Iterative probe design and detailed expression profiling with flexible in-situ synthesis arrays

Abstract

Methods and compositions are provided that are useful for detecting and reporting a plurality of different target polynucleotide sequences in a sample, such as polynucleotides corresponding to a plurality of different genes expressed by a cell or cells. In particular, the invention provides methods for screening a plurality of candidate polynucleotide probes to evaluate both the sensitivity and the specificity with which each candidate probe hybridizes to a target polynucleoide sequence. Candidate polynucleotide probes can then be ranked according to both their sensitivity and specificity, and probes that have optimal sensitivity and specificity for a target polynucleotide sequence can be selected. In one embodiment, polynucleotide probes can be selected according to the methods described herein to prepare “screening chips” wherein a large number of target polynucleotide sequences are detected using a single microarray have a few (e.g., 1-5) probes for each target polynucleotide sequence. In a particularly preferred embodiment, the invention provides a screening chip that can detect genetic transcripts from the entire genome of an organism. In an alternative embodiment, polynucleotide probes can be selected according to the methods described herein to prepare “signature chips” to more accurately detect certain selected “signature genes” using several polynucleotide probes (e:g., 10-20) for each signature gene. The invention additionally provides microarrays containing polynucleotide probes for a large number of genes expressed by a cell or organism. Further, methods for detecting a plurality of polynucleotide molecules, including a large number of genes expressed by a cell or organism, are also provided.

1 - 212 . (canceled)  
     
     
         213 . A method for selecting one or more different polynucleotide probes for detecting a target polynucleotide, 
 said target polynucleotide comprising a nucleotide sequence of a gene or gene product expressed by a cell or organism,    said one or more different polynucleotide probes being selected from a plurality of different candidate polynucleotide probes, each different candidate polynucleotide probe comprising a different sequence that is complementary and hybridizable to a nucleotide sequence of said target polynucleotide,    said method comprising:    (a) selecting from said plurality of different candidate polynucleotide probes a plurality of polynucleotide probes that have a magnitude of binding energy for hybridization to said target polynucleotide that is above a selected threshold;    (b) ranking the plurality of polynucleotide probes selected in step (a) according to 
 (b1) probe length, and  
 (b2) distance of the complementary sequence for each probe in the target polynucleotide from one end of said target polynucleotide;  
   (c) de-overlapping said ranked polynucleotide probes by a method which comprises 
 (c1) selecting the top ranked polynucleotide probe from said ranked polynucleotide probes, and  
 (c2) repeatedly selecting the next polynucleotide probe from said ranked polynucleotide probes which comprises a nucleotide sequence complementary to a sequence in said target polynucleotide that overlaps the complementary sequence of the previously selected polynucleotide probe in said target polynucleotide by no more than a selected number of nucleotide bases; and  
   (d) ranking said de-overlapped polynucleotide probes according to the most negative binding energy with which each of said de-overlapped polynucleotide probes cross-hybridizes to one or more other polynucleotide sequences expressed by said cell or organism,    wherein said one or more different polynucleotide probes for detecting said target polynucleotide are selected from said ranked, de-overlapped polynucleotide probes.    
     
     
         214 . The method of  claim 213  wherein the nucleotide sequences of said target polynucleotide that are complementary to said candidate polynucleotide probes are within a selected distance from one end of said target polynucleotide.  
     
     
         215 . The method of  claim 213  further comprising, before said step of selecting polynucleotide probes having a magnitude of binding energy for hybridization to said target polynucleotide that is above said selected threshold, steps of: 
 (i) rejecting candidate polynucleotide probes which comprise one or more sequences corresponding to a repetitive element, a simple repeat or a polyX repeat; and    (ii) rejecting candidate polynucleotide probes having a fraction of one or more selected nucleotide bases or a mathematical combination of fractions of one or more particular nucleotide bases which is not within a selected range of values.    
     
     
         216 . The method of  claim 213  wherein each said binding energy is calculated according to a nearest neighbor model.  
     
     
         217 . The method of  claim 213  wherein said one or more other polynucleotide sequences expressed by said cell or organism that cross-hybridize to a candidate polynucleotide probe are identified by a method which comprises identifying polynucleotide sequences expressed by said cell or organism comprising nucleotide sequences with a selected level of homology or identity to the complementary sequence of said candidate polynucleotide probe.  
     
     
         218 . The method of  claim 213 , wherein said step (b) is carried out by ranking the plurality of polynucleotide probes selected in step (a) according to a weighted combination of length and end-distance.  
     
     
         219 . A computer system for selecting one or more different polynucleotide probes for detecting a target polynucleotide, said computer system comprising: 
 (a) a memory; and    (b) a processor element interconnected with the memory,    wherein the memory encodes one or more programs causing the processor to perform the method of  claim 213 .    
     
     
         220 . A computer program product for use in conjunction with a computer having a memory and a processor, said computer program product comprising a computer readable storage medium having a computer program mechanism encoded thereon, wherein said computer program mechanism may be loaded into the memory of a computer and cause a processor of the computer to carry out the method of  claim 213 .  
     
     
         221 . A method for selecting one or more different polynucleotide probes for detecting a target polynucleotide, 
 said target polynucleotide comprising a nucleotide sequence of a gene or gene product expressed by a cell or organism,    said one or more different polynucleotide probes being selected from a plurality of different candidate polynucleotide probes, each different candidate polynucleotide probe comprising a different sequence that is complementary and hybridizable to a nucleotide sequence of said target polynucleotide,    said method comprising:    (a) selecting from said plurality of different candidate polynucleotide probes a plurality of polynucleotide probes that have a magnitude of binding energy for hybridization to said target polynucleotide that is above a selected threshold;    (b) ranking said plurality of polynucleotide probes selected in step (a) according to a minimax score, said minimax score for each said selected polynucleotide probe (b1) being the largest magnitude of binding energy with which said selected polynucleotide probes cross-hybridizes to one or more other polynucleotide sequences expressed by said cell or organism; or (b2) being determined using the product of the binding energy with which said selected polynucleotide probes cross-hybridizes to each of said one or more other polynucleotide sequences expressed by said cell or organism and a weighting factor; and    (c) selecting one or more different polynucleotide probes from said plurality of polynucleotide probes according to their ranks,    thereby selecting one or more different polynucleotide probes for detecting said target polynucleotide.    
     
     
         222 . The method of  claim 221 , further comprising prior to said step (a) selecting said plurality of different candidate polynucleotide probes such that said plurality of different candidate polynucleotide probes are complementary to nucleotide sequences in said target polynucleotide within a selected distance from one end of said target polynucleotide.  
     
     
         223 . The method of  claim 222 , wherein said end of said target polynucleotide is the preferentially labeled end.  
     
     
         224 . The method of  claim 223 , wherein said step (a) further comprises the steps of (i) ranking said selected plurality of polynucleotide probes according to distances of their respective complementary sequences in the target polynucleotide from the preferentially labeled end of said target polynucleotide; and (ii) selecting one or more polynucleotide probes from said ranked polynucleotide probes according to said rank.  
     
     
         225 . The method of  claim 221 , further comprising before said step (b) de-overlapping said plurality of polynucleotide probes by a method comprising 
 (i) ranking said plurality of polynucleotide probes according to binding energy for hybridization to said target polynucleotide;    (ii) selecting the top ranked polynucleotide probe from said ranked polynucleotide probes;    (iii) repeatedly selecting the next polynucleotide probe from said ranked polynucleotide probes which is complementary to a sequence in said target polynucleotide that overlaps the sequence complementary to the sequence of the previously selected polynucleotide probe in said target polynucleotide by no more than a selected number of nucleotide bases; and    (iv) rejecting polynucleotide probe or probes not selected in step (iii) from said plurality of polynucleotide probes.    
     
     
         226 . The method of  claim 221 , further comprising prior to said step (a) one or more of the following steps: 
 (a1) rejecting candidate polynucleotide probes which comprise one or more sequences corresponding to a repetitive element, a simple repeat or a polyX repeat; and    (a2) rejecting candidate polynucleotide probes having a fraction of one or more selected nucleotide bases or a mathematical combination of fractions of one or more selected nucleotide bases which is not within a selected range of values.    
     
     
         227 . The method of  claim 221 , wherein said plurality of different candidate polynucleotide probes comprises a set of successive overlapping probes tiled along a sequence region of said target polynucleotide at a tiling interval of between 1 and 10.  
     
     
         228 . The method of  claim 221 , wherein said one or more other polynucleotide sequences expressed by said cell or organism that cross-hybridize to a candidate polynucleotide probe comprise polynucleotide sequences expressed by said cell or organism comprising nucleotide sequences with a selected level of homology or identity to the complementary sequence of said candidate polynucleotide probe.  
     
     
         229 . The method of  claim 221 , wherein said selecting in step (c) is carried out by selecting one or more different polynucleotide probes that have the lowest minimax score or scores.  
     
     
         230 . The method of  claim 221 , wherein each of said plurality of different candidate polynucleotide probes consists of 40 to 70 nucleotides.  
     
     
         231 . A computer system for selecting one or more different polynucleotide probes for detecting a target polynucleotide, said computer system comprising: 
 (a) a memory; and    (b) a processor element interconnected with the memory,    wherein the memory encodes one or more programs causing the processor to perform the method of claim  22   1 .    
     
     
         232 . A computer program product for use in conjunction with a computer having a memory and a processor, said computer program product comprising a computer readable storage medium having a computer program mechanism encoded thereon, wherein said computer program mechanism may be loaded into the memory of a computer and cause a processor of the computer to carry out the method of  claim 221.