Customized oligonucleotide microchips that convert multiple genetic information to simple patterns, are portable and reusable

Abstract

This invention relates to using customized oligonucleotide microchips as biosensors for the detection and identification of nucleic acids specific for different genes, organisms and/or individuals in the environment, in food and in biological samples. The microchips are designed to convert multiple bits of genetic information into simpler patterns of signals that are interpreted as a unit. Because of an improved method of hybridizing oligonucleotides from samples to microchips, microchips are reusable and transportable. For field study, portable laser or bar code scanners are suitable.

We claim:  
     
         1 . A method for using a reusable microchip to identify at least one nitrogen base sequence in nucleic acids of a sample, said method comprising: 
 (a) providing a customized matrix of oligonucleotides on the microchip in an arrangement that is an ordered scheme designed to present a detectable pattern as a whole that identifies the nitrogen base sequences of nucleic acids in the sample;    (b) hybridizing nucleic acids from the sample on said microchip; and    (c) identifying at least one nitrogen base sequence in said sample by the pattern detected as a whole on the microchip, said pattern resulting from the locations of oligonucleotides which hybridized to the sample nucleic acids.    
     
     
         2 . The method of  claim 1 , wherein said nitrogen base sequence is in a DNA molecule.  
     
     
         3 . The method of  claim 1 , wherein said nitrogen base sequence is in a 16S RNA, mRNA or other RNA molecule.  
     
     
         4 . The method of  claim 1 , wherein the customized matrix of oligonucleotides on the reusable microchip is formed by a plurality of gel elements, wherein the number of elements is determined by the number of oligonucleotides in the matrix and wherein each gel element contains one oligonucleotide of a desired nitrogen base sequence length and concentration, each gel element being separated from another by hydrophobic glass spaces, and wherein the gel elements have a vertical height above the plane of the interstitial spaces of not more than about 30 μm, said oligonucleotides being positioned in specific locations.  
     
     
         7 . The method of  claim 1 , further comprising: 
 (e) adding a label to the nitrogen base sequences in said sample before hybridizing them to oligonucleotides on the microchip.    
     
     
         8 . The method of  claim 7 , wherein the label is a fluorescent dye.  
     
     
         9 . The method of  claim 7 , wherein the label is a plurality of different dyes.  
     
     
         11 . A diagnostic assay for the presence of a mutation in a gene in a sample said assay comprising: 
 (d) designing a customized reusable microchip biosensor comprising at least one oligonucleotide that hybridizes to a gene having the mutation, said designing producing an overall pattern that is detectable in the presence of said hybridization of the mutation;    (e) contacting the sample to the customized microchip biosensor under conditions that allow hybridization of the gene sequence that is a mutation to oligonucleotides on the microchip; and    (f)determining whether hybridization occurs by analyzing the microchip pattern from which presence of the mutation in the gene is determined.    
     
     
         12 . A method using non-equilibrium melting curves to detect mismatches between a nitrogen base sequence on a microchip and a nitrogen base sequence to be tested, said method comprising: 
 (g) generating the non-equilibrium melting curves by simultaneously monitoring hybridization between the nitrogen base sequence that matches or mismatches with sequences on the microchip at a series of temperatures;    (h) selecting the temperature at which maximum discrimination occurs between the match and the mismatch; and    (i) determining the degree of mismatch of the nitrogen base sequence to be tested at the selected temperature.    
     
     
         13 . The method of  claim 12 , wherein the selected temperature is that temperature at which the signal intensity of a mismatched sequence is at least one tenth of the signal intensity of a matched sequence.  
     
     
         14 . A customized oligonucleotide microchip for the detection of a betaglobin mutation, said microchip containing seven oligonucleotides having the following nitrogen base sequences:  
       
         
           
                 
                 
                 
               
                     
                 
                     
                 
                   Identifier 
                   Sequences 
                   SEQ ID NO: 
                 
                     
                 
                     
                 
                 
                 
                 
               
                   IVS (N) 
                   CCTGGGCAGGTTGGTATCA; 
                   48 
                 
                     
                 
                   IVS I/2 T/A 
                   CCTGGGCAGGaTGGTATCA; 
                   49 
                 
                     
                 
                   IVS I/1 G/A 
                   CCTGGGCAGaTTGGTATCA; 
                   50 
                 
                     
                 
                   IVS I/6 T/C 
                   CCTGGGCAGGTTGcTATCA; 
                   51 
                 
                     
                 
                   IVS 1/5 G/T 
                   CCTGGGCAGGTTGtTATCA; 
                   52 
                 
                     
                 
                   CD26 (N) 
                   GTTGGTGGTGAGGCCCTGG; 
                   53 
                 
                     
                 
                   CD26 G/A 
                   GTTGGTGGTaAGGCCCTGG 
                   54. 
                 
                     
                 
                     
                 
             
                
                
                
                
               
               
                
               
            
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         15 . A reusable customized oligonucleotide microchip for quantitation of the expression of a gene, wherein the design of the microchip presents a pattern corresponding to the degree of expression.  
     
     
         16 . A customized oligonucleotide microchip for the detection of HLA polymorphism, wherein the design of the microchip presents a pattern in which HLA probes are positioned to answer a specific question when hybridization occurs.  
     
     
         17 . The customized oligonucleotide microchip of  claim 16 , comprising the following oligonucleotides;  
       
         
           
                 
                 
                 
                 
                 
               
                     
                     
                 
                     
                   AGGCAACGTG 
                    (1); 
                   (SEQ 1D:55) 
                     
                 
                     
                     
                 
                     
                   AGGCGACGTG 
                    (2); 
                   (SEQ ID:56) 
                 
                     
                     
                 
                     
                   GGTGAACTGG 
                    (3); 
                   (SEQ ID:57) 
                 
                     
                     
                 
                     
                   TAAATCTGCG 
                    (4); 
                   (SEQ ID:58) 
                 
                     
                     
                 
                     
                   AGGCAACATG 
                    (5); 
                   (SEQ ID:59) 
                 
                     
                     
                 
                     
                   CAAAACCTCC 
                    (6); 
                   (SEQ ID:60) 
                 
                     
                     
                 
                     
                   GCAAACACCA 
                    (7); 
                   (SEQ ID:61) 
                 
                     
                     
                 
                     
                   TACACCATAA 
                    (8); 
                   (SEQ ID:62) 
                 
                     
                     
                 
                     
                   ACTGCTCATC 
                    (9); 
                   (SEQ ID:63) 
                 
                     
                     
                 
                     
                   CAATGTCTTC 
                   (10); 
                   (SEQ ID:64) 
                 
                     
                     
                 
                     
                   CTCCTCATCT 
                   (11); 
                   (SEQ ID:65) 
                 
                     
                     
                 
                     
                   TGCCGGTCAA 
                   (12); 
                   (SEQ ID:66) 
                 
                     
                     
                 
                     
                   TTAGGACAGC 
                   (13); 
                   (SEQ ID:67) 
                 
                     
                     
                 
                     
                   ACACCACAAG 
                   (14); 
                   (SEQ ID:68) 
                 
                     
                     
                 
                     
                   CACAATGCCT 
                   (15); 
                   (SEQ ID:69) 
                 
                     
                     
                 
                     
                   CAGCAGTAGA 
                   (16); 
                   (SEQ ID:70) 
                 
                     
                     
                 
                     
                   TGCGGGTCAA 
                   (17); 
                   (SEQ ID:71) 
                 
                     
                     
                 
                     
                   TTAGCACACC 
                   (18). 
                   (SEQ TD:72) 
                 
                     
                     
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         18 . A method for designing a reusable genetic microchip to answer a specific question about a test sample, said answer provided by detection of hybridization patterns of the nitrogen base sequences of nucleic or ribonucleic acids of molecules present in the sample, on the microchip as a whole, said method comprising: 
 (a) determining the specific question; and    (b) designing the microchip so that hybridization of nucleic acids present in the sample provides a pattern that when detected as a whole provides an answer to the question; and    (c) detecting the pattern.    
     
     
         19 . The method of  claim 18 , wherein the sample is water, and the question is what pathogens are present.  
     
     
         20 . The method of  claim 18 , wherein the sample is blood, and the question is what genetic mutations are present.  
     
     
         21 . The method of  claim 18 , wherein the sample is a food, and the specific question is what containments are present.  
     
     
         22 . The method of  claim 18 , wherein the pattern is a bar code pattern and detecting the pattern is done by a bar code laser scanner.