Process and apparatus for forming a dry DNA transfer film, a transfer film product formed thereby and an analyzing process using same

Abstract

Pivotable jigs or tables facilitate inversion or reciprocation of one or more well plates relative to a dry DNA transfer sheet to effect deposit of DNA gene solution as spaced spots on the surface of the transfer media sequentially to produce after drying of the DNA gene solution transfer of the dry DNA material from the spots by forcible impact or rubbing pressure through a printing mechanism of minute dry DNA dots onto a test substrate such as a glass plate for subsequent analysis optically via fluorescent labels to determine the presence or absence of mutations and a further identification of the mutation itself.

What is claimed:  
     
         1 . Process of forming a dry DNA transfer media comprising: 
 placing liquid DNA solutions within respective upwardly open spaced cup-shaped wells within a well plate;    causing localized momentary contact between an ink printing substrate and the DNA solutions within respective wells to create wet DNA solution spots at corresponding locations on said ink printing substrate; and    drying said spots to form said dry DNA transfer media for use in forcible impact or rubbing printing of dry DNA dots onto a facing glass test substrate from said selected dry DNA spots on said media.    
     
     
         2 . The process as claimed in    claim 1   , wherein said process further includes sealing said ink printing substrate face-to-face against said well plate in a position covering the openings of said well to create a sealed assembly and inverting said sealed assembly to wet coat the face of said media to create DNA solution spots at positions corresponding to the positions of said wells.  
     
     
         3 . The process as claimed in    claim 1   , wherein said ink printing substrate comprises one material of the group consisting of plastic, glass, nitrocellulose, acetate and paper.  
     
     
         4 . The process as claimed in    claim 3   , wherein said ink printing substrate comprises a mylar sheet, and wherein said mylar sheet has a roughened surface facing said well openings.  
     
     
         5 . The process as claimed in    claim 1   , further comprising porous wicks fitted within each cell having one end contactable with the DNA solution within the wells and having another end of the wick at least flush with the surface of the well plate bearing the wells such that a layer of DNA solution forms on the end of the wick exposed at the surface of the well plate, and said process further comprises placing said ink printing substrate in contact with the coated ends of said wicks to form wet DNA liquid spots on said ink printing substrate.  
     
     
         6 . The process as claimed in    claim 5   , further comprising the step of inverting an assembly of said well plate and said ink printing substrate to ensure DNA solution spotting of the ink printing substrate at said well locations.  
     
     
         7 . A process forming a plurality of spaced dry DNA dots on a glass substrate for testing purposes, said process comprising: 
 placing liquid DNA solution within an array of spaced cup-shaped wells within a well plate;    causing localized, momentary contact between an ink printing substrate and the liquid DNA within respective wells to create a corresponding series of spaced wet DNA spots on said ink printing substrate;    drying said spots to form a dry DNA transfer media and placing said dry DNA transfer media in proximity to said glass substrate, with said dry DNA spots facing a surface of said glass substrate; and    selectively, locally pressuring impacting the opposite face of said dry DNA transfer media behind said dry DNA spots to cause mechanical transfer of dry DNA dots from said media spots to the facing surface of said glass plate.    
     
     
         8 . A high throughput process for forming a dry DNA transfer film comprising: 
 placing separated, replicated DNA genes solubilized in a solution within respective wells of a generally rigid well plate having within an upper surface thereof, a plurality of closely spaced wells;    fixedly mounting a thin flexible resilient film against said upper surface and sealed to the upper surface of the well plate and effecting a rigid film plate assembly;    inverting said rigid assembly to cause the DNA solution to physically, locally wet coat the roughened surface of the film while preventing the DNA gene solution from running radially from one spot to another;    reinverting said assembly to its initial position;    removing the DNA gene solution spotted film slowly from the well plate;    drying the DNA gene solution spot coating thereon and thereby forming a dry DNA transfer film capable of physical and chemical dry transfer of DNA to a test substrate.    
     
     
         9 . The process as claimed in    claim 8   , wherein said step of drying the DNA gene solution spot coating comprises air drying.  
     
     
         10 . The process as claimed in    claim 8   , wherein said step sealing said thin flexible resilient film to said well plate top surface comprises forming a vacuum seal between said well plate and said thin flexible resilient film.  
     
     
         11 . A dry DNA transfer film formed by the process comprising: 
 placing separated, replicated DNA genes solubilized in a solution within a plurality of spaced cup-shaped wells within a generally rigid well plate, with said cells opening to a top surface of said well plate;    fixing a flexible resilient film having a roughened surface to said top surface of the well plate, with said roughened surface facing said well plate top surface;    effecting a rigid film plate assembly;    inverting said assembly to cause the DNA solution to physically, locally wet coat the roughened surface of the film, with the roughened surface causing the DNA gene solution to cling to the film as spaced dots corresponding to the well position while preventing the DNA gene solution from running radially from one spot to another;    reinverting the assembly to its initial position;    removing the DNA gene solution spotted film from the well plate; and    drying the DNA gene solution spots to form a dry DNA transfer film capable of physical and chemical dry transfer of the DNA to a test substrate.    
     
     
         12 . A dry form DNA analyzing process comprising the following steps: 
 prefilling respective separated, replicated DNA genes solubilized in a solution within a plurality of upwardly open spaced wells within an upper surface of said rigid well plate;    sealing a flexible resilient film at a predetermined position on the upper surface of said rigid well plate, with said film covering the prefilled wells;    forming a fixed, sealed assembly between the well plate and the overlying thin flexible film;    inverting the assembly to transfer DNA gene solution as spaced spots to the facing surface of the film over localized areas of said film defined by respective wells;    reinverting the assembly;    removing the film and drying the transferred DNA gene solution spots to thereby form a dry DNA gene transfer film;    placing the dry DNA gene transfer film in a position facing a flat glass test substrate and momentary pressing the face of the dry DNA gene transfer film opposite that bearing said spots at spot locations, causing pieces of dry DNA to locally contact the test substrate such that portions of the dry DNA spots and mechanically transferring from the transfer film to the test substrate by physical action and chemical attraction;    sequentially repeating the last step at different localized dried DNA spot locations on the dry DNA gene transfer film to complete a dry DNA gene test dot array on the test substrate; and    bathing the dots with two or more total genomic tag fluoresces to hybridize the tag to a particular gene of the array and optically scanning the array to identify dots containing fluorescently labeled DNA to determine the existence of mutants or lack of the same.    
     
     
         13 . The process as claimed in  12 , wherein the step of causing dry DNA to locally contact the test substrate comprises feeding the film in a first direction while moving a multi-pin print head across the film at right angles to the direction of feed of the film and impacting a selected print head pin against the film at the back of a preselected DNA spot location to forcibly imprint a portion of the DNA spot onto the facing surface of the glass test substrate.  
     
     
         14 . The process as claimed in    claim 13   , wherein said dry DNA gene transfer film is supported on a print tractor for movement in a direction of the film longitudinal axis via perforations extending along laterally offset edges of the film engaging motor driven sprocket wheels, wherein the film underlies the print head and overlies said glass test substrate, and wherein the print head moves transversely across the top of the DNA transfer film.  
     
     
         15 . The process as claimed in    claim 14   , further comprising moving said glass test substrate incrementally towards and away from the plane of said film, cyclically timed to the projection of print head pins within said print head, such that said glass test substrate is placed immediately adjacent to the surface of the dry DNA transfer film at the moment of print head pin impact, with the surface of the DNA transfer film opposite to that of selected projected print head pins.  
     
     
         16 . Apparatus for forming a dry DNA transfer media comprising: 
 a table, said table including an elongated table base extending horizontally, a vertical riser extending upwardly from the base intermediate of ends of the base, a horizontal flat table top hinged at one end to said vertical riser, a fixed stop mounted upright to said base and underlying said table top remote from said first hinge, a cover overlying said table and hinged by a second hinge to said table top so as to extend parallel to said table top overlying the same in first position and being rotatable 180° to an open position extending parallel to the table top and to the side of said center leg opposite that of said table top, a pivotable stop mounted to said base to the side of said center post opposite that of said fixed stop and underlying said cover when said cover is pivoted from said first position to said second position and being of a vertical height so as to maintain said cover horizontal when in said second position, the bottom surface of said cover being flat and including location pins for locating and fixing the position of said media on said bottom surface of said cover, means for positioning at least one well plate in fixed lateral and longitudinal positions on the upper surface of said table top at a position aligned with that of said transfer media, clamping means for fixing said cover in spaced overlying position on said table top, with said transfer media overlying said well plate, said well plate including a plurality of spaced upwardly open wells adapted to carry separated, replicated DNA genes solubilized in a solution within respective wells, said apparatus further comprising means for sealing the transfer media against the upper surface of said well plate and covering said upwardly open wells, whereby with said cover in said second position said transfer media may be fixed in a designated position on the bottom surface of said cover via location means, and said at least one well plate may be fixedly positioned at a predetermined position on the upper surface of said hinged table top, wherein by rotation of said cover 180° from said second position to said first position and by operation of said clamping means for fixing said cover in spaced overlying positions with respect to said table top, a fixed assembly may be achieved, whereupon subsequent rotation of said fixed assembly including said table top through a 180° from its position against the fixed stop in a direction towards said movable stop and by moving said movable stop to an inclined position on said table base, said assembly is inverted to cause the DNA solution to physically, locally wet coat the surface of said media creating spaced spots of DNA solution, whereupon the assembly may be pivoted about said first end through 180° to the extent of said table top contacting the fixed stop, whereupon the clamping means may be released permitting the cover to rotate from its first position to its second position to allow the media with the wet DNA coated spots to be removed from the table top and said spots to dry to thereby form said dry DNA transfer media.    
     
     
         17 . The apparatus as claimed in    claim 16   , further comprising a resilient foam sheet interposed between the bottom surface of the hinged cover and said transfer media and being of a thickness such that with the clamping means fixing the cover in spaced overlying position with said table top, said resilient foam sheet compresses the transfer media against the upper surface of the at least one well plate to seal said transfer media against the well plate surface.  
     
     
         18 . The apparatus as claimed in    claim 16   , wherein said at least one well plate comprises a plurality of well plates, and wherein said table top includes a like number of grooves within which respective well plates are positioned for positively locating said well plates on the upper surface of said table top.  
     
     
         19 . The apparatus as claimed in    claim 16   , wherein a vacuum groove is provided within the bottom surface of said hinged cover underlying said transfer media, and wherein said vacuum groove is connected to a source of vacuum via a vacuum line to fix the transfer media in position as determined by said location means.  
     
     
         20 . The apparatus as claimed in    claim 16   , wherein said location means comprises location pins projecting outwardly of the bottom surface of said hinged cover, and said transfer media includes holes sized to said pins and positioned to receive the pins.  
     
     
         21 . Apparatus for forming a dry DNA transfer media capable of physical and chemical dry transfer of DNA to a test substrate, said apparatus comprising: 
 a table, said table including a flat horizontal base, location means for locating a transfer media at a fixed position on an upper surface of said table top, riser means adjacent one end of said transfer media, a flat rectangular well plate holder pivoted at one end to an upper end of said riser means for pivoting of said well plate holder through an arc of at least 90° between a position overlying said transfer media and extending parallel thereto and a raised position, a locking pin and support means at an end of said well plate holder remote from said hinge and including a stop for supporting said well plate holder in a fixed position overlying said transfer media and parallel thereto, at least one elongated well plate hole within said well plate holder for receiving a well plate, said well plate including a plurality of spaced upwardly open cup-shaped wells for receiving separated, replicated DNA genes solubilized in a solution within respective wells, and means carried within said well for sealing off the upper open ends of said wells and for facilitating locally wet coating the facing surface of said transfer media when said at least one well plate is inverted and inserted within a correspondingly sized opening within said well plate holder to the extent of said means closing off the open ends of said wells contacting the surface of said transfer media, whereupon release of said clamping means and rotating of said pivotable well plate holder to said raised position moves the well plates away from the wet DNA coated surface of said transfer media to permit removal of said transfer media from the upper surface of said table and to allow replacement thereof by a new uncoated transfer media and the process repeated.    
     
     
         22 . The apparatus as claimed in    claim 21   , wherein said means mounted within said wells comprise: 
 dispensing valves, said dispensing valves each comprising a cylindrical upwardly open valve body having an axial bore and a counterbore, said axial bore extending through a bottom face of said valve body, said counterbore opening outwardly of a top face of said body, a movable valve plunger mounted coaxially within said bore and counterbore, a triangularly shaped stem guide and fill opening plate mounted within said counterbore adjacent said top face and including an axial bore receiving one end of said stem, said stem forming a part of said plunger and including a radially enlarged tapered valve stopper seated on an end of said axial bore for closing off said axial bore, biasing means biasing said plunger in valve closed position, said plunger terminating in a reduced diameter tip projecting axially below said bottom face, and wherein said dispenser valves are inverted and sealably positioned within the open end of said wells within said well plate, such that when the well plate is inverted after positioning of the dispenser valves within respective wells and with DNA solution within the wells, the tapered valve stoppers prevent release of DNA solution until the projecting tips of said movable valve plunger contact the facing surface of said transfer media to open said valve plunger against said biasing means to effect local wetting of the transfer media at positions corresponding to the positions of the spaced wells within the well plate.    
     
     
         23 . The apparatus as claimed in    claim 21   , wherein the means closing off the openings within the space wells of the well plate comprise applicator wicks, the wicks being sized slightly larger than the internal diameter of the wells and being fitted thereto and having one end projecting outwardly from the upper surface of the well plate and having another opposite end projecting downwardly within the well for contact with the DNA solution therein, and said one end being wetted by capillary action upon wick contact with the DNA solution such that a liquid DNA film forms on said one end of the applicator wick projecting outwardly from the upper surface of the well plate such that upon inversion of the well plate the DNA solution film on the projecting end of the applicator wick is contactable with the facing surface of the transfer media to create said wet spots of DNA solution during depression of the well plate within the well plate opening of the well plate holder.  
     
     
         24 . The apparatus as claimed in    claim 21   , wherein a vacuum groove is formed within the upper face of the table top beneath the transfer media, and a vacuum line couples the vacuum groove to a source of vacuum so as to vacuum fix the transfer media in a position determined by the location means.  
     
     
         25 . A dry DNA transfer media comprising one member of the group consisting of a flexible film, paper, nitrocellulose, plastic, glass for overlying a top surface of a generally rigid well plate carrying separated, replicated DNA genes solubilized in a solution within respective spaced upwardly open cells within said well plate, said media including DNA solution barriers at spaced locations within said media at locations corresponding to said wells to permit DNA solution local wet coating upon sealing of the transfer media to the surface of the well plate bearing said upwardly open well, but preventing the DNA gene solution from running radially from one spot to another as defined by the barrier means.  
     
     
         26 . The dry DNA transfer media as claimed in    claim 25   , wherein said media is paper, and wherein said barriers comprises mechanical indentions sized to and configured to said wells at respective well locations.  
     
     
         27 . The dry DNA transfer media as claimed in    claim 25   , wherein said media comprises a composite structure including a non-porous sheet bearing spaced cutouts corresponding to said well openings at respective locations corresponding to said wells and carrying coplanar porous inserts within respective cutouts.  
     
     
         28 . The dry DNA transfer media as claimed in    claim 27   , wherein said inserts are formed of porous paper.