DNA chip, PNA chip, and their preparation methods

Abstract

A DNA chip (or PNA chip) composed of a solid carrier and plural DNA fragments (or PNA fragments) fixed onto the solid carrier at each one end, wherein a plurality of short chain spacer molecules having a hydrophilic moiety at each one end are fixed at each another end onto a surface of the solid carrier having no DNA fragments (or no PNA fragments) on its surface is effective for high sensitive quantitative analysis of a nucleic acid fragment complementary to the DNA fragment (or PNA fragment).

What is claimed is:  
     
         1 . A DNA chip comprising a solid carrier and a plurality of DNA fragments fixed onto the solid carrier at each one end, wherein a plurality of short chain spacer molecules having a hydrophilic moiety at each one end are fixed at each another end onto a surface area of the solid carrier having no DNA fragments thereon.  
     
     
         2 . The DNA chip of  claim 1 , wherein the solid carrier is an electro-conductive substrate.  
     
     
         3 . The DNA chip of  claim 1 , wherein the DNA fragments are fixed onto the solid carrier in an amount of 10 −20  to 10 −12  mol./mm 2 .  
     
     
         4 . The DNA chip of  claim 1 , wherein the hydrophilic moiety of the spacer molecule is a hydroxyl group.  
     
     
         5 . The DNA chip of  claim 1 , wherein the spacer molecule is fixed onto the solid carrier through a mercapto moiety attached to the end of the spacer molecule.  
     
     
         6 . The DNA chip of claim.  1 , wherein the spacer molecule is derived from a compound selected from the group consisting of 2-mercaptoethanol, 3-mercaptoethanol; 6-mercaptoethanol, and N,N′-di(3-hydroxy-n-propyl)-imidazole-2-thione.  
     
     
         7 . A process for preparing a DNA chip of  claim 1  which comprises the steps of: 
 applying onto a solid carrier an aqueous solution of a plurality of DNA fragments dissolved or dispersed in an aqueous medium to fix the DNA fragments onto the solid carrier; and  
 applying onto the solid carrier having thereon the fixed DNA fragments an aqueous solution of short chain spacer molecules having at each one end a hydrophilic moiety and at each another end a moiety reactive to fix to the solid carrier.  
 
     
     
         8 . A method of quantitative analysis of a nucleic acid fragment contained in a sample liquid which is complementary to the DNA fragments of the DNA chip of  claim 2 , which comprises the steps of: 
 adjusting the concentration of the nucleic acid fragment in the sample liquid so that a droplet of the sample liquid applied to the DNA chip should contain 10 −20  to 10 −16  mol. of the nucleic acid fragment per 1 mm 2  of the surface of the electro-conductive substrate of the DNA chip;    bringing the nucleic acid concentration-adjusted sample liquid into contact with the DNA chip, whereby hybridizing the nucleic acid with the DNA fragment on the DNA chip;    bringing an electrochemically active molecule in contact with the hybridized nucleic acid and DNA fragment, whereby attaching the electrochemically active molecule to the hybridized nucleic acid and DNA fragment;    applying a potential to the DNA chip; and    measuring an electric current flowing from or to the electro-conductive substrate through the attached electrochemically active molecule.    
     
     
         9 . A kit for conducting quantitative analysis of a nucleic acid fragment contained in a sample liquid which is complementary to the DNA fragments of the DNA chip of  claim 2 , which comprises the DNA chip of  claim 2  and an electrochemically active molecule which is attachable to a hybridized nucleic acid and DNA fragment.  
     
     
         10 . A PNA chip comprising a solid carrier and a plurality of PNA fragments fixed onto the solid carrier at each one end, wherein a plurality of short chain spacer molecules having a hydrophilic moiety at each one end are fixed at each another end onto a surface area of the solid carrier having no PNA fragments thereon.  
     
     
         11 . The PNA chip of  claim 10 , wherein the solid carrier is an electro-conductive substrate.  
     
     
         12 . The PNA chip of  claim 10 , wherein the DNA fragments are fixed onto the solid carrier in an amount of 10 −20  to 10 −12  mol./mm 2 .  
     
     
         13 . The PNA chip of  claim 10 , wherein the hydrophilic moiety of the spacer molecule is a hydroxyl group.  
     
     
         14 . The PNA chip of  claim 10 , wherein the spacer molecule is fixed onto the solid carrier through a mercapto moiety attached to the end of the spacer molecule.  
     
     
         15 . The PNA chip of  claim 10 , wherein the spacer molecule is derived from a compound selected from the group consisting of 2-mercaptoethanol, 3-mercaptoethanol, 6-mercaptoethanol, and N,N′-di(3-hydroxy-n-propyl)-imidazole-2-thione.  
     
     
         16 . A process for preparing a PNA chip of  claim 10  which comprises the steps of: 
 applying onto a solid carrier an aqueous solution of a plurality of PNA fragments dissolved or dispersed in an aqueous medium to fix the PNA fragments onto the solid carrier; and  
 applying onto the solid carrier having thereon the fixed PNA fragments an aqueous solution of short chain spacer molecules having at each one end a hydrophilic moiety and at each another end a moiety reactive to fix to the solid carrier.  
 
     
     
         17 . A method of quantitative analysis of a nucleic acid fragment contained in a sample liquid which is complementary to the PNA fragments of the PNA chip of  claim 11 , which comprises the steps of: 
 adjusting the concentration of the nucleic acid fragment in the sample liquid so that a droplet of the sample liquid applied to the PNA chip should contain  10   −20  to 10 −16  mol. of the nucleic acid fragment per 1 mm 2  of the surface of the electro-conductive substrate of the PNA chip;    bringing the nucleic acid concentration-adjusted sample liquid into contact with the PNA chip, whereby hybridizing the nucleic acid with the PNA fragment on the PAX chip;    bringing an electrochemically active molecule in contact with the hybridized nucleic acid and PNA fragment, whereby attaching the electrochemically active molecule to the hybridized nucleic acid and PNA fragment;    applying a potential to the PNA chip; and    measuring an electric current flowing from or to the electro-conductive substrate through the attached electrochemically active molecule.    
     
     
         18 . A kit for conducting quantitative analysis of a nucleic acid fragment contained in a sample liquid which is complementary to the PNA fragments of the PNA chip of  claim 11 , which comprises the PNA chip of  claim 11  and an electrochemically active molecule which is attachable to a hybridized nucleic acid and PNA fragment.