets.  the in vitro'cleavage bubble' model presented is sequence-adaptable as determined by the binding oligonucleotide, and hence, has the potential to be tailored to discriminate between any two or more snps. furthermore, the demonstrated fluorometric assay has broad application potential, offering a rapid, sensitive and high-throughput means to determine tm and annealing rates as an alternative to conventional hybridisation detection strategies. 