Method of analyzing mRNA splice variants

A method for distinguishing splice variants in a mixed mRNA sample, said method comprising the steps of:

• (a) providing a mixed mRNA sample, said mixed mRNA sample comprising a plurality of splice variants, each one of said plurality of splice variants containing a distinct exon-exon junction not found in each other of said plurality of splice variants;
• (b) providing an array, said array comprising a plurality of different primers immobilized on a solid support at distinct locations thereon, with each of said plurality of different primers selectively hybridizing to a corresponding one of said distinct exon-exon junctions to form a duplex therebetween;
• (c) contacting said mixed mRNA sample to said array so that a duplex is formed between each different primer and each corresponding splice variant if said corresponding splice variant is included in said mixed mRNA sample;
• (d) subjecting said duplexes to a primer extension reaction so that the primers in said duplexes are extended with at least one labeled base;
• and then
• (e) detecting the presence or absence of said labeled base in each of said plurality of primers, the presence of said at least one labeled base indicating the presence of the splice variant to which said primer selectively binds in said mixed mRNA sample.

An array useful for determining the exons present in a potentially variantly spliced mRNA, said mRNA encoded by a DNA, said DNA comprising a plurality of exons, each of which plurality of exons may or may not be included in said mRNA, said array comprising:

• a solid support;
• and
• a plurality of different primers immobilized on said solid support at distinct locations thereon, with each of said plurality of different primers selectively hybridizing to a corresponding one of said plurality of exons under predetermined hybridization conditions;
• wherein said primers are immobilized to said solid support by the 5′ end thereof;
• and wherein said mRNA is CD44 mRNA.

An array useful for distinguishing splice variants in a mixed mRNA sample, said mixed mRNA sample comprising a plurality of splice variants, each one of said plurality of splice variants containing a distinct exon-exon junction, each of said distinct exon-exon junctions comprising a common exon segment coupled to a variable exon segment, with said common exon segment being the same among said plurality of splice variants and said variable exon segments being different among said plurality of splice variants, said array comprising:

• a solid support;
• and
• a plurality of different primers immobilized on said solid support at distinct locations thereon, with each of said plurality of different primers selectively hybridizing to a corresponding one of said distinct exon-exon junctions under predetermined hybridization conditions to form a duplex therebetween;
• wherein each of said plurality of primers contains a common primer segment coupled to a variable primer segment, with said common primer segment corresponding to said common exon segment and being the same among said plurality of primers, and with said variable primer segment corresponding to said variable exon segment and being different among said plurality of primers;
• and wherein said common primer segments are from 8 to 50 nucleotides in length and are positioned at the 5′ end of said primers, and said variable primer segments are from 2 to 7 nucleotides in length and are positioned at the 3′ end of said primers.

A system for determining the exons present in a potentially variantly spliced mRNA with a plurality of different primers, each of said plurality of primers selectively hybridizing to a corresponding one of said plurality of exons, and each of said plurality of primers being extended with at least one labeled base when said corresponding one of said plurality of exons is present, said system comprising:

• (a) a detector for detecting the presence or absence of said labeled base from each of said plurality of primers;
• (b) a signal generator operatively associated with said detector for generating a plurality of values, each of said values indicating the presence or absence of each of said exons in said mRNA from said detected presence or absence of said labeled base from each of said plurality of primers;
• and
• (c) a processor operatively associated with said signal generator for generating a code representing the exons present in said mRNA from said plurality of generated values.

A system for distinguishing splice variants in a mixed mRNA sample with a plurality of different primers, each of said plurality of primers selectively hybridizing to a distinct exon-exon junction in each of said plurality of splice variants, and each of said plurality of primers being extended with at least one labeled base when said corresponding one of said plurality of exons is present, said system comprising:

• (a) a detector for detecting the presence or absence of said labeled base from each of said plurality of primers;
• (b) a signal generator operatively associated with said detector for generating a plurality of values, each of said values indicating the presence or absence of each of said splice variants in said mixed mRNA sample from said detected presence or absence of said labeled base from each of said plurality of primers;
• and
• (c) a processor operatively associated with said signal generator for generating a determination of the splice variants present in said mixed mRNA sample from said plurality of generated values.