BACKGROUND
cancer is a group of diseases characterized by uncontrolled cell growth.
according to the world cancer report, cancer rates could further increase by 50% to  <dig> million new cases in the year  <dig>  <cit> .
the chances of surviving the onset of some common cancers depend largely on how early they are detected in addition to how well they are treated.
the progression of mammalian cells towards malignancy is an evolutionary process that involves an accumulation of mutations at the molecular and chromosomal level.
a candidate for involvement in this process is the tumor suppressor gene tp <dig>  which encodes a transcription factor  with cancer inhibiting properties.
this gene is the most frequently mutated gene in human cancer.
between 30% and 70% of cancers of almost every organ and histological subtype have a point mutation in one of the two tp <dig> gene copies together with loss of the other copy  <cit> .
in many cancers the distribution of mutations along the tp <dig> gene is tumor-specific, clustered between exons  <dig>   <dig> and  <dig>  this region is highly conserved throughout evolution  <cit> .
in this domain, six mutational "hotspots" have been identified at codons  <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig>  the mutational profile of the tp <dig> gene is different between cancers, for example, codons  <dig>   <dig>   <dig>   <dig>  and  <dig> have been designated as tp <dig> mutational hotspots in lung cancer  <cit> , while in breast cancer the mutational hotspots are in codons,  <dig>   <dig>   <dig>   <dig> and  <dig>  <cit> .
on the contrary, in cervical cancer the frequency of the p <dig> mutations is very low.
<cit> .
because mutations in the tp <dig> gene are so common in human tumors and have been widely reported in the literature, extensive mutational databases exist, such as that maintained by iarc  <cit> .
walker et al.
<cit> , have utilized the iarc tp <dig> mutation database to define  <dig> "hotspots" for mutation in tp <dig> related to changes in protein structure and function.
martin et al.
<cit>  performed a systematic automated analysis of the effects of tp <dig> mutations on the structure of the core domain of the p <dig> protein to functionally classify the various types of p <dig> mutants according to predicted effects on protein folding or dna-protein interactions.
most mutations are currently identified by conventional methods of polymorphism detection and dna sequencing  <cit> .
a growing number of nucleic acid hybridization techniques have recently been applied to biomedical problems, including development of dna probe arrays for detection of tp <dig> mutations  <cit> .
in many applications, dna chips containing surface-bound oligonucleotides  are used to interrogate sample  sequence information through complementary recognition  in a highly parallel fashion  <cit> .
major applications of dna microarrays include gene expression profiling  <cit>  and gene mutation analysis  <cit> .
these techniques allow parallel analysis of multiple dna samples.
mutation studies with dna microarrays are still at an early stage and are in continuous development  <cit> .
although the field of microarray assays is still in a relatively early stage, it is generally anticipated that microarray assays will offer decreased cost and faster results, compared with the traditional, more labor intensive dideoxy sequencing approach.
we recently reported a novel tandem hybridization strategy and its application for identification of mutations.
reliable discrimination of point mutations has been achieved by double tandem hybridization with a set of seven-mer probes  <cit> .
in the present work we designed a novel small format dna chip  to search for nine point mutations in codons  <dig>   <dig> and  <dig> of the tp <dig> gene.
RESULTS
pcr products and reference hybridization patterns
pcr products from exon  <dig>  and exon  <dig>  were obtained from each type of dna sources listed in materials and methods.
figure 2a shows gel electrophoretic analysis of the fragments of  <dig> and  <dig> bp corresponding to exons  <dig> and  <dig>  respectively.
to prepare ssdna targets pcr products were used as templates for cycle synthesis of a target strand, using the reverse pcr primer in single or multiplexed reactions cycled  <dig> times.
the production of single-stranded target dna's was assessed by its change in electrophoretic mobility in 4% agarose gels .
the reference hybridization patterns produced using synthetic dna targets are shown in figures.
3a and 3b.
as expected, all the synthetic targets, including those of the negative controls, yielded hybridization signals with their respective  probes.
pairs of wild-type with each mutant synthetic target sequences  were also hybridized to reproduce heterozygous conditions.
all gave the expected signals, .
hybridization of dna targets
hybridization of individual or multiplexed ssdna targets prepared from the  <dig> dna samples  was performed with similar results.
base substitutions were seen in  <dig> out of the  <dig> samples tested .
one, two or three codons were altered in  <dig> ,  <dig>  and  <dig>  samples, respectively.
in the cases of the samples having two or three codons affected, at least one of the base substitutions was silent.
in the other  <dig> samples only wt sequences were identified .
representative hybridization patterns are shown in figure.
4a and the overall results are summarized in table  <dig>  the dna samples hybridized with one or two probes from each codon, corresponding to the homozygous or heterozygous condition, respectively.
among the  <dig> different samples multiplexed or individually tested in the microarray, six single base susbstitutions in codon  <dig> were found by sequencing the pcr product.
all of them corresponded to base substitutions keeping the original aminoacid .
the same number of point mutations were also seen in codon  <dig> by dna sequencing.
however, no probes to search for these base substitutions were placed in the dna microarray.
six point mutations in codon  <dig> were obtained.
all of them, except the cgt→cct change observed in plasmid p <dig>  were searched with the set of probes contained in the dna microarray.
in this codon all the base substitutions are producers of aminoacid substitution.
discussion
the tp <dig> tumor suppressor gene has proven to be one of the cellular genes most often mutated in human neoplasias.
analysis of the mutational events that target the tp <dig> gene has revealed evidence for both exogenous and endogenous mutational mechanisms.
this gene mutational spectrum suggests evidence for a direct causal effect of tobacco smoke in lung cancer  <cit> , aflatoxin b <dig> in liver cancer  <cit> .
therefore, identification of mutations in the tp <dig> gene may play an important role in the diagnosis, staging, and management of the cancer patients.
more than 70% of the molecular studies have focused on the central region of the tp <dig> gene, more specifically on exons  <dig> through  <dig> which encode the dna binding domain  <cit> .
thus, missense mutations in this region can cause a modification that may alter dna binding.
it is therefore essential that clinical trials aim to accurately establish the effect of tp <dig> mutations on all clinical parameters.
one approach is the p <dig> genechip of affymetrix used to identify mutations in this gene  <cit> .
however, this technology is expensive and poorly available to many clinicians, which makes it necessary to develop new mutation detection methods  <cit> .
the updated iarc tp <dig> mutation database  <cit>  shows that more than  <dig> different point mutations, which are associated to cancers, are distributed along the tp <dig> gene.
tp <dig> somatic mutations are found in most types of sporadic human cancers at various frequencies   <cit> .
the database also shows that approximately 20% of all mutations are located in the five codons  in the core domain:  <dig>   <dig>   <dig>   <dig> and  <dig>  therefore a primary screen of these codons would be attractive.
most snp diagnostic arrays use probes varying from  <dig> to  <dig> nt in length with similar tm values  <cit> .
however, under this circumstance some of the mutant sequences are difficult to discriminate, due to the relatively small destabilizing effect of certain single mismatches in the probe/target duplex  <cit> .
the tandem hybridization method described here offers several advantages over the traditional oligonucleotide array configuration in which each interrogated target sequence is represented by a single surface-tethered probe.
because the long stacking probe targets the analysis to a single unique site within a nucleic acid, direct hybridization analysis of nucleic acid samples of high genetic complexity using short capture probes may be enabled  <cit> .
recently, we successfully used a double tandem hybridization strategy in the genesensor chip system to discriminate point mutations in codon  <dig> of ret oncogene in individuals affected by medullary thyroid carcinoma  <cit> .
this dna chip format involves a target sequence which is annealed with a pair of labeled oligonucleotides  prior to hybridization to the array of capture probes, forming a single-stranded gap between the stabilizing oligonucleotides and corresponding to the length of the capture probes.
the partially duplex product contains the sequences interrogated by the capture probes, and when the sequences between the target dna and the capture probe  are perfectly complementary, the stability of the hybridization is enhanced due to coaxial stacking of bases between the contiguous ends of the probe and the stabilizing oligonucleotides.
the energy involved varies with the identity of the base pairs and the coaxial stacking  <cit> .
tandem hybridization uses shorter capture probes than traditional oligonucleotide array hybridization, which may contribute to increased discrimination of point mutations because the single base mismatch acts on a less stable duplex formed between the target dna and the shorter probe.
two additional attractive features inherent to tandem hybridization are that, i) the probability that formation of secondary structure within the single stranded dna  <cit>  will block accessibility of the target sequence is reduced, and ii) the label needed to reveal the hybridization can be introduced into the two stabilizing oligonucleotides, avoiding the extra step of labeling individual target strands  <cit> .
the goal of the present work was to develop a simple format array able to perform a primary screening of the mutations occurring in the most frequently affected codons of the tp <dig> gene.
it has been recently reported that detection of mutations yields variable results depending on the type of dna sample  <cit> .
is clear that, the sensitivity to detect mutations in tp <dig>  may also differ depending on the method used.
in the current study the double tandem hybridization assay was applied to genomic dna samples extracted from a variety of sources, including blood cells, cell lines and plasmids.
there were not any differences in the ability of our system to detect mutations with respect to the different dna sources.
in the experiments employing synthetic dna targets hybridized with arrays of mutant and wt probes the expected hybridization signals were seen both in homozygous and heterozygous conditions .
this result indicates that the probes are working properly.
the hybridization of natural targets was done both with isolated and multiplexed pcr products with similar results.
multiplexing was applied in all  the steps.
so these results are suggesting that multiplexing can be confidently employed for this analysis.
base changes were found in  <dig> dna samples.
two were from plasmid, seven from cell lines and two from blood samples.
in  <dig> of these samples a single codon was affected, in other  <dig> samples two codons were altered, and in the other sample three codons were affected.
therefore a total of  <dig> codon alterations were detected.
in five of the  <dig> codon alterations  both the absence of hybridization signal with the wild type probe and the production of signal with the appropriated probe was seen.
in eight cases  no hybridization signal was seen even with the wild type sequence.
this was due to the absence of the corresponding mutant probe in the array.
in three cases  having either homozygous  or heterozygous  genotypes  no hybridization signal with the wild type probe and, an additional false hybridization signal with the probe corresponding to the substitution of guanine for thymine in codon  <dig>  was seen, which could be relatively stable under the conditions tested.
therefore an additional probe, searching by the aga sequence, should be added in the future array to discriminate between the two alternative silent  and missense  genotypes.
in the other two codons affected  the presence of signal with the wild type probe was seen even when the sequencing was showing a base change .
in both cases a g:n mismatch is formed which, according to santalucia  <cit> , is relatively stable.
so, as in the previous two cases the incorporation of additional probes in the array should help to improve performance of the dna microarray to confidently identify the genotypes.
the base substitution observed in b <dig> and b <dig> blood samples is a mutation that substitutes arginine by leucine.
the presence of this mutation in blood suggests that it could be from germinal origin.
in all the mutations  observed an arginine was involved, which can be expected because it is coded by  <dig> codons and it frequently produces non conservative aminoacid substitutions.
the array is able to confidently detect most of the mutations searched, however, additional probes should be added to improve its performance.
CONCLUSIONS
our results demonstrate that a simple tp <dig> microarray using short  probes, used in combination with a double tandem hybridization approach and a simple multiplex fragment preparation method, can identify common tp <dig> point mutations from different dna sources.
all the previous data confirms that the tandem hybridization technology, with the appropriated set of probes, is highly specific and can be confidently employed for diagnostic purposes.
this system could provide rapid, economical and accurate mutational screening in the tp <dig> gene.
we are presently improving our system by adding probes for these and other tp <dig> mutational hot spots.

 cancer is a group of diseases characterized by uncontrolled cell growth. according to the world cancer report, cancer rates could further increase by 50% to million new cases in the year. the chances of surviving the onset of some common cancers depend largely on how early they are detected in addition to how well they are treated. the progression of mammalian cells towards malignancy is an evolutionary process that involves an accumulation of mutations at the molecular and chromosomal level. a candidate for involvement in this process is the tumor suppressor gene tp which encodes a transcription factor with cancer inhibiting properties. this gene is the most frequently mutated gene in human cancer. between 30% and 70% of cancers of almost every organ and histological subtype have a point mutation in one of the two tp gene copies together with loss of the other copy. in many cancers the distribution of mutations along the tp gene is tumor-specific, clustered between exons and this region is highly conserved throughout evolution. in this domain, six mutational "hotspots" have been identified at codons and the mutational profile of the tp gene is different between cancers, for example, codons and have been designated as tp mutational hotspots in lung cancer, while in breast cancer the mutational hotspots are in codons, and. on the contrary, in cervical cancer the frequency of the p mutations is very low.. because mutations in the tp gene are so common in human tumors and have been widely reported in the literature, extensive mutational databases exist, such as that maintained by iarc. walker et al., have utilized the iarc tp mutation database to define "hotspots" for mutation in tp related to changes in protein structure and function. martin et al. performed a systematic automated analysis of the effects of tp mutations on the structure of the core domain of the p protein to functionally classify the various types of p mutants according to predicted effects on protein folding or dna-protein interactions. most mutations are currently identified by conventional methods of polymorphism detection and dna sequencing. a growing number of nucleic acid hybridization techniques have recently been applied to biomedical problems, including development of dna probe arrays for detection of tp mutations. in many applications, dna chips containing surface-bound oligonucleotides are used to interrogate sample sequence information through complementary recognition in a highly parallel fashion. major applications of dna microarrays include gene expression profiling and gene mutation analysis. these techniques allow parallel analysis of multiple dna samples. mutation studies with dna microarrays are still at an early stage and are in continuous development. although the field of microarray assays is still in a relatively early stage, it is generally anticipated that microarray assays will offer decreased cost and faster results, compared with the traditional, more labor intensive dideoxy sequencing approach. we recently reported a novel tandem hybridization strategy and its application for identification of mutations. reliable discrimination of point mutations has been achieved by double tandem hybridization with a set of seven-mer probes. in the present work we designed a novel small format dna chip to search for nine point mutations in codons and of the tp gene.  pcr products and reference hybridization patterns pcr products from exon and exon were obtained from each type of dna sources listed in materials and methods. figure 2a shows gel electrophoretic analysis of the fragments of and bp corresponding to exons and respectively. to prepare ssdna targets pcr products were used as templates for cycle synthesis of a target strand, using the reverse pcr primer in single or multiplexed reactions cycled times. the production of single-stranded target dna's was assessed by its change in electrophoretic mobility in 4% agarose gels. the reference hybridization patterns produced using synthetic dna targets are shown in figures. 3a and 3b. as expected, all the synthetic targets, including those of the negative controls, yielded hybridization signals with their respective probes. pairs of wild-type with each mutant synthetic target sequences were also hybridized to reproduce heterozygous conditions. all gave the expected signals,. hybridization of dna targets hybridization of individual or multiplexed ssdna targets prepared from the dna samples was performed with similar results. base substitutions were seen in out of the samples tested. one, two or three codons were altered in, and samples, respectively. in the cases of the samples having two or three codons affected, at least one of the base substitutions was silent. in the other samples only wt sequences were identified. representative hybridization patterns are shown in figure. 4a and the overall results are summarized in table the dna samples hybridized with one or two probes from each codon, corresponding to the homozygous or heterozygous condition, respectively. among the different samples multiplexed or individually tested in the microarray, six single base susbstitutions in codon were found by sequencing the pcr product. all of them corresponded to base substitutions keeping the original aminoacid. the same number of point mutations were also seen in codon by dna sequencing. however, no probes to search for these base substitutions were placed in the dna microarray. six point mutations in codon were obtained. all of them, except the cgt→cct change observed in plasmid p were searched with the set of probes contained in the dna microarray. in this codon all the base substitutions are producers of aminoacid substitution. discussion the tp tumor suppressor gene has proven to be one of the cellular genes most often mutated in human neoplasias. analysis of the mutational events that target the tp gene has revealed evidence for both exogenous and endogenous mutational mechanisms. this gene mutational spectrum suggests evidence for a direct causal effect of tobacco smoke in lung cancer, aflatoxin b in liver cancer. therefore, identification of mutations in the tp gene may play an important role in the diagnosis, staging, and management of the cancer patients. more than 70% of the molecular studies have focused on the central region of the tp gene, more specifically on exons through which encode the dna binding domain. thus, missense mutations in this region can cause a modification that may alter dna binding. it is therefore essential that clinical trials aim to accurately establish the effect of tp mutations on all clinical parameters. one approach is the p genechip of affymetrix used to identify mutations in this gene. however, this technology is expensive and poorly available to many clinicians, which makes it necessary to develop new mutation detection methods. the updated iarc tp mutation database shows that more than different point mutations, which are associated to cancers, are distributed along the tp gene. tp somatic mutations are found in most types of sporadic human cancers at various frequencies. the database also shows that approximately 20% of all mutations are located in the five codons in the core domain: and therefore a primary screen of these codons would be attractive. most snp diagnostic arrays use probes varying from to nt in length with similar tm values. however, under this circumstance some of the mutant sequences are difficult to discriminate, due to the relatively small destabilizing effect of certain single mismatches in the probe/target duplex. the tandem hybridization method described here offers several advantages over the traditional oligonucleotide array configuration in which each interrogated target sequence is represented by a single surface-tethered probe. because the long stacking probe targets the analysis to a single unique site within a nucleic acid, direct hybridization analysis of nucleic acid samples of high genetic complexity using short capture probes may be enabled. recently, we successfully used a double tandem hybridization strategy in the genesensor chip system to discriminate point mutations in codon of ret oncogene in individuals affected by medullary thyroid carcinoma. this dna chip format involves a target sequence which is annealed with a pair of labeled oligonucleotides prior to hybridization to the array of capture probes, forming a single-stranded gap between the stabilizing oligonucleotides and corresponding to the length of the capture probes. the partially duplex product contains the sequences interrogated by the capture probes, and when the sequences between the target dna and the capture probe are perfectly complementary, the stability of the hybridization is enhanced due to coaxial stacking of bases between the contiguous ends of the probe and the stabilizing oligonucleotides. the energy involved varies with the identity of the base pairs and the coaxial stacking. tandem hybridization uses shorter capture probes than traditional oligonucleotide array hybridization, which may contribute to increased discrimination of point mutations because the single base mismatch acts on a less stable duplex formed between the target dna and the shorter probe. two additional attractive features inherent to tandem hybridization are that, i) the probability that formation of secondary structure within the single stranded dna will block accessibility of the target sequence is reduced, and ii) the label needed to reveal the hybridization can be introduced into the two stabilizing oligonucleotides, avoiding the extra step of labeling individual target strands. the goal of the present work was to develop a simple format array able to perform a primary screening of the mutations occurring in the most frequently affected codons of the tp gene. it has been recently reported that detection of mutations yields variable results depending on the type of dna sample. is clear that, the sensitivity to detect mutations in tp may also differ depending on the method used. in the current study the double tandem hybridization assay was applied to genomic dna samples extracted from a variety of sources, including blood cells, cell lines and plasmids. there were not any differences in the ability of our system to detect mutations with respect to the different dna sources. in the experiments employing synthetic dna targets hybridized with arrays of mutant and wt probes the expected hybridization signals were seen both in homozygous and heterozygous conditions. this result indicates that the probes are working properly. the hybridization of natural targets was done both with isolated and multiplexed pcr products with similar results. multiplexing was applied in all the steps. so these results are suggesting that multiplexing can be confidently employed for this analysis. base changes were found in dna samples. two were from plasmid, seven from cell lines and two from blood samples. in of these samples a single codon was affected, in other samples two codons were altered, and in the other sample three codons were affected. therefore a total of codon alterations were detected. in five of the codon alterations both the absence of hybridization signal with the wild type probe and the production of signal with the appropriated probe was seen. in eight cases no hybridization signal was seen even with the wild type sequence. this was due to the absence of the corresponding mutant probe in the array. in three cases having either homozygous or heterozygous genotypes no hybridization signal with the wild type probe and, an additional false hybridization signal with the probe corresponding to the substitution of guanine for thymine in codon was seen, which could be relatively stable under the conditions tested. therefore an additional probe, searching by the aga sequence, should be added in the future array to discriminate between the two alternative silent and missense genotypes. in the other two codons affected the presence of signal with the wild type probe was seen even when the sequencing was showing a base change. in both cases a g:n mismatch is formed which, according to santalucia, is relatively stable. so, as in the previous two cases the incorporation of additional probes in the array should help to improve performance of the dna microarray to confidently identify the genotypes. the base substitution observed in b and b blood samples is a mutation that substitutes arginine by leucine. the presence of this mutation in blood suggests that it could be from germinal origin. in all the mutations observed an arginine was involved, which can be expected because it is coded by codons and it frequently produces non conservative aminoacid substitutions. the array is able to confidently detect most of the mutations searched, however, additional probes should be added to improve its performance.  our results demonstrate that a simple tp microarray using short probes, used in combination with a double tandem hybridization approach and a simple multiplex fragment preparation method, can identify common tp point mutations from different dna sources. all the previous data confirms that the tandem hybridization technology, with the appropriated set of probes, is highly specific and can be confidently employed for diagnostic purposes. this system could provide rapid, economical and accurate mutational screening in the tp gene. we are presently improving our system by adding probes for these and other tp mutational hot spots. 
our results demonstrate that a simple tp microarray employing short probes, used in combination with single or double tandem hybridization approach and a simple or multiplex target preparation method, can identify common tp missense mutations from a variety of dna sources with good specificity. in the case of multiplexed sspcr products, only two stacking oligonucleotides were added per target, therefore the gap for the pcr products having two consecutive codons to be interrogated in exon was nt long, so only single tandem hybridization was produced with these respective probes. we have developed an oligonucleotide microarray utilizing a double tandem hybridization technique to search for point mutations located in the most frequently altered codons of the tp gene. independent ssdna's were annealed with the corresponding pairs of labeled stacking oligonucleotides to create partially duplex dna having a 7-nt gap, which contains the sequence that will be interrogated by the capture probes forming double tandem hybridization. in other cases a mutation was falsely suggested by the combination of the absence of the wild type signal along with a false signal in the other probe. in the other cases the presence of the mutation was not detected due to the production of a false hybridization signal with the wild type probe. isolated and multiplexed pcr products, and bp long, from exons and respectively, were obtained from different samples. single-stranded target dna was then prepared from isolated or multiplexed pcr products, through cyclic dna synthesis.
 we have developed an oligonucleotide microarray utilizing a double tandem hybridization technique to search for point mutations located in the most frequently altered codons of the tp gene. isolated and multiplexed pcr products, and bp long, from exons and respectively, were obtained from different samples. single-stranded target dna was then prepared from isolated or multiplexed pcr products, through cyclic dna synthesis. independent ssdna's were annealed with the corresponding pairs of labeled stacking oligonucleotides to create partially duplex dna having a 7-nt gap, which contains the sequence that will be interrogated by the capture probes forming double tandem hybridization. in the case of multiplexed sspcr products, only two stacking oligonucleotides were added per target, therefore the gap for the pcr products having two consecutive codons to be interrogated in exon was nt long, so only single tandem hybridization was produced with these respective probes.  codon substitutions were found by dna sequencing. in of them a perfect correlation with the pattern of hybridization was seen. in other cases a mutation was falsely suggested by the combination of the absence of the wild type signal along with a false signal in the other probe. in the other cases the presence of the mutation was not detected due to the production of a false hybridization signal with the wild type probe. in both cases relatively stable mismatched target/probe duplexes should be formed. these problems could be avoided by the addition of probes to improve the performance of the array.  our results demonstrate that a simple tp microarray employing short probes, used in combination with single or double tandem hybridization approach and a simple or multiplex target preparation method, can identify common tp missense mutations from a variety of dna sources with good specificity. 