Methods and probes for identifying a nucleotide sequence

Abstract

The present invention provides a method for identifying a set of target nucleotide sequences capable of identifying a member of a group of related nucleotide sequences, the method comprising the step of dividing the nucleotide sequence of each member of the group into a plurality of subsequences, wherein at least two of the subsequences overlap. The method is useful in generating probe sets capable of assigning alleles at HLA or KIR loci.

1 . A method for identifying a set of target nucleotide sequences capable of identifying a member of a group of related nucleotide sequences, the method comprising the step of dividing the nucleotide sequence of each member of the group into a plurality of subsequences, wherein at least two of the subsequences overlap. 
     
     
         2 . A method according to  claim 1  wherein at least three of the subsequences overlap with each other. 
     
     
         3 . A method according to  claim 1  wherein at least four of the subsequences overlap with each other. 
     
     
         4 . A method according to  claim 1  wherein at least five of the subsequences overlap with each other. 
     
     
         5 . A method according to  claim 1  wherein the overlap is complete overlap. 
     
     
         6 . A method according to  claim 1  comprising the step of analyzing at least a portion of the subsequences for redundancy. 
     
     
         7 . A method according to  claim 1  wherein one or more of the subsequences does not contain one or more polymorphic sites at, or toward, the 5′ and/or 3′ ends of the one or more subsequences. 
     
     
         8 . A method according to  claim 1  wherein one or more of the subsequences contains one or more polymorphic sites at, or toward, the center of the one or more subsequences. 
     
     
         9 . A method according to  claim 1  wherein one or more of the subsequences contain one polymorphic site at the center of the one or more subsequences. 
     
     
         10 . A method according to  claim 1  wherein the related sequences differ by the presence of one or more nucleotide polymorphisms. 
     
     
         11 . A method according to  claim 10  wherein the nucleotide polymorphisms are single nucleotide polymorphisms. 
     
     
         12 . A method according to  claim 1  wherein the subsequences are probe-length. 
     
     
         13 . A method according to  claim 1  wherein the subsequences are from about 10 to about 50 nucleotides in length. 
     
     
         14 . A method according to  claim 1  wherein the subsequences are from about 15 to about 35 nucleotides in length. 
     
     
         15 . A method according to  claim 1  wherein the subsequences are about 25 nucleotides in length. 
     
     
         16 . A method according to  claim 1  wherein all subsequences are of the same or similar length. 
     
     
         17 . A method according to  claim 1  wherein the related nucleotide sequences have a sequence identity of at least 50%, 60%, 70%, 80%, 90%, 95% or 99%. 
     
     
         18 . A method according to  claim 1  wherein the related sequences exhibit SNPs at a high density. 
     
     
         19 . A method according to  claim 1  wherein the related sequences are protein coding, non-coding, or a combination of protein coding and non-coding. 
     
     
         20 . A method according to  claim 1  wherein the related sequences are directed to the same region of a genome. 
     
     
         21 . A method according to  claim 1  wherein the related nucleotide sequences are alleles of a gene. 
     
     
         22 . A method according to  claim 1  wherein the number of related nucleotide sequences in the group of related nucleotide sequences is more than 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000. 
     
     
         23 . A method according to  claim 1  wherein the related nucleotide sequences are part of a gene locus involved in the immune system. 
     
     
         24 . A method according to  claim 23  wherein the locus is a locus of the Major Histocompatability Complex (MHC), the T-cell receptor, the B-cell receptor, the Killer Inhibitory Receptor, or an immunoglobulin. 
     
     
         25 . A method according to  claim 23  wherein the locus is a locus of the Human Leukocyte Antigen (HLA) system. 
     
     
         26 . A method according to  claim 23  wherein the wherein the locus is a Class I or Class II MHC transmembrane protein. 
     
     
         27 . A method according to  claim 23  wherein the locus is a DR, DQ or DP locus. 
     
     
         28 . A method according to  claim 6  comprising removal or non-inclusion of at least one redundant sequence from the set of target nucleotide sequences. 
     
     
         29 . A method according to  claim 28  wherein the method reduces the number of sequences in the set of target nucleotide sequences by a multiple of at least about 5, 10 or 20 from the number of probes expected by theory. 
     
     
         30 . A method according to  claim 28  wherein the method reduces the probe number by at least about 50%, 60%, 70%, 80%, 90% or 95%. 
     
     
         31 . A method according to  claim 28  wherein substantially all redundant sequences are removed, or are not included, in the probe set. 
     
     
         32 . A method according to  claim 1 , wherein the method is amenable to automation. 
     
     
         33 . A method according to  claim 1 , wherein the method is capable of identifying new polymorphic sites, or new combinations of polymorphic sites in the related sequences. 
     
     
         34 . A probe set capable of specifically hybridizing to target nucleotide sequences identified by a method according to  claim 1 . 
     
     
         35 . A probe set according to  claim 34  wherein at least one probe comprises a label selected from the group consisting of Cy5, Cy3, FITC, rhodamine, biotin, DIG and a radioisotope. 
     
     
         36 . A solid matrix including an immobilized probe set according to  claim 34 . 
     
     
         37 . A solid matrix according to  claim 36 , wherein the solid matrix is a microarray chip. 
     
     
         38 . A method of identifying a member of a group of related nucleotide sequences using a probe set according to  claim 34 . 
     
     
         39 . A method according to  claim 38  comprising the step of recovering a member of a group of related nucleotide sequences using a probe set according to  claim 34 . 
     
     
         40 . A method according to  claim 39  comprising the steps of exposing the probe set to the group of related nucleotide sequences under conditions allowing a probe of the probe set to bind to a nucleotide sequence of the group of related nucleotide sequences to form a probe/nucleotide sequence complex, and substantially isolating the probe/nucleotide sequence complex. 
     
     
         41 . A method of definitive allele assignment comprising use of a probe set according to  claim 34 . 
     
     
         42 . A method of transplantation tissue typing based on the HLA system comprising use of a probe set according to  claim 34 . 
     
     
         43 . A method of identifying a new allele comprising use of a probe set according to  claim 34 . 
     
     
         44 . A computer executable code capable of executing a method according to  claim 1 . 
     
     
         45 . A method according to  claim 1  substantially as herein before described with reference to any of the Figures or Examples.