the sequential deletion method and other biological and biochemical experiments are generally used to locate the functional domain of a protein .
for example , a previous study ( 1 ) used the sequential deletion method accompanied by manual pcr primers design to generate the n - terminal truncated mutants of different lengths ( figure 1 ) , which in turn were used in further biological experiments to decipher the functional domain of the 5s rna - protein complex ( 5s rrnp ) .
the 5s rrnp is believed to be formed by a co - translation event leading to the binding of the 5s rrna to the nascent ribosomal protein l5 .
lin et al . ( 1 ) used an in vitro translation system to investigate how and when 5s rrna triggers the formation of the eukaryotic 5s rrnp .
the l5 and truncated l5 mutant mrnas were prepared on a large scale for their investigation and a great amount of time was needed to manually modify the in - frame pattern of atg start codon for conventional pcr and truncated mutant translation experiments . 

the illustration of the produced n - terminal truncated mutants . in order to save time and money needed in the traditional sequential deletion method
, this web - based application system ntmg is proposed to automatically do the multiplex pcr assays design in order to generate the various n - terminal truncated mutants . given a protein cdna sequence ,
the ntmg will first find those atg - like codons that are suitable to act as the starting positions of truncated mutants .
then , the ntmg will design the forward primers for all possible truncated mutants . finally , with all these primers
, the ntmg will choose those primers that can be divided into the least number of groups such that each group constitutes a multiplex pcr assay .
in this section , we describe the input to the ntmg , the methodology of the ntmg and the output of the ntmg . since the primer design and the multiplex pcr primer design are two important parts of the ntmg , some factors concerning the primer design such as the primer length and the melting temperature are input as parameters .
there are also some factors that may affect the multiplex pcr amplification with multiple primers in the same tube ( 2,3 ) .
these factors include the cross - dimerization , the melting temperature , the products co - existence and others .
figure 2 gives the input screen of the ntmg . on the top of the screen ,
the cdna sequence , the start codon address and the stop codon address are input . then comes four classes of parameters : 
 primer criteria ( 47)these include the forward primer length ( default 2030  bp ) , the reverse primer length ( default 1428  bp ) and the gc content ( default 40 - 60%).temperature criteria ( 5,810)these include melting temperature ( default 5160c ) , the melting temperature range for each group ( default 5c ) , the molar concentration of monovalent cation ( default 50 
mmol / l ) , the molar concentration of mg ( default 1.25  mmol / l ) and the dntps concentration ( default 0.02  mmol / l).complementary criteria ( 4,8,1012)these include the terminal repeated sequence ( default 3  bp ) , the intra self - complementary sequence ( default 3  bp ) , the specificity ( default 65% ) , the cross - dimer distance ( default 10  bp ) , the cross - dimer  total similarity ( default 50% ) and the cross - dimer  terminal similarity ( default 3  bp).grouping criteriathese include the product length difference ( default 80  bp ) and the maximum number of primers in each group ( default 16 ) . 
 figure 2.the input screen of the ntmg . primer criteria ( 47 )
these include the forward primer length ( default 2030  bp ) , the reverse primer length ( default 1428  bp ) and the gc content ( default 40 - 60% ) .
temperature criteria ( 5,810 ) these include melting temperature ( default 5160c ) , the melting temperature range for each group ( default 5c ) , the molar concentration of monovalent cation ( default 50  mmol / l ) , the molar concentration of mg ( default 1.25  mmol / l ) and the dntps concentration ( default 0.02  mmol / l ) .
complementary criteria ( 4,8,1012 ) these include the terminal repeated sequence ( default 3  bp ) , the intra self - complementary sequence ( default 3  bp ) , the specificity ( default 65% ) , the cross - dimer distance ( default 10  bp ) , the cross - dimer  total similarity ( default 50% ) and the cross - dimer  terminal similarity ( default 3  bp ) .
these include the product length difference ( default 80  bp ) and the maximum number of primers in each group ( default 16 ) .
first , a cdna sequence is input , then the ntmg searches the sequence with in - frame criterion in order to find all the atg - like codons .
an atg - like codon is a xtg , an axg or an atx with x representing a , t , c or g. for each atg - like codon , the ntmg generates candidate primers using the sliding window .
the ntmg also modifies the atg - like codon into the atg codon in each candidate primer .
hence , each candidate primer contains the atg start codon with in - frame criterion that can translate correctly to truncated mutants .
after that , the ntmg applies the primary criteria and the secondary criteria to check if the candidate primers satisfy all the criteria .
those candidate primers that pass the criteria checking are then divided into classes according to the melting temperature .
each class contains primers whose melting temperatures are within the same range ( default 5c ) .
so the primers chosen from the same class may be used in the same pcr experiment . for candidate primers in the same class
, the ntmg does the cross - dimer check and the co - existence check and builds the cross - dimer matrix and the co - existence matrix .
these two matrices are then anded to produce a new matrix which acts as an adjacency matrix of a graph .
previous studies ( 13,14 ) proposed the transformation of minimizing the number of primers into the finding of the maximum clique in a graph .
next , each class is further divided into subclasses according to the positions of the atg - like codons .
the proposed genetic algorithm is then applied for each class in order to choose a set of primers , one from each subclass and the objective is to divide these primers into as few groups as possible such that each group of primers can be put in a tube in the pcr experiment .
that is , the genetic algorithm tries to find a good multiplex pcr assay design .
a heuristic maximum clique algorithm is proposed to calculate the fitness of a chromosome in the genetic algorithm .
this heuristic algorithm tries to find the maximum clique in the graph previously mentioned and the maximum clique corresponds to the minimum number of grouping .
the ntmg is written in java using java 2 platform standard edition 5.0 development kit ( j2sdk ) and employs the java server page ( jsp ) on the apache tomcat server ( http://tomcat.apache.org/ ) .
the ntmg outputs the number of primer groups , the number of forward primers , the number of reverse primers and the primers in each group ( figure 4 shows a solution report ) . 

figure 2 gives the input screen of the ntmg . on the top of the screen ,
the cdna sequence , the start codon address and the stop codon address are input . then comes four classes of parameters : 
 primer criteria ( 47)these include the forward primer length ( default 2030  bp ) , the reverse primer length ( default 1428  bp ) and the gc content ( default 40 - 60%).temperature criteria ( 5,810)these include melting temperature ( default 5160c ) , the melting temperature range for each group ( default 5c ) , the molar concentration of monovalent cation ( default 50  mmol / l ) , the molar concentration of mg ( default 1.25  mmol / l ) and the dntps concentration ( default 0.02  mmol / l).complementary criteria ( 4,8,1012)these include the terminal repeated sequence ( default 3  bp ) , the intra self - complementary sequence ( default 3  bp ) , the specificity ( default 65% ) , the cross - dimer distance ( default 10  bp ) , the cross - dimer  total similarity ( default 50% ) and the cross - dimer  terminal similarity ( default 3  bp).grouping criteriathese include the product length difference ( default 80  bp ) and the maximum number of primers in each group ( default 16 ) . 
 figure 2.the input screen of the ntmg . primer criteria ( 47 )
these include the forward primer length ( default 2030  bp ) , the reverse primer length ( default 1428  bp ) and the gc content ( default 40 - 60% ) .
temperature criteria ( 5,810 ) these include melting temperature ( default 5160c ) , the melting temperature range for each group ( default 5c ) , the molar concentration of monovalent cation ( default 50  mmol / l ) , the molar concentration of mg ( default 1.25  mmol / l ) and the dntps concentration ( default 0.02  mmol / l ) .
complementary criteria ( 4,8,1012 ) these include the terminal repeated sequence ( default 3  bp ) , the intra self - complementary sequence ( default 3  bp ) , the specificity ( default 65% ) , the cross - dimer distance ( default 10  bp ) , the cross - dimer  total similarity ( default 50% ) and the cross - dimer  terminal similarity ( default 3  bp ) .
these include the product length difference ( default 80  bp ) and the maximum number of primers in each group ( default 16 ) . the input screen of the ntmg .
first , a cdna sequence is input , then the ntmg searches the sequence with in - frame criterion in order to find all the atg - like codons .
an atg - like codon is a xtg , an axg or an atx with x representing a , t , c or g. for each atg - like codon , the ntmg generates candidate primers using the sliding window .
the ntmg also modifies the atg - like codon into the atg codon in each candidate primer .
hence , each candidate primer contains the atg start codon with in - frame criterion that can translate correctly to truncated mutants .
after that , the ntmg applies the primary criteria and the secondary criteria to check if the candidate primers satisfy all the criteria .
those candidate primers that pass the criteria checking are then divided into classes according to the melting temperature .
each class contains primers whose melting temperatures are within the same range ( default 5c ) . so the primers chosen from the same class may be used in the same pcr experiment . for candidate primers in the same class
, the ntmg does the cross - dimer check and the co - existence check and builds the cross - dimer matrix and the co - existence matrix .
these two matrices are then anded to produce a new matrix which acts as an adjacency matrix of a graph .
previous studies ( 13,14 ) proposed the transformation of minimizing the number of primers into the finding of the maximum clique in a graph .
next , each class is further divided into subclasses according to the positions of the atg - like codons .
the proposed genetic algorithm is then applied for each class in order to choose a set of primers , one from each subclass and the objective is to divide these primers into as few groups as possible such that each group of primers can be put in a tube in the pcr experiment .
that is , the genetic algorithm tries to find a good multiplex pcr assay design .
a heuristic maximum clique algorithm is proposed to calculate the fitness of a chromosome in the genetic algorithm .
this heuristic algorithm tries to find the maximum clique in the graph previously mentioned and the maximum clique corresponds to the minimum number of grouping .
the ntmg is written in java using java 2 platform standard edition 5.0 development kit ( j2sdk ) and employs the java server page ( jsp ) on the apache tomcat server ( http://tomcat.apache.org/ ) .
the ntmg outputs the number of primer groups , the number of forward primers , the number of reverse primers and the primers in each group ( figure 4 shows a solution report ) . 

a web - based application system called the ntmg is provided for researches who need to apply the sequential deletion method to locate the functional domain of a protein .
each group of primers can be put in a tube and all tubes can be accommodated in a single batch of the multiplex pcr amplification under the same condition .
we conducted a wet laboratory experiment on the multiplex pcr assay design proposed by the ntmg on input hl5 cdna .
the ntmg found 48 forward primers and one reverse primer and it divided them into 8 groups . in the wet pcr experiment , 44 pcr products had been found and the success rate is 91.7% ( see supplementary data ) .
hence , the ntmg is of practical use to researchers who need to apply the sequential deletion method . as a future work
, we plan to develop the more general multiplex pcr assay design . given a set of pcr experiment requirements
, we plan to develop a system that can automatically find the primers and try to divide the primers into as few groups as possible such that the primers in each group can be put in a tube and all tubes can be accommodated in a single batch of the multiplex pcr experiment .
 the sequential deletion method is generally used to locate the functional domain of a protein . with this method , in order to find the various n - terminal truncated mutants , researchers have to investigate the atg - like codons , to design various multiplex polymerase chain reaction ( pcr ) forward primers and to do several pcr experiments . this web server ( n - terminal truncated mutants generator for cdna ) 
 will automatically generate groups of forward pcr primers and the corresponding reverse pcr primers that can be used in a single batch of a multiplex pcr experiment to extract the various n - terminal truncated mutants . 
 this saves much time and money for those who use the sequential deletion method in their research . 
 this server is available at http://oblab.cs.nchu.edu.tw:8080/websdl/. 
INTRODUCTION
SYSTEM
Input Parameters
Methodology
Environment
Output
CONCLUSION AND FUTURE WORK
SUPPLEMENTARY DATA