Microarray system with improved sequence specificity

Abstract

The invention provides a novel array method for nucleic acid sequence detection with improved specificity which allows for detection of genetic variation, from simple SNPs (where the variation occurs at a fixed position and is of limited allelic number) to more complex sequence variation patterns (such as with multigene families or multiple genetic strains of an organism where the sequence variation between the individual members is neither fixed nor consistent). The array is comprised of short, synthetic oligonucleotide probes attached to a solid surface which are hybridized to single-stranded targets. Single stranded targets can be produced using a method that employs primers modified on the 5′ end to prohibit degradation by a 5′-exonuclease that is introduced to degrade the unprotected strand. The invention further provides for printing buffers/solutions for the immobilization of oligonucleotide probes to an array surface. The invention also provides hybridization and wash buffers and conditions to maximize hybridization specificity and signal intensity, and reduce hybridization times.

1 . An array comprising a surface comprising epoxide moieties and a plurality of oligonucleotides each comprising a 5′ hydrazide linker attached to the surface of the array through a bond formed between the linker and an epoxide moiety. 
     
     
         2 . A method of forming a microarray on a surface comprising epoxide moieties, comprising:
 depositing a plurality of samples onto the surface in discrete domains, each sample comprising a spotting buffer and an oligonucleotide comprising a 5′ hydrazide linker, under conditions that allow formation of a bond between the hydrazide linker and the epoxide moiety.   
     
     
         3 . The method of  claim 2 , wherein the spotting buffer has a pH of less than about 8.5 and comprises monobasic sodium phosphate, an ethylene oxide based nonionic detergent, and ethylene glycol. 
     
     
         4 . The method of  claim 3 , wherein the nonionic detergent is Nonidet P-40. 
     
     
         5 . The method of  claim 3 , wherein the pH is between about 4.0 and about 8.0. 
     
     
         6 . The method of  claim 3 , wherein the pH is between about 4.5 and about 5.5.