Probe design methods and microarrays for comparative genomic hybridization and location analysis

Abstract

Methods and systems for identifying and selecting nucleic acid probes for detecting a target with a nucleic acid probe array or comparative genome hybridization microarray, comprising selecting a plurality of potential target sequences, generating a plurality of candidate probes from the target sequences, filtering the plurality of candidate probes by analyzing candidate probes for selected probe properties in silico. Microarrays comprising probes selected by the methods of the invention are particularly useful for comparative genome hybridization and location analysis.

1 . A microarray comprising: 
 a) a solid support: and    b) a plurality of polynucleotide probes attached to said support, said plurality of polynucleotide probes having a corresponding plurality of different nucleotide sequences, at least 50% of said polynucleotide probes have a duplex T m  within a delta T m  of less than 4° C.    
   
   
       2 . The microarray of  claim 1 , wherein at least 1,000 said polynucleotide probes are surface bound to said support.  
   
   
       3 . The microarray of  claim 2 , wherein at least 2,000 said polynucleotide probes are surface bound to said support.  
   
   
       4 . The microarray of  claim 2 , wherein at least 10,000 said polynucleotide probes are surface bound to said support.  
   
   
       5 . The microarray of  claim 2 , wherein at least 40,000 said polynucleotide probes are surface bound to said support.  
   
   
       6 . The microarray of  claim 2 , wherein at least 20,000 said polynucleotide probes are surface bound to said support.  
   
   
       7 . The microarray of  claim 2 , wherein at least 80% of said polynucleotide probes have a duplex T m  within a delta T m  of less than 4° C.  
   
   
       8 . The microarray of  claim 2 , wherein at least 90% of said polynucleotide probes have a duplex Tm within a delta Tm of less than 4° C.  
   
   
       9 . The microarray of  claim 2 , wherein said delta Tm is less than 3° C.  
   
   
       10 . The microarray of  claim 7 , wherein said delta T m  is less than 3° C.  
   
   
       11 . The microarray of  claim 8 , wherein said delta T m  is less than 3° C.  
   
   
       12 . The microarray of  claim 2 , wherein said delta T m  is less than 2° C.  
   
   
       13 . The microarray of  claim 7 , wherein said delta T m  is less than 2° C.  
   
   
       14 . The microarray of  claim 8 , wherein said delta T m  is less than 2° C.  
   
   
       15 . The microarray of  claim 2 , wherein said delta T m  is less than 1° C.  
   
   
       16 . The microarray of  claim 7 , wherein said delta T m  is less than 1.5° C.  
   
   
       17 . The microarray of  claim 2 , wherein said delta T m  is less than 0.5° C.  
   
   
       18 . The microarray of  claim 2 , wherein the nucleotide length of said polynucleotide probes are in the range of at least 20 nucleotides to 100 nucleotides.  
   
   
       19 . The microarray of  claim 2 , wherein the nucleotide length of said polynucleotide probes are in the range of at least 20 nucleotides to 100 nucleotides.  
   
   
       20 . The microarray of  claim 2 , wherein at least 50% of said polynucleotide probes on said solid support have the same nucleotide length.  
   
   
       21 . The microarray of  claim 2 , wherein said at least 50% of said polynucleotide probes are of 60 nucleotides in length.  
   
   
       22 . The microarray of  claim 2 , wherein at least 5% of said polynucleotide probes on said solid support hybridize to regulatory regions of a nucleotide sample of interest.  
   
   
       23 . The microarray of  claim 2 , wherein at least 30% of said polynucleotide probes on said solid support hybridize to exonic regions of a nucleotide sample of interest.  
   
   
       24 . The microarray of  claim 2 , wherein at least 50% of said polynucleotide probes on said solid support hybridize intergenic regions of a nucleotide sample of interest.  
   
   
       25 . The microarray of  claim 2 , wherein said polynucleotide probes are structured and configured for analysis of a nucleotide sample by comparative genome hybridization.  
   
   
       26 . The microarray of  claim 2 , wherein said nucleotide sequences of said polynucleotide probes hybridize to nucleotide samples from the human genome.  
   
   
       27 . The microarray of  claim 2 , wherein said nucleotide sequences of said polynucleotide probes hybridize to nucleotide samples from the mouse genome.  
   
   
       28 . A microarray comprising: 
 a) a solid support; and    b) at least 1,000 polynucleotide probes attached to said support, said plurality of polynucleotide probes having a corresponding plurality of different nucleotide sequences, at least 50% of said polynucleotide probes have a duplex T m  within a delta T m  of less than 4° C.    
   
   
       29 . A microarray comprising: 
 a) a solid support; and    b) a plurality of polynucleotide probes attached to said support, said plurality of polynucleotide probes having a corresponding plurality of different nucleotide sequences, where at least 70% of said polynucleotide probes have a percent GC content within a delta percent GC of 10%.    
   
   
       30 . The microarray of  claim 29 , wherein at least 1,000 said polynucleotide probes are surface bound to said support.  
   
   
       31 . The microarray of  claim 30 , wherein at least 2,000 said polynucleotide probes are surface bound to said support.  
   
   
       32 . The microarray of  claim 30 , wherein at least 10,000 said polynucleotide probes are surface bound to said support.  
   
   
       33 . The microarray of  claim 30 , wherein at least 40,000 said polynucleotide probes are surface bound to said support.  
   
   
       34 . The microarray of  claim 30 , wherein at least 20,000 said polynucleotide probes are surface bound to said support.  
   
   
       35 . A microarray comprising: 
 a) a solid support; and    b) a plurality of polynucleotide probes attached to said support, said plurality of polynucleotide probes having a corresponding plurality of different nucleotide sequences, where at least 60% of said polynucleotide probes have a percent GC content within a delta percent GC of 5%.    
   
   
       36 . The microarray of  claim 30 , wherein at least 75% of said polynucleotide probes have a percent GC content within a delta percent GC of 10%.  
   
   
       37 . The microarray of  claim 30 , wherein at least 85% of said polynucleotide probes have a percent GC content within a delta percent GC of 10%.  
   
   
       38 . The microarray of  claim 35 , where at least 70% of said polynucleotide probes have a percent GC content within a delta percent GC of 5%.  
   
   
       39 . A microarray comprising: 
 a) a solid support; and    b) a plurality of polynucleotide probes attached to said support, said plurality of polynucleotide probes having a corresponding plurality of different nucleotide sequences, where at least 40% of said polynucleotide probes have a percent GC content within a delta percent GC of 3%.    
   
   
       40 . The microarray of  claim 39 , where at least 50% of said polynucleotide probes have a percent GC content within a delta percent GC of 3%.  
   
   
       41 . The microarray of  claim 39 , where at least 60% of said polynucleotide probes have a percent GC content within a delta percent GC of 3%.  
   
   
       42 . The microarray of  claim 30 , wherein the nucleotide length of said polynucleotide probes are in the range of at least 20 nucleotides to 100 nucleotides.  
   
   
       43 . The microarray of  claim 30 , wherein the nucleotide length of said polynucleotide probes are in the range of at least 20 nucleotides to 100 nucleotides.  
   
   
       44 . The microarray of  claim 30 , wherein at least 50% of said polynucleotide probes on said solid support have the same nucleotide length.  
   
   
       45 . The microarray of  claim 30 , wherein said at least 50% of said polynucleotide probes are of 60 nucleotides in length.  
   
   
       46 . The microarray of  claim 30 , wherein at least 5% of said polynucleotide probes on said solid support hybridize to regulatory regions of a nucleotide sample of interest.  
   
   
       47 . The microarray of  claim 30 , wherein at least 30% of said polynucleotide probes on said solid support hybridize to exonic regions of a nucleotide sample of interest.  
   
   
       48 . The microarray of  claim 30 , wherein at least 50% of said polynucleotide probes on said solid support hybridize intergenic regions of a nucleotide sample of interest.  
   
   
       49 . The microarray of  claim 30 , wherein said polynucleotide probes are structured and configured for analysis of a nucleotide sample by comparative genome hybridization.  
   
   
       50 . The microarray of  claim 30 , wherein said nucleotide sequences of said polynucleotide probes hybridize to nucleotide samples generated the human genome.  
   
   
       51 . The microarray of  claim 30 , wherein said nucleotide sequences of said polynucleotide probes hybridize to nucleotide samples from the mouse genome.  
   
   
       52 . A microarray comprising: 
 a) a solid support; and    b) at least 1,000 polynucleotide probes attached to said support, said plurality of polynucleotide probes having a corresponding plurality of different nucleotide sequences, where at least 60% of said polynucleotide probes have a percent GC content within a delta percent GC of 10%.    
   
   
       54 . A method for generating candidate probes, comprising: 
 a) selecting target sequences from a chromosome of interest;    b) repeat-masking said target sequences to form non-repeat masked regions; c) tiling sequences across said non-repeat masked regions to generate said candidate probes; and    d) screening said candidate probes according to at least one in silico parameter.    
   
   
       55 . The method of  claim 54 , further comprising identifying restriction cut sites in the chromosome of interest, and selecting target sequences that exclude said restriction sites.  
   
   
       56 . The method of  claim 54 , further comprising screening said candidate probes according to at least one experimentally measurable property.  
   
   
       57 . The method of  claim 54 , further comprising validating said candidate probes by target hybridization experiment.  
   
   
       58 . The method of  claim 54 , wherein said screening according to said in silico parameters comprises annotating said candidate probes for expression and association with said chromosome of interest.  
   
   
       59 . The method of  claim 54 , wherein said screening according to said in silico parameters comprises analyzing said candidate probes for target structural stability.  
   
   
       60 . The method of  claim 54 , wherein said screening according to said in silico parameters comprises thermodynamically annotating said candidate probes.  
   
   
       61 . The method of  claim 54 , further comprising applying pairwise probe selection to said candidate probes  
   
   
       62 . The method of  claim 54 , further comprising applying biased pairwise probe selection to said candidate probes.  
   
   
       63 . The method of  claim 54 , wherein said screening according to said in silico parameters comprises determining a homology signal-to-background score for each said candidate probe.  
   
   
       64 . The method of  claim 60 , wherein said in silico parameters comprise a parameter for duplex melting temperature for said candidate probes.  
   
   
       65 . The method of  claim 60 , wherein said in silico parameters comprise a parameter for hairpin structural stability for said candidate probes.  
   
   
       66 . The method of  claim 64 , wherein said parameter for duplex melting temperature is determined by GC content of said candidate probes.  
   
   
       67 . The method of  claim 60 , wherein said in silico parameters comprise a parameter for duplex stability for said candidate probes.  
   
   
       68 . The method of  claim 67 , wherein said parameter for duplex stability evaluates said candidate probes for a property selected from the group consisting of melting temperature, entropy, enthalpy and Gibb's free energy.  
   
   
       69 . The method of  claim 65 , wherein said parameter for hairpin structural stability evaluates said candidate probes for a property selected from the group consisting of melting temperature, entropy, enthalpy and Gibb's free energy.  
   
   
       70 . The method of  claim 59 , wherein said parameter for target structural stability evaluates said candidate probes for a property selected from the group consisting of melting temperature, entropy, enthalpy and Gibb's free energy.  
   
   
       71 . The method of  claim 54 , wherein said screening according to said in silico parameters comprises analyzing said candidate probes for target specificity.  
   
   
       72 . The method of  claim 54 , wherein said in silico parameters comprise a parameter for kinetic properties of said candidate probes.  
   
   
       73 . The method of  claim 54 , wherein said in silico parameters comprise a parameter for intergenicity of said candidate probes.  
   
   
       74 . The method of  claim 54 , wherein said in silico parameters comprise a parameter for expression of said candidate probes.  
   
   
       75 . The method of  claim 73 , wherein said parameter for intergenicity evaluates said candidate probes for a property selected from the group consisting of said candidate probe sequence is within a gene, said candidate probe sequence is in between a gene, and said candidate probe sequence is within a coding region of a gene.  
   
   
       76 . The method of  claim 74 , wherein said parameter for expression evaluates said candidate probes for a property selected from the group consisting of said candidate probe sequence is within a gene, said candidate probe sequence is within an expression region of a gene and said candidate probe sequence is within a coding region of a gene.  
   
   
       77 . The method of  claim 54 , comprising eliminating said target sequences which contain a restriction enzyme site.  
   
   
       78 . The method of  claim 61 , wherein said applying pairwise selection analysis comprises, 
 selecting a plurality of probe pairs, each said probe pair comprising a first probe sequence and a second probe sequence which are adjacent probe sequences within the chromosome of interest,    evaluating said first and said second probe sequences for at least one probe property,    assigning at least one score for each said probe property to said first and second probe sequences, and determining which probe sequence of each said probe pair comprises the optimum probe characteristics for said microarray.    
   
   
       79 . The method of  claim 78 , wherein said probe pairs are randomly selected for pairwise analysis.  
   
   
       80 . The method of  78 , wherein said probe pairs are selected for said pairwise analysis by the order in which they target the chromosome sequence of interest.  
   
   
       81 . The method of  78 , wherein said probe pairs are selected for said pairwise analysis in order of the base pair gap between said first and second probe sequence.  
   
   
       82 . The method of  claim 62 , wherein said applying biased pairwise selection algorithm comprises; 
 analyzing neighboring probe sequences within a genomic region of interest, evaluating said neighboring probe sequences for a first group of probe properties,    evaluating said neighboring probe sequences for a second group of probe properties and    scoring said neighboring probe sequences for said first group of probe properties and biasing said scoring by said second group of probe properties.    
   
   
       83 . The method of  claim 82 , wherein said probe property is selected from the group consisting of duplex melting temperature, hairpin stability, GC content, probe is within an exon, probe is within a gene, probe is within an intron and probe is within a intergenic region.  
   
   
       84 . The method of  claim 56 , wherein said experimentally measurable parameter is selected from the group consisting of signal intensity, reproducibility of signal intensity, dye bias, susceptibility to non-specific binding, wash stability and persistence of probe hybridization.  
   
   
       85 . The method of  claim 57 , wherein said validating said candidate probes comprises; 
 hybridizing said candidate probes to a plurality of target sets, evaluating said candidate probes for a probe property for each said target set and comparing the values for said probe property of each said candidate probe across said plurality of target sets.    
   
   
       86 . A computer readable medium carrying one or more sequences of instructions for identifying and selecting nucleic acid probes for detecting a target with a probe array, wherein execution of one or more sequences of instructions by one or more processors causes the one or more processors to perform the steps of: 
 a) repeat-masking said target sequences to form non-repeat masked regions; and    b) tiling sequences across said non-repeat masked regions to generate said candidate probes.    
   
   
       87 . The computer readable medium of  claim 86 , further comprising the steps of; 
 a) identifying restriction cut sites in a chromosome of interest; and    b) selecting target sequences that exclude said restriction sites.