Reagents and methods for improving reproducibility and reducing mispriming in PCR amplification

Abstract

An additive for preventing mispriming in polymerase chain reaction (PCR) amplifications and assays comprising a hairpin oligonucleotide having a stem duplex greater than six nucleotides in length and a stabilized stem terminus. The additive improves PCR amplifications, including LATE-PCR amplifications when added to initial amplification reaction mixtures. It can be included in oligonucleotide sets and in kits for PCR amplification and assays.

1 . A reagent capable of preventing at least one manifestation of mispriming in a polymerase chain reaction (PCR) amplification to produce at least one amplified DNA product when added at a concentration of not more than 650 nM to a PCR amplification mixture that includes 1.25 units of a thermostable DNA polymerase per 25 μl of reaction mixture, said reagent being a non-extendable oligonucleotide that has a stem-loop structure, that is not a hybridization probe for said at least one amplified DNA product, that has a stem that is greater than six nucleotides in length, that is stabilized at its terminus away from the loop, and that has a calculated stem melting temperature (Tm) below 94° C. 
     
     
         2 . The reagent according to  claim 1  wherein the stem is terminally stabilized by interacting chemical moieties covalently attached to the 3′ and 5′ nucleotides of the stem. 
     
     
         3 . The reagent according to  claim 2  wherein the interacting chemical moieties are non-fluorescent quencher moieties. 
     
     
         4 . The reagent according to  claim 3  wherein the loop is an oligonucleotide comprising at least three nucleotides. 
     
     
         5 . The reagent according to  claim 4  wherein the stem comprises a double-stranded sequence of 9-12 base pairs. 
     
     
         6 . The reagent according  claim 3  wherein the stem comprises a double-stranded region of 9-12 base pairs. 
     
     
         7 . The reagent according to  claim 2  wherein the stem has a calculated melting temperature (Tm) is in the range of 72-85° C. 
     
     
         8 . The reagent according to  claim 2  wherein the stem has a calculated melting temperature (Tm) in the range of 50-71° C. 
     
     
         9 . The reagent according to  claim 2  wherein the stem comprises a double-stranded region of 9-12 base pairs. 
     
     
         10 . The reagent according to  claim 1  wherein the loop comprises an oligonucleotide comprising at least three nucleotides. 
     
     
         11 . The reagent according to  claim 10  wherein the stem comprises a double-stranded region of 9-12 base pairs. 
     
     
         12 . The reagent according to  claim 1  wherein the loop is a non-nucleotide chemical linker. 
     
     
         13 . The reagent according to  claim 12  wherein the chemical linker is a hydrocarbon chain. 
     
     
         14 . The reagent according to  claim 13  wherein the hydrocarbon chain is a methylene chain of 3-6 carbon atoms. 
     
     
         15 . The reagent according to  claim 13  wherein the stem is terminally stabilized by interacting chemical moieties covalently attached to the 3′ and 5′ nucleotides of the stem. 
     
     
         16 . The reagent according to  claim 15  wherein the interacting chemical moieties are non-fluorescent quencher moieties. 
     
     
         17 . The reagent according to  claim 15  wherein the calculated melting temperature of the stem is at least 50° C. 
     
     
         18 . The reagent according to  claim 1  wherein the stem is stabilized by inclusion therein of non-natural terminal nucleotides. 
     
     
         19 . The reagent according to  claim 18  wherein the loop is an oligonucleotide comprising at least three nucleotides. 
     
     
         20 . The reagent according to  claim 18  wherein the calculated melting temperature of the stem is at least 50° C. 
     
     
         21 . The reagent according to  claim 20  wherein the stem comprises a double-stranded region of 9-12 base pairs. 
     
     
         22 . The reagent according to  claim 18  wherein the loop is a non-nucleotide chemical linker. 
     
     
         23 . The reagent according to  claim 1  wherein the calculated melting temperature of the stem is at least 50° C. 
     
     
         24 . The reagent according to  claim 23  wherein the calculated melting temperature of the stem is in the range of 72-85° C. 
     
     
         25 . A method for preventing at least one manifestation of mispriming in a polymerase chain reaction (PCR) amplification of a reaction mixture that includes a thermostable DNA polymerase, comprising adding to said reaction mixture at least one reagent according to  claim 1 . 
     
     
         26 . The method of  claim 25  wherein said reagent has a stem that is terminally stabilized by interacting chemical moieties covalently attached to the 3′ and 5′ nucleotides of the stem. 
     
     
         27 . The method of  claim 26  wherein said reagent has an oligonucleotide loop comprising at least three nucleotides and said reagent is added to the reaction mixture at a concentration of not more than 300 nM. 
     
     
         28 . The method of  claim 26  wherein said reagent has a stem of 9-12 base pairs in length. 
     
     
         29 . The method of  claim 36  wherein said reagent has a loop that is a non-nucleotide chemical linker. 
     
     
         30 . The method of  claim 29  wherein said reagent is added to the reaction mixture at a concentration of not more than 1000 nM. 
     
     
         31 . The method of  claim 29  wherein said linker is an alkylene chain. 
     
     
         32 . The method of  claim 29  wherein said linker comprises a chain of 3-6 carbon atoms. 
     
     
         33 . The method of  claim 25  wherein said reagent has an oligonucleotide loop comprising at least three nucleotides. 
     
     
         34 . The method of  claim 25  wherein said reagent has a loop that is a non-nucleotide chemical linker. 
     
     
         35 . The method of  claim 25  wherein the PCR amplification comprises a liner-after-the exponential PCR (LATE-PCR) assay including a low-temperature detection step. 
     
     
         36 . The method of  claim 25  wherein the PCR amplification is followed by end-point detection. 
     
     
         37 . A kit of reagents for performing a polymerase chain reaction (PCR) amplification comprising a thermostable DNA polymerase, at least one pair of PCR primers, dNTP's and at least one reagent according to  claim 1 . 
     
     
         38 . The kit according to  claim 37  further comprising reagents for nucleic acid isolation. 
     
     
         39 . The kit according to  claim 37  wherein the reagent has an oligonucleotide loop of at least three nucleotides. 
     
     
         40 . The kit according to  claim 37  wherein the reagent has a loop that is a non-nucleotide chemical linker. 
     
     
         41 . The kit according to  claim 37  wherein the reagent has a stem that is terminally stabilized by interacting chemical moieties covalently attached to the 3′ and 5′ nucleotides of the stem. 
     
     
         42 . A set of oligonucleotides for performing a polymerase chain reaction (PCR) amplification comprising at least one pair of PCR primers and at least one reagent according to  claim 1 . 
     
     
         43 . The set according to  claim 42  further including at least one fluorescently labeled probe that hybridizes to the amplified product defined by said at least one pair of PCR primers.