because the functional and morphological diversities of an 
 organism represent the value of the organism itself , the traditional biological 
 techniques used to characterize these properties provide 
 indispensable information . conventional biology techniques face 
 difficulties , however , such as classifying characterless organisms 
 like microbes   and analyzing communities composed of huge numbers of various organisms , owing to both the instability of phenotypes , which are easily affected by 
 environmental factors , and an insufficient number of experts .
a potential solution to these problems has been to characterize an 
 organism according to the sequence of the small subunit ribosomal 
 rna ( 16s/18s rrna ) , an approach that has been applied to various 
 organisms [ 57 ] .
similarly , cytochrome oxygenase subunit 1 ( cod1 ) , gyrase , and other genes have been used for this purpose .
the superiority of these approaches is that they 
 are based on popular and well - established sequencing technology 
 and can provide the determinate result of nucleotide sequence , 
 which can be further computer - analyzed and can fuel the activity 
 of bioinformatics .
nevertheless , this approach can not be said to 
 be a readily usable method for classifying species because ( i ) it 
 is rather costly and time - consuming for application to a large 
 number of species ( e.g. , > 100 ) , especially for scientists in 
 general all over the world , and ( ii ) it often results in an 
 insufficient amount of information for identifying and classifying 
 species .
the latter problem can be overcome by sequencing additional genes [ 810 ] ; however , this makes the approach 
 more complicated and less accessible . here
, we present a solution for the universal classification of 
 species together with a demonstration of its effectiveness , which 
 has been tested by applying it to taxonomically well - established 
 organisms such as plants , fish , and insects .
genome profiling ( gp ) 
 has already been established as a method for the identification of 
 species , and has sometimes been applied to clustering of organisms . due to the nature of the samples used in gp 
 ( mostly , bacteria , fungi , and protozoa , which taxonomically can 
 sometimes be subject to debate ) , it has not been possible to 
 establish gp as a technique for classification up till 
 now .
however , here we show for the first time that gp 
 [ 13 , 14 ] is successfully applied to the purpose of classification . owing to its convenience and its highly 
 informative nature ,
this technique of classification by gp can be 
 widely applied to biological research , including botanical 
 research .
the plant , insect , and fish species used in this study are listed 
 in table 1 .
all samples were well washed with distilled water prior to dna 
 extraction . in particular , the legs of insects were vigorously 
 washed with sds detergent to remove contaminating microbes .
dnas 
 of plants were prepared according to the conventional alkaline 
 extraction method , whereas those of fish and insects were extracted by the phenol - chloroform - proteinase k - method 
  using a tiny portion ( 0.05 mg ) of 
 caudal fins or legs . for convenience , here we define genomic dna 
 as the whole set of dnas contained in cell .
gp is a well - established method   comprising the three following major steps : random pcr , temperature gradient gel electrophoresis ( tgge ) ,   and data - normalization by computer - processing .
random pcr has the ability to pick 
 up , for example , the top ten dna fragments that are generated by 
 more stable hybridization of the primer dna .
the random pcr 
 solution ( 50 l ) contained 200 m dntps ( n = g , 
 a , t , c ) , 0.5 m primer , 10 mm tris - hcl ( ph 9.0 ) , 50 mm kcl , 2.5 mm mgcl2 , 0.02 unit/l taq dna polymerase ( takara bio , japan ) and template dna ( arbitrary amount ) .
random pcr was carried out with 30 cycles of denaturation ( 94c , 30 s ) , annealing ( 28c , 2 min ) and extension ( 47c , 2 min ) using a ptc-100tm pcr 
 machine ( mj research , inc , usa ) .
dna samples , together with the 
 internal reference dna , were subjected to tgge 
 ( micro - temperature gradient gel electrophoresis )   ( one inch square size ) using a -tg apparatus ( taitec , japan ) with 
 a linear temperature gradient of 15c to 60c . 

the gel used was 6% acrylamide ( acrylamide : bis   = 19 : 1 ) 
 containing 90 mm tris - hcl , 90 mm boric acid , 2 mm edta ( ph 8.0 ) , and 8 m urea .
detection of dna bands 
 was carried out either by monitoring fluorescence with a 
 fluorescence imager , molecular imager fx ( biorad , usa ) , or by 
 staining with silver . from the resulting band patterns , which were rather complicated , characteristic or  featuring  

points ( e.g. , kinked points ) were extracted and then processed 
 with the aid of computer to generate spiddos ( species 
 identification dots ) [ 11 , 18 ] .
sets of spiddos are able to provide a sufficient amount of information for provisionally identifying species , which is usually done by 
 calculating the pattern similarity score ( pass ) between two 
 genomes . using spiddos
, we can define pass of the genomes between two species as follows : 
 ( 1)pass=11ni=1n|pipi||pi|+|pi| , 0  pass  1 , 
 where pi and pi 
 correspond to the normalized positional vectors ( composed of two elements , mobility , and 
 temperature ) for spiddos pi and pi collected from two genome profiles ( discriminated with or without a prime ) , respectively , and i denotes the serial number of 
 spiddos .
gp needs to be carried out with the following specific precautions 
 to get successful results .
( i ) during random pcr , contamination by 
 other organisms should be carefully avoided . in particular
, the 
 entire random pcr solution without the template dna should be 
 uv - irradiated prior to the pcr reaction in order to inactivate any 
 contaminating dnas that could act as the template .
( ii ) the random pcr reaction should be terminated before the primer 
 molecules are consumed in order to attain a 
double - strand 
 stop ,  which means that the major pcr products are in a 
 double - stranded state and are suitable for tgge analysis ( i.e.
, the 
 melting transition of double stranded dna to single stranded one 
 can be detected ) .
( iii ) the gp pattern should be strictly checked 
 from the viewpoint of  quality score  in order to rule out false 
 positives : the number of bands ( usually more than eight are 
 required ) and the clearness of bands and background should be 
 sufficient ( note that random pcr generates dna products at 
 different molecular ratios ( eventually , the sum of them is 
 equivalent to that of the input primer ) and , sometimes , 
 overexpression of highly expressed dnas ( where the primer binds 
 strongly for forward and reverse extension ) suppresses the 
 appearance of lower expressed ones , leading to less than eight 
 observable bands ) .
gp patterns that are sufficient in both the 
 number and the clarity of bands are categorized  quality a  and 
 used for the further analysis ; those that are sufficient in only 
 one of the two ( but the other is still permissible ) are 
 categorized  quality b  and can be used with caution ( note that 
 quality b patterns were not used in this study ) ; and the remaining 
 patterns (  quality c  ) should be discarded and the whole 
 experiment should be retried .
we developed a clustering and displaying program termed 
  free lighter  on the basis of ward 's method [ 22 , 23 ] , which is a type of nearest neighbor method with an objective function of minimizing the  error sum of squares . 
we 
 also tested other derivative methods such as the group 
 average method , median method , furthest neighbor method , as well as ward 's method , thereby confirming the well - known phenomenon of occasional minor changes in 
 phylodendrons .
these methods are based on the distance 
 defined in ( 2 ) which implies that clusters a and b 
 are to be merged into c , and x is an arbitrary cluster : 
 ( 2)dc = adxa + adxb + dxb + |dxadxb| , 

 where a , b , a , and  are weighing parameters , dc , dxa , dxb , and dab 
 represent distances between relevant clusters such as cluster x 
 and cluster a for dxa .
the parametric differences among these methods are 
 a = na / nc , b = nb / nc , b = 0 ,  = 0 for the group average method ; a = 0.5 , b = 0.5 ,  = 0.25 ,  = 0 for the median method ; a = 0.5 , b = 0.5 ,  = 0 ,  = 0.5 for the furthest neighbor method ; and a = ( nx + na)/(nx + nc ) , b = ( nx + nb)/(nx + nc ) ,  = nx/(nx + nc ) ,  = 0 for ward 's method . in this experiment ,
the clustering results were found to be rather robust against changes in the above parameters , although there was a slight change in the 
 order of neighbor joining in several cases .
the first step is to extract dna fragments specifically from the 
 genomic dna of an organism through random pcr , resulting in the specific reduction of the amount of dna to be analyzed .
the 
 second step is tgge to obtain the sequence - related 
 information ( i.e. , a property related to the melting temperature 
 of dna ) .
the final step is to computer - process the experimental raw data ( i.e. , the band patterns in the gel ) to 
 obtain a normalized digital pattern ( spiddos ) that can be 
 used for further analyses such as species identification and 
 clustering [ 12 , 18 ] .
figure 1 outlines the whole procedure used in this 
 study to classify species by gp .
random pcr is a process in which 
 dna fragments are sampled at random from genomic dna through the 
 hybridization of a mismatch - containing primer to the template dna 
 during pcr . in other words ,
this process is equivalent to the statistical approach used in random - sampling in a public 
 opinion poll , from which an unbiased image of the whole can be 
 grasped .
tgge   is used to get information related to the melting temperature ( tm ) of the sampled dnas which is sequence - dependent .
importantly , an internal reference dna should be co - migrated on the gels in order to obtain 
 experimental fluctuation - free ( or normalized ) data by the 
 subtraction method .
those featuring points that appear in electrophoretic band patterns ( i.e. , start of the melting 
 transition ; see figure 2 ) are marked and converted to 
 provide the coordinates of spiddos ( species 
 identification dots ) . by calculating a pattern similarity score ( pass ) between two 
 genomes using spiddos as defined in materials and 
 methods , we can get the information on how closely the two 
 relevant genomes ( organisms ) are related ( 0  pass  
 1 ; pass = unity for a complete match ) .
the pass value is 
 known to be strongly correlated with the relatedness between two 
 genomes although pass itself is based on a stochastic process 
 .
this means that the random - sampling method can not rule 
 out the possibility of selecting a biased subpopulation ; thus , the 
 larger the sampling number of sampling becomes , the closer to the 
 true image the sampled one will be .
this is also the case with the 
 gp approach ; however , the sampling number of sampling can be 
 increased by carrying out another random pcr using a different 
 primer , resulting in the accumulation of information on a genome 
 .
as far as the experiments that we have done are concerned ( several hundreds of species and strains ) , a strong 
 correlation between the pass value and the relatedness of two 
 genomes has been observed [ 12 , 18 , 26 ] , which can be 
 theoretically rationalized   and is also experimentally verified in this paper ( taking into consideration all of these 
 facts , the pass value can be assumed to be semiquantitative as discussed later ) . here , to test our method , we collected and used three domains of 
 organisms  namely , plants , fish , and insects  that are 
 taxonomically well established ( table 1 ) .
figures 
 figure 3(a)figure 3(c ) shows the species that we tested here 
 and the pass values obtained among them , respectively .
to 
 illustrate the technique , some of the results of gp and 
 spiddos representations for plant and fish are shown in 
 figure 2 , where the pairs of panels a / b ( a1 : 
 typha orientalis , a9 : viola xwittrockiana ) and 
 panels e / f ( c1 : oncorhynchus masou , c4 : salmo 
 trutta ) apparently represent closer relationships than do the 
 pairs of panels c / d ( a11 : davidia involucrata , a12 : 
 hydrangea macrophylla ) and g / h ( c8 : osmerus 
 eperlanus mordax , c12 : barbatula barbatula ) , as 
 expected . the results obtained here for plants , fish , and insects 
 were clustered ( nearest neighbor method ) to determine intra - domains .
on the basis of taxonomical knowledge , which has 
 been established according to phenotypic traits , and the 
 clustering results , two phylogenetic trees were constructed as 
 shown in figure 4 .
all of the organisms examined were 
 classified topologically to the same position in both the 
 phenotype- and the genotype - based trees ( note that , throughout 
 this study , no arbitrary selection of data was made except for 
 ruling out a few low - quality data samples , meaning that the 
 correspondence between the phenotype - based and the genotype - based 
 classifications was perfect so far as tested ) .
it is surprising that such a limited amount of information ( gp 
 obtained with only a single oligonucleotide probe ) can provide 
 such perfect results for all organisms tested .
although it had 
 been believed that gp should be able to classify species 
 , it was considered that it would be better to use three 
 or more probes per genome .
of course , another surprise is the 
 strong correspondence between the results obtained by two quite 
 different approaches : the traditional phenotype - based taxonomy 
 ( which by its nature is based on the well - considered but rather 
 arbitrarily selected traits ) and a genome sequence - based one 
 ( which is not directly based on sequence information itself ) . 

theoretically , some ranks of hierarchy must be interconvertible in 
 phenotype - based taxonomy so that such a correspondence is not a 
 matter of course .
apart from the rrna and the other sequencing approaches 
 described above , this is the first report describing the 
 procedure of making phylogenetic trees of mutually - distant 
 organisms based on the same criterion . because the rrna approach 
 needs different pairs of primers to analyze a wide range of 
 organisms , our simple approach is advantageous and can be used to 
 complement the rrna one .
notably , the length of branches in the 
 phenotype - based tree is arbitrary and mainly implies topological 
 meaning , whereas those in the genotype - based tree have a 
 quantitative meaning : the longer the tree is , the more distantly 
 related the organisms are . the results obtained here indicate that 
 the quantitative expression of pass is very effective to some 
 extent , even though the accuracy of the measure given by pass is 
 thought to be limited , a priori , due to its stochastic nature   ( i.e. , there are some steps that are stochastic in nature and can influence determination of the pass value : for example , random pcr may or may not select a dna 
 fragment containing mutations , and the degree of displacement 
 caused by a point mutation depends on the type of mutation such as 
 a to g or a to c substitution , among others ; further consideration will lead to the conclusion that this is the case 
 not only with the gp approach but also with other approaches that 
 depend on the comparison of a particular gene sequence ) . in other words , even though the clustering of the organisms 
 considered here was done , for the sake of simplicity , on the basis 
 of a single experimental result obtained with a common 
 oligonucleotide probe ( dagaacgcgcctg , pfm12 ) , the results 
 were taxonomically consistent . at the current level of data ( i.e. , 
 relatively small - scale sampling ) , we may be able to suggest 
 conservatively that fish are widely classified on the basis of a 
 more limited number of genes than are other organisms such 
 as plants and insects , as can be seen in figures 2 and 
 4 , where relatively small differences in the genome 
 sequence ( measured by pass ) are observed among species of fish , 
 although the possibility of biased sampling can not be completely 
 ruled out .
evidently , by using more kinds of probes , taxonomically more 
 reliable results can be obtained , as we have demonstrated 
 experimentally for other organisms such as fungi and rice ( to be 
 published elsewhere ) .
an excellent feature of this methodology is 
 that the amount of information used for classification can be 
 increased on demand   without limitation , simply by 
 repeatedly performing an additional random pcr with a different 
 oligonucleotide probe and thus obtaining additional 
 spiddos ( which can be expressed as 
  information - scalable  ) .
thus , this methodology has   the potential to become a highly accurate classification tool , as well 
 as a convenient , universal one .
moreover , it has been already 
 demonstrated that dnas that provide spiddos can be 
 collected and sequenced if necessary [ 26 , 30 ] . as mentioned above ,
gp has proved to be useful for identifying 
 species [ 12 , 14 ] , in addition to classifying species as shown 
 here .
it could therefore be said that the ability of gp to 
 classify species was indirectly supported by the earlier studies , 
 but it had not been explicitly demonstrated .
we can not but feel 
 the splendor of the correspondence between the phenotypic world 
 and the genotypic one , although its meaning needs to be considered 
 more deeply hereafter .
 traditionally , organisms have been classified on the basis of their phenotype . recently , genotype - based classification has become possible through the development of sequencing technology . however , it is still difficult to apply sequencing approaches to the analysis of a large number of species due to the cost and labor . in most biological fields , 
 the analysis of complex systems comprising various species has become an important theme , demanding an effective method for handling a vast number of species . in this paper , we have demonstrated , using plants , fish , and insects , that genome profiling , a compact technology for genome analysis , can classify organisms universally . 
 surprisingly , in all three of the domains of organisms tested , the phylogenetic trees generated from the phenotype topologically matched completely those generated from the genotype . 
 furthermore , a single probe was sufficient for the genome profiling , thereby demonstrating that this methodology is universal and compact . 
1. INTRODUCTION
2. MATERIALS AND METHODS
3. RESULTS AND DISCUSSION