BACKGROUND
the role of submicroscopic dna copy number variation in genetic pathologies has been established now for two decades  <cit> .
early investigations recognised the importance of deletions or duplications in specific genes as causative mutations in clinical conditions, such as brca <dig> in familial breast and ovarian cancer  <cit>  and dmd in duchenne/becker muscular dystrophy  <cit> .
furthermore, subtelomeric chromosomal rearrangements leading to copy number changes have been associated with learning disability and other developmental abnormalities  <cit> .
recent advances in the diagnostic technologies applied to such conditions have also proven to be useful tools in elucidating the dynamic model of the human genome, with copy number variation recently recognised as an important component of human polymorphism  <cit> .
as the significance of copy number variation in genetic analysis becomes more widely recognised so does the need to improve and extend the range of techniques available.
in particular, for scanning multi-exon genes for deletions or duplications, maph and mlpa  <cit>  have been used to assess the dosage of small  regions to high accuracy , properties not easily implemented using, for example, current array-cgh methods.
one feature shared by both multiplex amplifiable probe hybridization   <cit>  and multiplex ligation-dependent probe amplification   <cit>  is the limit to multiplicity set by the resolution of probes distinguished by their length, using capillary electrophoresis.
in maph, the requirement for specific and stable hybridization sets a lower limit on probe size of around  <dig> bp, and the requirement for comparable co-amplification of different probes sets an upper limit of around  <dig> bp; thus the largest single probe set could contain  <dig> probes spaced  <dig> bp apart.
while this does not pose a limitation on applications using relatively small probe sets, such as the  <dig> probes used in brca <dig> testing  <cit> , where many exons are involved, such as dmd  <cit> , or where screening is not targeted to single gene loci, probe sets of this magnitude would be inadequate.
the most obvious route to breaking these limits is to employ dna microarray technology to replace capillary gel electrophoresis as the final analytical step in maph, as discussed in the original maph publication  <cit>  and in a more recent review  <cit> .
a microarray based on oligonucleotides complementary to individual maph probes would allow detection and quantification based on sequence rather than on size.
microarray detection and quantification should therefore allow the simultaneous measurement of copy number at a larger number of loci.
the initial stages of microarray maph, up to the recovery of specifically bound probes, remain the same as capillary maph.
the subsequent amplification of these probes is modified to allow the incorporation of labels suitable for a standard two-channel photometric array analysis.
thus, cy3-pza primer is used for unknown samples and cy5-pza is used to generate a reference sample from a sub-population of normal males for comparison.
the oligonucleotide spots of the array are co-hybridised with cy3-probes from test samples and cy5-probes from reference samples.
copy number can then be calculated by measuring the ratio of cy3:cy <dig> intensities, reflecting the amount of probe in the test  samples relative to the reference  samples.
thus, an elevated copy number would be revealed by a cy3/cy <dig> ratio greater than one, and less than one for a reduced copy number.
pamgene's flow-through microarrays use a novel format in which probe is actively pumped repeatedly across the plane of the array ; diffusion is no longer the rate-limiting step, allowing faster hybridization kinetics than in diffusion-limited systems  <cit> .
in this study we used the flow-through arrays in conjunction with the olympus fd <dig> microarray instrument, which automates the required hybridization, pumping and image capture.
applications of the fd <dig> instrument in mutation detection and gene expression analyses have been published  <cit> .
using pamgene's 3-dimensional, flow-through microarray platform we present a new format for maph: microarray maph, using pmp <dig> gene dosage measurements in normal controls and cmt1a patients as an example.
in this paper we demonstrate for first time successful combination of microarray technology with maph, and show that the accuracy of microarray-based maph is comparable to established electrophoretic methods.
RESULTS
normalised data from microarray images were analysed in groups of normal and cmt1a samples for an initial global comparison of each endpoint of the mean returned copy number for each probe .
the cmt1a group has an elevated mean copy number for pmp <dig> probes, consistent with heterozygous duplication, in both microarray and standard capillary assays.
the standard deviations for the majority of probes are similar for each format, suggesting that the microarray format reports the amounts of each probe as accurately as capillary electrophoresis.
however, probes b <dig> and da show unusually high standard deviations in the microarray maph, suggesting an additional source of variation in this format.
high variation in reported dosage for probe b <dig> was traced to low spot intensity.
both the cy <dig> and cy <dig> intensities for each of the three b <dig> complementary oligonucleotides for all samples were similar to that of the non-human control probes  and were thus no greater than background.
this effect was independent of the oligonucleotide used, the position on the array and the sample analysed and therefore is probably due to an unusual property of this specific probe resulting in its poor capture on the array.
we were, however, unable to identify obvious areas of self-complementarity in this sequence that might prevent capture by the array.
in contrast, the high variation seen with probe da is entirely due to two individual results that returned higher than expected intensities in the cy <dig> images.
these are most likely to be array image artefacts and are consistent with the tendency of cy <dig> images to be more susceptible to substrate and dust particle fluorescence compared to cy <dig> images, as can be seen to some extent in figure  <dig>
the accuracy and precision of both formats, represented by the distribution of "mrd6" values  in the normal and cmt1a populations are very similar .
this is particularly clear when directly comparing the mean mrd <dig> value and mrd <dig> standard deviation for each population shown in table  <dig>  the close agreement between the results of each format is confirmed by a correlation coefficient of  <dig>  for mrd <dig> values for the full data set .
we conclude that the pamchip microarray format for maph is able to determine copy number for pmp <dig> as accurately as the previous format using capillary electrophoresis.
discussion
in this study we have successfully demonstrated the combination of two powerful technologies, maph and the pamgene 3d microarray.
by taking the same source material through analyses with both capillary maph and array maph we have shown both endpoints to be in close agreement.
this conclusion was based on three key observations.
firstly, each sample was correctly partitioned into one of two distinct populations on the basis of the mrd <dig> score: for normal samples the mrd <dig> ranged between  <dig>  and  <dig> ; for duplicated  samples, mrd <dig> ranged between  <dig>  and  <dig> .
secondly, the average normalised copy number for each probe  across the sample set was very similar.
thirdly, the mrd <dig> values for the two assays were closely correlated.
the potential for non-systematic noise in array maph was illustrated by the unusually high cy <dig> intensity of two data points for spots complementary to the da probe.
these high intensity 'spikes' are caused by dust particles settling onto the array surface which tend to fluoresce more intensely in the cy <dig> emission spectrum than in the cy <dig>  we did not take any particular precautions to ensure a dust-free environment for our experiments, and it is possible that these additional steps may have eliminated dust particles.
although it is doubtful whether dust contamination can be completely eliminated, especially when using the more open fd <dig>  the more enclosed design of more recently released array instruments such as the pamstation  <dig> and pamstation  <dig> should also help reduce contamination.
it is clear that applications where diagnosis may depend on the result of a single probe, such as the subtelomeric sets, it will be necessary to screen and control very carefully for spurious signals due to this kind of noise.
for example, employing duplicate pcrs from samples, or duplicate spots on each array would distinguish false-positive signals from true copy-number change, and the data from this study show that signals from duplicate hybridizing spots have a correlation coefficient of  <dig> , and a mean pairwise difference of about 11%.
thus gross anomalies  might be efficiently detected as poorly correlated signals, and removed from the data set.
however, in order to present a clear picture of the potential of the system, the data presented in this report were analysed without any prior selection.
as we have seen with probe b <dig> the potential for systematic noise also exists in array maph.
as hybridisation is a key step of maph we can anticipate a high rate of success in using maph probe sets in microarray analysis without further modification, but it is unrealistic to expect that all probes will perform equally well.
it is likely then that development of future applications of array maph may involve some minor modifications of existing probe sets.
in comparison with other established and commercially available technologies, there are some clear advantages and disadvantages to the technology we describe in this report.
roma  <cit>  uses competitive hybridization of test and reference "representations", made by amplification of restriction fragments, to arrays of oligonucleotides.
detection of putative copy number changes by allele-specific microarray methods  <cit> , including recent work using molecular inversion probes  <cit> , depends on analysis of allele ratios from genome-wide snp analyses.
published work on copy number measurement using molecular inversion probes  <cit>  can involve not only information from allele ratios, but also from total signal .
the relative standard deviation from mip "copy sum" analyses is of the order of  <dig> – <dig>  for most probes, but data analysis procedures including the removal of outliers, and "smoothing" procedures combining data from neighbouring loci, make it difficult to derive a fair comparison with the data presented here.
"smoothing" across neighbouring loci does improve the sensitivity, but at a cost of resolution.
furthermore, while in principle it is possible to use custom molecular inversion probes based on invariant positions , the published data makes use of established assays based on snps  <cit> .
the resolution of mip analyses is therefore limited by the spacing of the probes, but even if placed at very high density in custom sets, the requirement for data smoothing inevitably makes the effective resolution lower.
thus while mip technology may have real advantages for truly genome-wide scans for copy number change, it is much less suitable for targeted analysis of  exonic deletion and duplication in multi-exon genes, where the accuracy of single-probe measurement is critical.
maph has the advantage over mlpa  <cit> , mip and roma that maph results are not sensitive to unexpected substitutional variation in critical positions.
in contrast to roma, mip, and other allele-specific technologies which "smooth" data over neighbouring loci, the true effective resolution of maph is given by the size of the probe .
maph probes can be designed for nearly all single-copy sequences, so that a direct test can be made of very short sequences of interest .
by contrast, deduction of copy number changes from roma and allele-specific snp arrays depends on indirect inference from the states of neighbouring markers, and the placement of these markers is dictated not by the biological question but by their inclusion in a subgenomic representation  or by the existence of common snp variation .
maph also analyses relatively small amounts  of genomic dna without prior manipulation or amplification, both conserving genomic dna resources and reducing the opportunity for distorting representation before analysis.
nevertheless, for other well-established methods capable of full whole-genome copy number measurement, current commercial availability and the capacity to screen the whole genome in a single assay currently outweighs their limitations.
furthermore, in some analyses there may be a real advantage to allele-specificity, making methods like roma and maph less advantageous.
for all these reasons, the particular advantages of microarray maph may make it most suitable for a targeted analysis of an intermediate number of loci at very high precision and resolution.
examples could include interrogating a set of 50– <dig> exons from selected "target" genes, or a set of 400– <dig> single-copy loci for analysis of karyotypic changes at relatively low resolution, but with high signal:noise ratios at each locus tested, and in a simple, fast and inexpensive assay.
maph is constrained by core elements of the assay, including probe sequence specificity, pcr efficiency and hybridisation kinetics.
probe design for capillary maph is further complicated by the electrophoretic end-point used to determine relative probe dosage.
removing these restrictions has implications for current and future applications of maph as an analytical tool.
probe sets can now be refined, if necessary, to allow simultaneous use of probes of similar lengths.
one of the great strengths of maph, its multiplicity, has also been limited by the resolution of capillary electrophoresis.
probe sets that have been too large to be accommodated in a single gel run can now be combined to allow inexpensive and efficient analysis of larger numbers of loci simultaneously.
we chose to base array maph on pamgene's flow-through microarray platform.
the superior hybridisation kinetics at the heart of the pamchip technology has shortened the hybridisation step from overnight to less than an hour.
even so, the implementation of the technology used here allows only four samples to be tested in the time it would take to resolve around thirty two samples on capillary analysis.
since this study was begun pamgene have developed the pamstation  <dig> which, as the name implies, increases the sample throughput of the technology via a 96-well format.
in parallel with improvements in the pamchip technology we have also been successful in evolving the core maph protocol into a 96-well format.
together, these advances have the potential to unlock the true power of maph in combination with pamgene's microarray systems.
CONCLUSIONS
we have been able to use oligonucleotide microarrays to display the products from maph for the analysis of dna copy number.
the accuracy of the copy number determinations compares favourably with results obtained with standard electrophoretic separation of fluorescently-labelled fragments.
microarray readout removes the upper limit imposed by electrophoretic mobility on the multiplicity, and in principle will allow the extension of maph as a highly accurate, high-resolution method for simultaneous copy number determination at larger numbers of loci.

 the role of submicroscopic dna copy number variation in genetic pathologies has been established now for two decades. early investigations recognised the importance of deletions or duplications in specific genes as causative mutations in clinical conditions, such as brca in familial breast and ovarian cancer and dmd in duchenne/becker muscular dystrophy. furthermore, subtelomeric chromosomal rearrangements leading to copy number changes have been associated with learning disability and other developmental abnormalities. recent advances in the diagnostic technologies applied to such conditions have also proven to be useful tools in elucidating the dynamic model of the human genome, with copy number variation recently recognised as an important component of human polymorphism. as the significance of copy number variation in genetic analysis becomes more widely recognised so does the need to improve and extend the range of techniques available. in particular, for scanning multi-exon genes for deletions or duplications, maph and mlpa have been used to assess the dosage of small regions to high accuracy, properties not easily implemented using, for example, current array-cgh methods. one feature shared by both multiplex amplifiable probe hybridization and multiplex ligation-dependent probe amplification is the limit to multiplicity set by the resolution of probes distinguished by their length, using capillary electrophoresis. in maph, the requirement for specific and stable hybridization sets a lower limit on probe size of around bp, and the requirement for comparable co-amplification of different probes sets an upper limit of around bp; thus the largest single probe set could contain probes spaced bp apart. while this does not pose a limitation on applications using relatively small probe sets, such as the probes used in brca testing, where many exons are involved, such as dmd, or where screening is not targeted to single gene loci, probe sets of this magnitude would be inadequate. the most obvious route to breaking these limits is to employ dna microarray technology to replace capillary gel electrophoresis as the final analytical step in maph, as discussed in the original maph publication and in a more recent review. a microarray based on oligonucleotides complementary to individual maph probes would allow detection and quantification based on sequence rather than on size. microarray detection and quantification should therefore allow the simultaneous measurement of copy number at a larger number of loci. the initial stages of microarray maph, up to the recovery of specifically bound probes, remain the same as capillary maph. the subsequent amplification of these probes is modified to allow the incorporation of labels suitable for a standard two-channel photometric array analysis. thus, cy3-pza primer is used for unknown samples and cy5-pza is used to generate a reference sample from a sub-population of normal males for comparison. the oligonucleotide spots of the array are co-hybridised with cy3-probes from test samples and cy5-probes from reference samples. copy number can then be calculated by measuring the ratio of cy3:cy intensities, reflecting the amount of probe in the test samples relative to the reference samples. thus, an elevated copy number would be revealed by a cy3/cy ratio greater than one, and less than one for a reduced copy number. pamgene's flow-through microarrays use a novel format in which probe is actively pumped repeatedly across the plane of the array; diffusion is no longer the rate-limiting step, allowing faster hybridization kinetics than in diffusion-limited systems. in this study we used the flow-through arrays in conjunction with the olympus fd microarray instrument, which automates the required hybridization, pumping and image capture. applications of the fd instrument in mutation detection and gene expression analyses have been published. using pamgene's 3-dimensional, flow-through microarray platform we present a new format for maph: microarray maph, using pmp gene dosage measurements in normal controls and cmt1a patients as an example. in this paper we demonstrate for first time successful combination of microarray technology with maph, and show that the accuracy of microarray-based maph is comparable to established electrophoretic methods.  normalised data from microarray images were analysed in groups of normal and cmt1a samples for an initial global comparison of each endpoint of the mean returned copy number for each probe. the cmt1a group has an elevated mean copy number for pmp probes, consistent with heterozygous duplication, in both microarray and standard capillary assays. the standard deviations for the majority of probes are similar for each format, suggesting that the microarray format reports the amounts of each probe as accurately as capillary electrophoresis. however, probes b and da show unusually high standard deviations in the microarray maph, suggesting an additional source of variation in this format. high variation in reported dosage for probe b was traced to low spot intensity. both the cy and cy intensities for each of the three b complementary oligonucleotides for all samples were similar to that of the non-human control probes and were thus no greater than background. this effect was independent of the oligonucleotide used, the position on the array and the sample analysed and therefore is probably due to an unusual property of this specific probe resulting in its poor capture on the array. we were, however, unable to identify obvious areas of self-complementarity in this sequence that might prevent capture by the array. in contrast, the high variation seen with probe da is entirely due to two individual results that returned higher than expected intensities in the cy images. these are most likely to be array image artefacts and are consistent with the tendency of cy images to be more susceptible to substrate and dust particle fluorescence compared to cy images, as can be seen to some extent in figure the accuracy and precision of both formats, represented by the distribution of "mrd6" values in the normal and cmt1a populations are very similar. this is particularly clear when directly comparing the mean mrd value and mrd standard deviation for each population shown in table the close agreement between the results of each format is confirmed by a correlation coefficient of for mrd values for the full data set. we conclude that the pamchip microarray format for maph is able to determine copy number for pmp as accurately as the previous format using capillary electrophoresis. discussion in this study we have successfully demonstrated the combination of two powerful technologies, maph and the pamgene 3d microarray. by taking the same source material through analyses with both capillary maph and array maph we have shown both endpoints to be in close agreement. this conclusion was based on three key observations. firstly, each sample was correctly partitioned into one of two distinct populations on the basis of the mrd score: for normal samples the mrd ranged between and; for duplicated samples, mrd ranged between and. secondly, the average normalised copy number for each probe across the sample set was very similar. thirdly, the mrd values for the two assays were closely correlated. the potential for non-systematic noise in array maph was illustrated by the unusually high cy intensity of two data points for spots complementary to the da probe. these high intensity'spikes' are caused by dust particles settling onto the array surface which tend to fluoresce more intensely in the cy emission spectrum than in the cy we did not take any particular precautions to ensure a dust-free environment for our experiments, and it is possible that these additional steps may have eliminated dust particles. although it is doubtful whether dust contamination can be completely eliminated, especially when using the more open fd the more enclosed design of more recently released array instruments such as the pamstation and pamstation should also help reduce contamination. it is clear that applications where diagnosis may depend on the result of a single probe, such as the subtelomeric sets, it will be necessary to screen and control very carefully for spurious signals due to this kind of noise. for example, employing duplicate pcrs from samples, or duplicate spots on each array would distinguish false-positive signals from true copy-number change, and the data from this study show that signals from duplicate hybridizing spots have a correlation coefficient of, and a mean pairwise difference of about 11%. thus gross anomalies might be efficiently detected as poorly correlated signals, and removed from the data set. however, in order to present a clear picture of the potential of the system, the data presented in this report were analysed without any prior selection. as we have seen with probe b the potential for systematic noise also exists in array maph. as hybridisation is a key step of maph we can anticipate a high rate of success in using maph probe sets in microarray analysis without further modification, but it is unrealistic to expect that all probes will perform equally well. it is likely then that development of future applications of array maph may involve some minor modifications of existing probe sets. in comparison with other established and commercially available technologies, there are some clear advantages and disadvantages to the technology we describe in this report. roma uses competitive hybridization of test and reference "representations", made by amplification of restriction fragments, to arrays of oligonucleotides. detection of putative copy number changes by allele-specific microarray methods, including recent work using molecular inversion probes, depends on analysis of allele ratios from genome-wide snp analyses. published work on copy number measurement using molecular inversion probes can involve not only information from allele ratios, but also from total signal. the relative standard deviation from mip "copy sum" analyses is of the order of – for most probes, but data analysis procedures including the removal of outliers, and "smoothing" procedures combining data from neighbouring loci, make it difficult to derive a fair comparison with the data presented here. "smoothing" across neighbouring loci does improve the sensitivity, but at a cost of resolution. furthermore, while in principle it is possible to use custom molecular inversion probes based on invariant positions, the published data makes use of established assays based on snps. the resolution of mip analyses is therefore limited by the spacing of the probes, but even if placed at very high density in custom sets, the requirement for data smoothing inevitably makes the effective resolution lower. thus while mip technology may have real advantages for truly genome-wide scans for copy number change, it is much less suitable for targeted analysis of exonic deletion and duplication in multi-exon genes, where the accuracy of single-probe measurement is critical. maph has the advantage over mlpa, mip and roma that maph results are not sensitive to unexpected substitutional variation in critical positions. in contrast to roma, mip, and other allele-specific technologies which "smooth" data over neighbouring loci, the true effective resolution of maph is given by the size of the probe. maph probes can be designed for nearly all single-copy sequences, so that a direct test can be made of very short sequences of interest. by contrast, deduction of copy number changes from roma and allele-specific snp arrays depends on indirect inference from the states of neighbouring markers, and the placement of these markers is dictated not by the biological question but by their inclusion in a subgenomic representation or by the existence of common snp variation. maph also analyses relatively small amounts of genomic dna without prior manipulation or amplification, both conserving genomic dna resources and reducing the opportunity for distorting representation before analysis. nevertheless, for other well-established methods capable of full whole-genome copy number measurement, current commercial availability and the capacity to screen the whole genome in a single assay currently outweighs their limitations. furthermore, in some analyses there may be a real advantage to allele-specificity, making methods like roma and maph less advantageous. for all these reasons, the particular advantages of microarray maph may make it most suitable for a targeted analysis of an intermediate number of loci at very high precision and resolution. examples could include interrogating a set of 50– exons from selected "target" genes, or a set of 400– single-copy loci for analysis of karyotypic changes at relatively low resolution, but with high signal:noise ratios at each locus tested, and in a simple, fast and inexpensive assay. maph is constrained by core elements of the assay, including probe sequence specificity, pcr efficiency and hybridisation kinetics. probe design for capillary maph is further complicated by the electrophoretic end-point used to determine relative probe dosage. removing these restrictions has implications for current and future applications of maph as an analytical tool. probe sets can now be refined, if necessary, to allow simultaneous use of probes of similar lengths. one of the great strengths of maph, its multiplicity, has also been limited by the resolution of capillary electrophoresis. probe sets that have been too large to be accommodated in a single gel run can now be combined to allow inexpensive and efficient analysis of larger numbers of loci simultaneously. we chose to base array maph on pamgene's flow-through microarray platform. the superior hybridisation kinetics at the heart of the pamchip technology has shortened the hybridisation step from overnight to less than an hour. even so, the implementation of the technology used here allows only four samples to be tested in the time it would take to resolve around thirty two samples on capillary analysis. since this study was begun pamgene have developed the pamstation which, as the name implies, increases the sample throughput of the technology via a 96-well format. in parallel with improvements in the pamchip technology we have also been successful in evolving the core maph protocol into a 96-well format. together, these advances have the potential to unlock the true power of maph in combination with pamgene's microarray systems.  we have been able to use oligonucleotide microarrays to display the products from maph for the analysis of dna copy number. the accuracy of the copy number determinations compares favourably with results obtained with standard electrophoretic separation of fluorescently-labelled fragments. microarray readout removes the upper limit imposed by electrophoretic mobility on the multiplicity, and in principle will allow the extension of maph as a highly accurate, high-resolution method for simultaneous copy number determination at larger numbers of loci. 
measurement of microarray-based endpoints for maph appears to be of comparable accuracy to electrophoretic methods, and holds the prospect of fully exploiting the potential multiplicity of maph. in this study, to improve the throughput associated with multiplex amplifiable probe hybridisation we aimed to develop a modification based on the 3-dimensional, flow-through microarray platform from pamgene international. current methods for measurement of copy number do not combine all the desirable qualities of convenience, throughput, economy, accuracy and resolution. copy number analysis of hybridised probes is based on a dual-label approach by comparing the intensity of cy3-labelled maph probes amplified from test samples co-hybridised with similarly amplified cy5-labelled reference maph probes. in this study we showed that microarray maph measurement of pmp gene dosage correlates well with pmp gene dosage determined by capillary maph and that copy number was accurately reported in analyses of dna from individuals, of which were known to have charcot-marie-tooth disease type 1a. the technology has the potential to simplify copy number assays for genes with a large number of exons, or of expanded sets of probes from dispersed genomic locations.
 current methods for measurement of copy number do not combine all the desirable qualities of convenience, throughput, economy, accuracy and resolution. in this study, to improve the throughput associated with multiplex amplifiable probe hybridisation we aimed to develop a modification based on the 3-dimensional, flow-through microarray platform from pamgene international. in this new method, electrophoretic analysis of amplified products is replaced with photometric analysis of a probed oligonucleotide array. copy number analysis of hybridised probes is based on a dual-label approach by comparing the intensity of cy3-labelled maph probes amplified from test samples co-hybridised with similarly amplified cy5-labelled reference maph probes. the key feature of using a hybridisation-based end point with maph is that discrimination of amplified probes is based on sequence and not fragment length.  in this study we showed that microarray maph measurement of pmp gene dosage correlates well with pmp gene dosage determined by capillary maph and that copy number was accurately reported in analyses of dna from individuals, of which were known to have charcot-marie-tooth disease type 1a.  measurement of microarray-based endpoints for maph appears to be of comparable accuracy to electrophoretic methods, and holds the prospect of fully exploiting the potential multiplicity of maph. the technology has the potential to simplify copy number assays for genes with a large number of exons, or of expanded sets of probes from dispersed genomic locations. 