Polymerase extension at 3' terminus of PNA-DNA chimera

Abstract

The invention provides methods and a kit for primer extension of PNA-DNA chimera from template nucleic acids using polymerases, nucleotide 5′-triphosphates, and primer extension reagents. Structural requirements of the chimera for primer extension include 5 to 15 contiguous PNA monomer units, 3 or more contiguous nucleotides, and a 3′ hydroxyl terminus. The chimera and/or a nucleotide is labelled with fluorescent dyes or other labels. The methods include DINA sequencing, DNA fragment analysis, reverse transcription, mini-sequencing, chromosome labelling, amplification, and single nucleotide polymorphism (SNP) detection.

We claim:  
     
         1 . A method of producing a non-radioisotopically labelled chimeric extension product comprising the step of enzymatically extending a PNA-DNA chimera in the presence of a template nucleic acid, a polymerase and a primer extension reagent, wherein said primer extension reagent comprises a nucleotide 5′-triphosphate capable of effecting enzymatic chimera primer extension, and wherein the chimera or the nucleotide 5′-triphosphate is labelled with a non-radioisotopic label.  
     
     
         2 . The method of  claim 1  in which the PNA-DNA chimera has the structure: 
       P x -L-N y 3′ wherein: 
 each P is independently a PNA monomer;  
 x is an integer from 5 to 15;  
 L represents a covalent linkage between P and N;  
 each N is independently a nucleotide; and  
 y is an integer from 3 to 15;  
 with the proviso that the 3′ terminal N has a 3′ hydroxyl group.  
   
     
     
         3 . The method of  claim 1  in which the primer extension reagent comprises a mixture of nucleotide 5′-triphosphates capable of effecting continuous primer extension.  
     
     
         4 . The method of  claim 3  in which the mixture comprises four different nucleotide 5′-triphosphates, and further wherein 
 a nucleotide 5′-triphosphate is ATP, dATP, 7-deaza dATP, 2-amino-ATP or 2-amino-dATP, and  
 another nucleotide 5′-triphosphate is GTP, dGTP, or 7-deaza dGTP, another nucleotide 5′-triphosphate is CTP, dCTP, 5-methyl-CTP, 5-methyl-dCTP or 5-propynyl dCTP, and  
 another nucleotide 5′-triphosphate is UTP, dUTP, dTTP, 5-Br-UTP, 5-Br-dUTP, 5-F-UTP, 5-F-dUTP or 5-propynyl-dUTP.  
 
     
     
         5 . The method of  claim 4  in which the mixture further includes a terminating nucleotide 5′-triphosphate.  
     
     
         6 . The method of  claim 1  in which the nucleotide 5′-triphosphate is a terminating nucleotide 5′-triphosphate.  
     
     
         7 . The method of  claim 5  or  6  in which the terminating nucleotide 5′-triphosphate is detectably labelled with a fluorescent dye.  
     
     
         8 . The method of  claim 1  in which the primer extension reagent further comprises a mixture of terminating nucleotide 5′-triphosphates.  
     
     
         9 . The method of  claim 8  in which the mixture comprises four different terminating nucleotide 5′-triphosphates, and further wherein a terminating nucleotide 5′-triphosphate is ddATP, 7-deaza ddATP, or 2′,3′-dideoxy-dehydro-ATP, 
 another terminating nucleotide 5′-triphosphate is ddGTP, 7-deaza ddGTP or 2′,3′-dideoxy-dehydro-GTP,  
 another terminating nucleotide 5′-triphosphate is ddCTP or 2′,3′-dideoxy-dehydro-CTP, and  
 another terminating nucleotide 5′-triphosphate is ddTTP, ddUTP, 2′,3′-dideoxy-dehydro-TTP or 2′,3′-dideoxy-dehydro-UTP.  
 
     
     
         10 . The method of  claim 9  wherein each different terminating nucleotide 5′-triphosphate is labelled with a different detectable label.  
     
     
         11 . The method of  claim 2  wherein P x  is a 2-aminoethylglycine peptide nucleic acid.  
     
     
         12 . The method of  claim 2  in which each N is independently a 2′-deoxyribonucleotide.  
     
     
         13 . The method of  claim 2  in which each N is independently a ribonucleotide.  
     
     
         14 . The method of  claim 2  wherein the nucleobases of N are selected from the group consisting of C-5-alkyl pyrimidine, 2.6-diaminopurine, 2-thiopyrimidine, C-5-propyne pyrimidine, phenoxazine, 7-deazapurine, isocytidine, pseudo-isocytidine, isoguanosine, hypoxanthine, 8-oxopurine, and 4(3 H)-pyrimidone.  
     
     
         15 . The method of  claim 2  wherein the sugars of N are selected from the group consisting of 29-O-alkyl-ribonucleotides, 2′-O-methyl-ribonucleotides, 2′-O-allyl-ribonucleotides, 2′-allyl ribonucleotides, 2′-halo-ribonucleotides, 2′-O-methoxyethyl-ribonucleotides, 4′-α-anomeric nucleotides, 1′-α-anomeric nucleotides, 2′,4′-linked nucleotides, and bicyclic nucleotides.  
     
     
         16 . The method of  claim 1  wherein the PNA-DNA chimera is labelled at the amino terminus of the PNA moiety.  
     
     
         17 . The method of  claim 1  wherein the nucleotide 5′-triphosphate is labelled at the nucleobase.  
     
     
         18 . The method of  claim 17  wherein the nucleobase label sites are the N-9 or C-8 positions of the purine or deazapurine, and the C-5 position of the pyrimidine.  
     
     
         19 . The method of  claim 1  wherein the label is selected from the group consisting of fluorescent dyes, fluorescence quenchers, hybridization-stabilizers, energy-transfer dye sets, electrophoretic mobility modifiers, chemiluminescent dyes, amino acids, proteins, peptides, enzymes, and affinity ligands.  
     
     
         20 . The method of  claim 19  where the fluorescent dyes are selected from the group consisting of FAM, TET, HEX, JOE, TAMPA, d-TAMRA, JODA, ROX, VIC, NED, dJON, dR139, 4,7-dichloro-fluoresceins, 4,7-dichloro-rhodamines, and cyanines.  
     
     
         21 . The method of  claim 19  where the fluorescence quenchers are selected from the group consisting of TAMRA, d-TAMRA, ROX, DABCYL, DABSYL, malachite green, NTB, and cyanines.  
     
     
         22 . The method of  claim 19  where the hybridization-stabilizers are minor groove binders.  
     
     
         23 . The method of  claim 19  where the minor groove binders are selected from the group consisting of Hoechst 33258, CDPI 1-3  , MGB1, netropsin, and distamycin.  
     
     
         24 . The method of  claim 19  where the affinity ligands are selected from the group consisting of biotin, 2.4-dinitrophenyl, digoxigenin, cholesterol, polyethyleneoxy, peptides, and fluorescein.  
     
     
         25 . The method of  claim 2  wherein L is selected from the group consisting of a covalent bond, alkyldiyl consisting of 1-20 carbon atoms, aryldiyl, 0 linker, and —(CH 2 CH 2 O) m — where m is 1 to 6.  
     
     
         26 . The method of  claim 1  in which the template nucleic acid is a DNA and the polymerase is selected from the group consisting of Klenow, T4, Bst, AmpliTaq, AmpliTaq Gold, AmpliTaq Stoffel fragment, Sequenase, Vent, Pfu, and bacteriophage T7.  
     
     
         27 . The method of  claim 1  in which the template nucleic acid is an RNA and the polymerase is a reverse transcriptase.  
     
     
         28 . The method of  claim 1  in which the template nucleic acid is a metaphase or interphase chromosome.  
     
     
         29 . The method of  claim 28  in which the chromosome is denatured.  
     
     
         30 . The method of  claim 1  in which the PNA-DNA chimera is immobilized on a solid substrate.  
     
     
         31 . The method of  claim 30  in which the chimera is covalently attached to the solid substrate, optionally with the aid of a linker.  
     
     
         32 . The method of  claim 1  in which the template nucleic acid is immobilized on a solid substrate.  
     
     
         33 . The method of  claim 32  in which the template nucleic acid is covalently attached to the solid substrate, optionally with the aid of a linker.  
     
     
         34 . The method of  claim 30  or  32  wherein the solid substrate is selected from the group consisting of polystyrene, controlled-pore-glass, silica gel, silica, polyacrylamide, magnetic beads, polyacrylate, hydroxyethylmethacrylate, polyamide, polyethylene, polyethyleneoxy, and copolymers and grafts of any of the above solid substrates.  
     
     
         35 . The method of  claim 30  or  32  wherein the solid substrate is selected from the group consisting of small particles, beads, membranes, frits, slides, plates, micromachined chips, alkanethiol-gold layers, non-porous surfaces, and polynucleotide-immobilizing media.  
     
     
         36 . A kit for primer extension comprising: 
 a PNA-DNA chimera primer, said primer comprising 5 to 15 contiguous PNA monomer units, 3 to 15 contiguous nucleotides, and a 3′ hydroxyl terminus; one or more nucleotide 5′-triphosphates and; a polymerase enzyme,    wherein the chimera primer or a nucleotide 5′-triphosphate is non-radioisotopically labelled.    
     
     
         37 . The kit of  claim 36  further comprising a template nucleic acid comprising a sequence complementary to the chimera primer or containing one or more mismatches to the chimera primer.  
     
     
         38 . A method of sequencing a template nucleic acid, comprising the steps of: 
 a. generating a labelled primer extension product by enzymatically extending a primer-template nucleic acid hybrid in the presence of a polymerase and a terminating nucleotide 5′-triphosphate, wherein said primer is a PNA-DNA chimera and either said primer or said terminating nucleotide 5′-triphosphate is detectably and non-radioisotopically labelled;    b. separating the labelled primer extension products based on size; and    c. determining the sequence of the template nucleic acid.    
     
     
         39 . The method of  claim 38  wherein a nested set of labelled primer extension products are generated by a mixture of enzymatically extendable nucleotide 5′-triphosphates capable of supporting continuous primer extension.  
     
     
         40 . The method of  claim 38  wherein the PNA-DNA chimera is labelled.  
     
     
         41 . The method of  claim 38  wherein the terminating nucleotide 5′-triphosphate is labelled.  
     
     
         42 . The method of  claim 41  wherein the labelled, terminating nucleotide 5′-triphosphate is selected from the group consisting of a labelled ddNTP, a labelled 2′-amino, 2′-deoxynucleotide, a labelled 2′-halo, 2′-deoxynucleotide, and a labelled 2′,3′-- dideoxy-dehydronucleotide.  
     
     
         43 . A method of reverse transcription comprising the step of generating labelled primer extension products by enzymatically extending a primer-template RNA hybrid in the presence of a reverse transcriptase, a mixture of enzymatically-extendable nucleotide 5′-triphosphates capable of supporting continuous primer extension, wherein said primer is a PNA-DNA chimera and either said primer or a nucleotide 5′-triphosphate is non-radioisotopically labelled.  
     
     
         44 . A method of DNA amplification comprising the steps of: 
 a. generating labelled amplification products by enzymatically extending a primer-template nucleic acid hybrid in the presence of two primers each of which is capable of hybridizing to the template and wherein one or both of which is a PNA-DNA chimera primer, a DNA polymerase and a mixture of enzymatically-extendable nucleotide 5′-triphosphates capable of supporting continuous primer extension, wherein either said primers or nucleotide 5′-triphosphates are non-radioisotopically labelled: and    b. cycling the temperature to effect denaturation, annealing, and primer extension to form an amplification product by extension of the primers with nucleotide 5′-triphosphates;    wherein one or both of the 5′ terminii of the amplification product bears the PNA sequence of the chimera primers.    
     
     
         45 . The method of  claim 44  in which the amplification product is immobilized by hybridization on a solid substrate comprising a nucleic acid with a sequence complementary to the PNA sequence of the amplification product.