Replica amplification of nucleic acid arrays

Abstract

Disclosed are improved methods of making and using immobilized arrays of nucleic acids, particularly methods for producing replicas of such arrays. Included are methods for producing high density arrays of nucleic acids and replicas of such arrays, as well as methods for preserving the resolution of arrays through rounds of replication. Also included are methods which take advantage of the availability of replicas of arrays for increased sensitivity in detection of sequences on arrays. Improved methods of sequencing nucleic acids immobilized on arrays utilizing single copies of arrays and methods taking further advantage of the availability of replicas of arrays are disclosed. The improvements lead to higher fidelity and longer read lengths of sequences immobilized on arrays. Methods are also disclosed which improve the efficiency of multiplex PCR using arrays of immobilized nucleic acids.

1 . A method of making an immobilized nucleic acid molecule array comprising: 
 a) providing an immobilized array of spots of a nucleic acid capture activity wherein: 
 i) said spots are separated by a distance greater than the diameter of said spots; and  
 ii) the size of said spots is less than the diameter of the excluded volume of said nucleic acid molecule to be captured; and  
   b) contacting said array of spots of a nucleic acid capture activity with an excess of nucleic acid molecules capable of being bound by said nucleic acid capture activity, said nucleic acid molecules having an excluded volume diameter greater than the diameter of said spots, resulting in an immobilized nucleic acid array in which each said spot of said nucleic acid capture activity can bind only one of said nucleic acid molecules having an excluded volume greater than the size of said spots.    
     
     
         2 . The method of  claim 1  wherein said nucleic acid capture activity is selected from the group consisting of: a hydrophobic compound; an oligonucleotide; an antibody or fragment of an antibody; a protein; a peptide; an intercalator; biotin; and avidin or streptavidin.  
     
     
         3 . The method of either one of claims  1  or  2  wherein said immobilized array of spots of a nucleic acid capture activity are arranged in a predetermined geometry.  
     
     
         4 . The method of any one of claims  1 - 3  wherein said spots of nucleic acid capture activity are aligned with other microfabricated features.  
     
     
         5 . A method of making a plurality of a nucleic acid array wherein said nucleic acid array is produced according to the method of either of claims  1 - 4 .  
     
     
         6 . A method for the detection of a nucleic acid on an array of nucleic acid molecules, said method comprising: 
 a) generating a plurality of a nucleic acid molecule array wherein the nucleic acid molecules of each member of said plurality occupy positions which correspond to those positions occupied by the nucleic acid molecules of each other member of said plurality of a nucleic acid array; and    b) subjecting one or more members of said plurality, but at least one less than the total number of said plurality to a method of signal detection comprising a signal amplification method which renders said member of said plurality of a nucleic acid array non-reusable.    
     
     
         7 . The method of  claim 6  wherein said signal amplification method comprises fluorescence measurement.  
     
     
         8 . The method of either one of claims  6  or  7  wherein said method of detection of a nucleic acid on an array of nucleic acid molecules detects the amount of an RNA expressed in a first RNA-containing nucleic acid population relative to that expressed in a second RNA-containing nucleic acid population, said method further comprising the steps of: 
 a) preparing a first fluorescently labeled cDNA population using said first population of RNA-containing nucleic acid as a template;  
 b) preparing a second fluorescently labeled cDNA population using said second population of RNA-containing nucleic acid as a template, said second fluorescently labeled cDNA population being labeled with a fluorescent label distinguishable from that used to label said first population;  
 c) contacting a mixture of said first fluorescently labeled cDNA population and said second fluorescently labeled cDNA population with a member of said plurality of nucleic acid arrays under conditions which permit hybridization of said fluorescently labeled cDNA populations with nucleic acids immobilized on said members of said plurality of nucleic acid arrays;  
 d) detecting the fluorescence of said first fluorescently labeled population of cDNA and the fluorescence of said second fluorescently labeled population of cDNA hybridized to said member of said plurality of nucleic acid arrays, wherein the relative amount of said first fluorescent label and said second fluorescent label detected on a given nucleic acid feature of said array indicates the relative level of expression of RNA derived from the nucleic acid of that feature in the mRNA-containing cDNA populations tested.  
 
     
     
         9 . The method of either of claims  6  or  7  wherein said method of detection of a nucleic acid on an array of nucleic acid molecules measures the amount of an mRNA expressed in a first mRNA-containing nucleic acid population relative to that expressed in a second mRNA-containing nucleic acid population, said method further comprising the steps of: 
 a) preparing a first fluorescently labeled cDNA population using said first population of mRNA-containing nucleic acid as a template;  
 b) preparing a second fluorescently labeled cDNA population using said second population of mRNA-containing nucleic acid as a template;  
 c) contacting said first fluorescently labeled cDNA population with one member of a plurality of immobilized nucleic acid arrays under conditions which permit hybridization of said fluorescently labeled cDNA population with nucleic acid immobilized on said member of a plurality of immobilized nucleic acid arrays;  
 d) contacting said second flourescently labeled cDNA population with another member of the same plurality of immobilized nucleic acid arrays used in step (c) under conditions which permit hybridization of said fluorescently labeled cDNA population with nucleic acid immobilized on said member of a plurality of immobilized nucleic acid arrays;  
 e) detecting the intensity of fluorescence on each member of said plurality contacted with a fluorescently labeled cDNA population in steps (c)-(d); and  
 f) comparing the intensity of fluorescence detected in step (e) on each member of said plurality of immobilized nucleic acid arrays so tested, to determine the relative expression of mRNA derived from those nucleic acids on the array in the mRNA-containing cDNA populations tested.  
 
     
     
         10 . A method of preserving the resolution of nucleic acid features on a first immobilized array during cycles of array replication, said method comprising the following steps: 
 a) amplifying the features of a first array to yield an array of features with a hemispheric radius, r, and a cross-sectional area, q, at the surface supporting said array, such that said features remain essentially distinct;    b) contacting said array of features with a radius, r, with a support, maintained at a fixed distance from said first array, said fixed distance less than r, and such that the cross-sectional area of the hemispheric feature, measured at said fixed distance from the surface supporting said first array is less than q, and such that at least a subset of nucleic acid molecules produced by said amplifying are transferred to said support;    c) covalently affixing said nucleic acid molecules to said support to form a replica of said first immobilized array, wherein the positions of said nucleic acid molecules on said replica correspond to the positions of said nucleic acid molecules of said first array from which they were amplified, and wherein the areas occupied on the surface of said support by the individual features of said replica are less than the areas occupied on the surface supporting said first immobilized array.    
     
     
         11 . The method of  claim 10  wherein said amplifying is performed by PCR.  
     
     
         12 . The method of either of claims  10  or  11  wherein cycles of said steps (a)-(c) are repeated.  
     
     
         13 . A method for determining the nucleotide sequence of the features of an immobilized nucleic acid array, said method comprising the steps of: 
 a) ligating a first double-stranded nucleic acid probe to one end of a nucleic acid of a feature of said array, said first double stranded nucleic acid probe having a restriction endonuclease recognition site for a restriction endonuclease whose cleavage site is separate from its recognition site and which generates a protruding strand upon cleavage;    b) identifying one or more nucleotides at the end of said polynucleotide by the identity of the first double stranded nucleic acid probe ligated thereto or by extending a strand of the polynucleotide or probe;    c) amplifying the features of said array using a primer complementary to said first double stranded nucleic acid probe, such that only molecules which have been successfully ligated with said first double stranded nucleic acid probe are amplified to yield an amplified array;    d) contacting said amplified array with support such that at least a subset of nucleic acid molecules produced by said amplifying are transferred to said support;    e) covalently attaching said subset of nucleic acid molecules transferred in step (d) to said support to form a replica of said amplified array;    f) cleaving the nucleic acid features of the array with a nuclease recognizing said nuclease recognition site of said probe such that the nucleic acid of the features is shortened by one or more nucleotides; and    g) repeating steps (a)-(f) until the nucleotide sequences of the features of said array are determined.    
     
     
         14 . The method of  claim 13  wherein said nucleic acid probe comprises four components, each component being capable of indicating the presence of a different nucleotide in said protruding strand upon ligation.  
     
     
         15 . The method of  claim 14  wherein each of said components of said probe is labeled with a different fluorescent dye and the different fluorescent dyes are spectrally resolvable.  
     
     
         16 . The method of any one of claims  13 - 15  wherein after said step (e) and before said step (f), the features of said array are amplified.  
     
     
         17 . The method of any one of claims  13 - 16  wherein amplification is performed by PCR.  
     
     
         18 . The method of any one of claims  13 - 17  wherein: 
 i) after one or more cycles using said first double stranded nucleic acid probe in step (a), a distinct nucleic acid probe is used, in place of said first double stranded nucleic probe in step (a), said distinct nucleic acid probe comprising a restriction endonuclease recognition site for a restriction endonuclease whose cleavage site is separated from its recognition site, said distinct nucleic acid probe also comprising sequences such that a primer complementary to said distinct nucleic acid probe will not hybridize with said first double stranded nucleic acid probe; and  
 ii) a primer complementary to said distinct nucleic acid probe is used in place of said primer complementary to said first double stranded nucleic acid probe in step (c), so that selective amplification of those features which successfully completed the previous cycle of restriction and ligation occurs.  
 
     
     
         19 . The method of  claim 18  wherein a new distinct nucleic acid probe is used after each cycle of restriction and ligation, said new distinct nucleic acid probe comprising a sequence such that a primer complementary to that sequence will not hybridize to any probe used in previous cycles.  
     
     
         20 . A method of determining the nucleotide sequence of the features of an array of immobilized nucleic acids comprising the steps of: 
 a) adding a mixture comprising an oligonucleotide primer and a template-dependent polymerase to an array of immobilized nucleic acid features under conditions permitting hybridization of the primer to the immobilized nucleic acids;    b) adding a single, fluorescently labeled deoxynucleoside triphosphate to the mixture under conditions which permit incorporation of the labeled deoxynucleotide onto the 3′ end of the primer if it is complementary to the next adjacent base in the sequence to be determined;    c) detecting incorporated label by monitoring fluorescence;    d) repeating steps (b)-(c) with each of the remaining three labeled deoxynucleoside triphosphates in turn; and    e) repeating steps (b)-(d) until the nucleotide sequence is determined.    
     
     
         21 . The method of  claim 20  wherein the primer, buffer and polymerase are cast into a polyacrylamide gel bearing the array of immobilized nucleic acids.  
     
     
         22 . The method of either of claims  20  or  21  wherein said single fluorescently labeled deoxynucleotide further comprises a mixture of the single deoxynucleoside triphosphate in labeled and unlabeled forms.  
     
     
         23 . The method of any one of claims  20 - 22  wherein after step (d) and before step (e) the additional step of photobleaching said array is performed.  
     
     
         24 . The method of any one of claims  20 - 23  wherein said fluorescently labeled deoxynucleoside triphosphates are labeled with a cleavable linkage to the fluorophore.  
     
     
         25 . The method of  claim 24  wherein after step (d) and before step (e) the additional step of cleaving said linkage to the fluorophore is performed.  
     
     
         26 . The method of any one of claims  20 - 25  wherein said oligonucleotide primer comprises sequences permitting formation of a hairpin loop.  
     
     
         27 . The method of any one of claims  20 - 26  wherein after a predetermined number of cycles of steps (b)-(d), a defined regimen of deoxynucleotide and chain-terminating deoxynucleotide analog addition is performed, such that out-of-phase molecules are blocked from further extension cycles, said regimen followed by continued cycles of steps (b)-(d) until said nucleotide sequence is determined.  
     
     
         28 . A method of determining the nucleotide sequence of the features of an array of immobilized nucleic acids comprising the steps of: 
 a) adding a mixture comprising an oligonucleotide primer and a template-dependent polymerase to an array of immobilized nucleic acid features under conditions permitting hybridization of the primer to the immobilized nucleic acids;    b) adding a first mixture of three unlabeled deoxynucleoside triphosphates under conditions which permit incorporation of deoxynucleotides to the end of the primer if they are complementary to the next adjacent base in the sequence to be determined;    c) adding a second mixture of three unlabeled deoxynucleoside triphosphates, said second mixture comprising the deoxynucleoside triphosphate not included in the mixture of step (b), under conditions which permit incorporation of deoxynucleotides to the end of the primer if they are complementary to the next adjacent base in the sequence to be determined;    d) repeating steps (b)-(c) for a predetermined number of cycles;    e) adding a single, fluorescently labeled deoxynucleoside triphosphate to the mixture under conditions which permit incorporation of the labeled deoxynucleotide onto the 3′ terminus of the primer if it is complementary to the next adjacent base in the sequence to be determined;    f) detecting incorporated label by monitoring fluorescence;    g) repeating steps (e)-(f), with each of the remaining three labeled deoxynucleoside triphosphates in turn; and    h) repeating steps (e) -(g) until the nucleotide sequence is determined.    
     
     
         29 . The method of  claim 28  wherein for said first or second mixtures of three unlabeled deoxynucleoside triphosphates, a mixture which comprises deoxyguanosine triphosphate further comprises deoxyadenosine triphosphate.  
     
     
         30 . The method of either of claims  28  or  29  wherein the primer and polymerase are cast into a polyacrylamide gel bearing the array of immobilized nucleic acids.  
     
     
         31 . The method of any one of claims  28 - 30  wherein said single fluorescently labeled deoxynucleotide further comprises a mixture of the single deoxynucleoside triphosphate in labeled and unlabeled forms.  
     
     
         32 . The method of any one of claims  28 - 31  wherein after step (g) and before step (h) the additional step of photobleaching said array is performed.  
     
     
         33 . The method of any one of claims  28 - 32  wherein said fluorescently labeled deoxynucleoside triphosphates are labeled with a cleavable linkage to the fluorophore.  
     
     
         34 . The method of  claim 33  wherein after step (g) and before step (h) the additional step of cleaving said linkage to the fluorophore is performed.  
     
     
         35 . The method of any one of claims  28 - 34  wherein said oligonucleotide primer comprises sequences permitting formation of a hairpin loop.  
     
     
         36 . The method of any one of claims  28 - 35  wherein after a predetermined number of cycles of steps (e)-(g), a defined regimen of deoxynucleotide and chain-terminating deoxynucleotide analog addition is performed, such that out-of-phase molecules are blocked from further extension cycles, said regimen followed by continued cycles of steps (e)-(g) until said nucleotide sequence of the features of the array is determined.  
     
     
         37 . A method of determining the nucleotide sequence of the features of a micro-array of nucleic acid molecules, said method comprising the following steps: 
 a) creating a micro-array of nucleic acid features in a linear arrangement within and along one side of a polyacrylamide gel, said gel further comprising one or more oligonucleotide primers, and a template-dependent polymerizing activity;    b) amplifying the microarray of step (a);    c) adding a mixture of deoxynucleoside triphosphates, said mixture comprising each of the four deoxynucleoside triphosphates dATP, dGTP, dCTP and dTTP, said mixture further comprising chain-terminating analogs of each of the deoxynucleoside triphosphates dATP, dGTP, dCTP and dTTP, and said chain-terminating analogs each distinguishably labeled with a spectrally distinguishable fluorescent moiety;    d) incubating said mixture with said micro-array under conditions permitting extension of said one or more oligonucleotide primers;    e) electrophoretically separating the products of said extension within said polyacrylamide gel; and    f) determining the nucleotide sequence of the features of said micro-array by detecting the fluorescence of the extended, terminated and separated reaction products within the gel.    
     
     
         38 . The method of  claim 37  wherein said amplifying is performed by PCR.  
     
     
         39 . The method of  claim 37  wherein said amplifying is performed by an isothermal method.  
     
     
         40 . The method of any of claims  37 - 39  wherein said microarray of nucleic acid features in a linear arrangement is derived as a replica of features arranged on a chromosome.  
     
     
         41 . The method of any one of claims  37 - 39  wherein said micro-array of nucleic acid features in a linear arrangement is derived as a replica of one linear subset of features on a separate, non-linear micro-array of nucleic acid features.  
     
     
         42 . A method of simultaneously amplifying a plurality of nucleic acids, said method comprising the steps of: 
 a) creating a micro-array of immobilized oligonucleotide primers;    b) incubating the microarray of step (a) with amplification template and a non-immobilized oligonucleotide primer under conditions allowing hybridization of said template with said oligonucleotide primers;    c) incubating the hybridized primers and template of step (b) with a DNA polymerase activity, and deoxynucleotide triphosphates under conditions permitting extension of the primers;    d) repeating steps (b) and (c) for a defined number of cycles to yield a plurality of amplified DNA molecules.    
     
     
         43 . The method of  claim 42  wherein said non-immobilized oligonucleotide primer comprises a pool of oligonucleotide primers comprised of 5′ and 3′ sequence elements, said 5′ sequence element identical in all members of said pool, and said 3′ sequence element containing random sequences.  
     
     
         44 . The method of  claim 43  wherein said 5′ sequence element comprises a restriction endonuclease recognition sequence.  
     
     
         45 . The method of either of claims  43  or  44  wherein said 5′ element comprises a transcriptional promoter sequence.  
     
     
         46 . The method of either of claims  42  or  43  wherein said immobilized primers are amplified before step (b).  
     
     
         47 . The method of any one of claims  42 - 44  wherein said immobilized oligonucleotide primers are generated from genomic DNA.  
     
     
         48 . The method of any one of claims  42 - 47  wherein the microarray, template, non-immobilized primer, and polymerase are cast in a polyacrylamide gel.