# US 2018/0002739 A1 · Reagents and methods of pcr

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- Number: US 2018/0002739 A1
- Document ID: US20180002739A1
- Status: Published application
- Kind: A1
- Application number: 15/698,992
- Filed: 2017-09-08
- Published: 2018-01-04
- Earliest claimed: 2009-03-12
- Assignees: Brandeis University
- Applicants: Brandeis University
- Inventors: Lawrence J. Wangh; John E. Rice; Nicholas Rice; Yanwei Jia
- Web page: https://patentgrep.com/patent/US20180002739A1

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6853: using modified primers or templates
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

Modified double-stranded oligonucleotides that have terminal regions on each of their strands, that have a hybrid length of 6-50 nucleotides long, that have a melting temperature Tm of at least 32° C., and that include 2-4 modifying groups, each covalently attached to a different terminal region, preferably to a terminal nucleotide, said modifying groups being polycyclic substituents that do not have bulky portions that are non-planar, said modified oligonucleotide being capable of binding to the 5′ exonuclease domains of DNA polymerases and, when included in a PCR or other primer-dependent DNA amplification reaction at a concentration, generally not more than 2000 nM, that is effective for at least one of the functions of suppressing mispriming, increasing polymerase selectivity against 3′ terminal mismatches. increasing polymerase selectivity against AT-rich 3′ ends, reducing scatter among replicates, suppressing polymerase 5′ exonuclease activity, and inhibiting polymerase activity; as well as amplification reaction mixtures containing such modified double-stranded oligonucleotides, and amplification reactions, amplification assays and kits that include such modified double-stranded oligonucleotides.

## Claims

### Claim 1

In a reaction mixture for a primer-dependent DNA amplification reaction including primer extension by a DNA polymerase for amplifying at least one DNA target sequence, said reaction mixture including at least one primer pair, a DNA polymerase and dNTP's, the improvement comprising including in the reaction mixture prior to the start of amplification at least one double-stranded oligonucleotide additive that has a hybrid length of 6-50 nucleotides long, that is at least fifty percent double-stranded at 32° C., that has terminal regions on each of its strands and includes 1-4 modifying groups, each covalently attached to a different terminal region, said modifying groups being polycyclic moieties that do not have bulky portions that are non-planar, wherein said at least one double-stranded oligonucleotide additive is included at a concentration relative to the concentration of said DNA polymeras that is effective for at least one of the functions of suppressing mispriming, increasing polymerase selectivity against hybrids having recessed 3′ terminal sequences that are not perfectly complementary, increasing polymerase selectivity against hybrids having recessed 3′ terminal sequences that are AT-rich, reducing scatter among replicate reactions, inhibiting polymerase 5′ exonuclease activity, and inhibiting polymerase activity; provided that, if the additive is a primer or detection probe for any target sequence, it includes at least three modifying groups.

### Claim 2 (depends on 1)

The amplification reaction mixture of claim 1 wherein the at least one modifying groups is 2-4 modifying groups.

### Claim 3 (depends on 2)

The amplification reaction mixture of claim 2 wherein the 2-4 modifying groups is three modifying groups.

### Claim 4 (depends on 3)

The amplification reaction mixture of claim 3 wherein the additive includes a first strand that is a primer or probe for said at least one target sequence and a reverse complement strand that is partially complementary to the first strand.

### Claim 5 (depends on 2)

The amplification reaction mixture of claim 2 wherein the 2-4 modifying groups is four modifying groups.

### Claim 6 (depends on 1, 2, 3, 4, 5)

The amplification reaction mixture of any of claims 1-5 wherein the modifying groups are covalently linked to terminal nucleotides of said at least one double-stranded oligonucleotide additive.

### Claim 7 (depends on 1, 2, 3, 4, 5, 6)

The amplification reaction mixture of any of claims 1-6 wherein the modifying groups are Dabcyl.

### Claim 8 (depends on 2)

The amplification reaction mixture of claim 2 wherein the at least one additive is a mixture of two additives.

### Claim 9 (depends on 8)

The amplification reaction mixture of claim 8 wherein the mixture consists of three strands.

### Claim 10 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9)

The amplification reaction mixture of any of claims 1-9, wherein the at least one double-stranded oligonucleotide additive consists of natural nucleotides.

### Claim 11 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9)

The amplification reaction mixture of any of claims 1-9 wherein the at least one double-stranded oligonucleotide additive is DNA.

### Claim 12 (depends on 1, 2, 3, 5, 6, 7, 8, 9, 10, 11)

The amplification reaction mixture according to any of claims 1-3 and 5-11 wherein the at least one additive is not a primer or probe for said at least one target sequence.

### Claim 13 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)

The amplification reaction mixture according to any of claims 1-12 wherein the concentration of said at least one double-stranded oligonucleotide additive is not more than 1000 nM.

### Claim 14 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)

The amplification reaction mixture according to any of claims 1-13, further including a reverse transcriptase.

### Claim 15 (depends on 1)

The amplification reaction mixture of claim 1 wherein the at least one double-stranded additive includes from one to four single-stranded overhangs.

### Claim 16 (depends on 15)

The amplification reaction mixture of claim 15 wherein the at least one double-stranded additive comprises at least one strand that, when not hybridized, forms a stem-loop structure.

### Claim 17 (depends on 1)

A method for amplifying at least one DNA target sequence comprising contacting said at least and DNA target sequence with an amplification reaction mixture according to claim 1 and subjecting the reaction mixture to a primer-dependent DNA amplification reaction having a primer annealing temperature and a primer extension temperature.

### Claim 18 (depends on 17)

The method of claim 17 wherein contacting the at least one DNA target sequence with the reaction mixture consists of adding the at least one DNA target sequence in single-stranded form to the reaction mixture.

### Claim 19 (depends on 17)

The method of claim 17 that includes reverse transcribing RNA to obtain the at least one DNA target sequence.

### Claim 20 (depends on 17)

The method of claim 17 wherein the at least one modifier is 2-4 modifiers.

### Claim 21 (depends on 20)

The method of claim 20 wherein the double-stranded oligonucleotide has a melting temperature, Tm, that is in the range of the primer annealing temperature to not more than 5° C. below the primer annealing temperature.

### Claim 22 (depends on 20)

The method of claim 20 wherein the double-stranded oligonucleotide has a melting temperature, Tm, that is higher than the primer annealing temperature.

### Claim 23 (depends on 17)

The method of claim 17 wherein the additive has three modifying groups and includes a first strand that is a primer or probe for said at least one target sequence and a reverse complement strand that is partially complementary to the first strand.

### Claim 24 (depends on 20)

The method of claim 20 wherein said at least one additive is a mixture of two additives.

### Claim 25 (depends on 22)

The method of claim 22 wherein the mixture includes a first additive having a double-stranded oligonucleotide that has a Tm that is in the range of the primer annealing temperature to not more than 5° C. below the primer annealing temperature and a second additive having a double-stranded oligonucleotide that has a Tm that is higher than the primer annealing temperature.

### Claim 26 (depends on 25)

The method of claim 25 wherein each additive includes 3-4 modifying groups.

### Claim 27 (depends on 17, 18, 19, 20, 21, 22, 23, 24, 25, 26)

The method according to any of claims 17-26, wherein the primer-dependent amplification reaction is a polymerase chain reaction (PCR) amplification reaction.

### Claim 28 (depends on 17, 18, 19, 20, 21, 22, 23, 24, 25, 26)

The method according to any of claims 17-26, wherein the primer-dependent amplification is a LATE-PCR amplification reaction.

### Claim 29 (depends on 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)

The method according to any of claims 17-28 wherein the at least one DNA target sequence is reverse transcribed from an RNA target sequence.

### Claim 30 (depends on 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)

An amplification assay that includes amplification according to any of claims 17-29 and fluorescence detection of single-stranded products of the reaction, double-stranded products of the reaction, or both, either in real time during amplification or end point following amplification, wherein double-stranded products of the reaction are detected with a fluorescent DNA dye, single-stranded products of the reaction are detected with at least one fluorescently labeled hybridization probe, or both.

### Claim 31 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)

A kit of reagents amplifying at least one DNA target sequence, said kit including the reagents for a reaction mixture according to any of claims 1-16.

### Claim 32 (depends on 31)

The kit according claim 31 further including reverse transcriptase.

### Claim 33 (depends on 31, 32)

The kit according to claim 31 or claim 32 further including a DNA dye.

### Claim 34 (depends on 31, 32, 33)

The kit according to any of claims 31-33 further including a fluorescently labeled detection probe for said at least one target sequence.

### Claim 35

A modified double-stranded oligonucleotide that has terminal regions on each of its strands, that has a hybrid length of 6-50 nucleotides long, that is at least fifty percent double-stranded at 32° C., and that includes 2-4 modifying groups, each covalently attached to a different terminal region, said modifying groups being polycyclic moieties that do not have bulky portions that are non-planar, said modified oligonucleotide being capable of inhibiting the 5′ exonuclease domains of DNA polymerases.

### Claim 36 (depends on 35)

The modified oligonucleotide of claim 35 wherein the double-stranded oligonucleotide is DNA.

### Claim 37 (depends on 35, 36)

The modified oligonucleotide of claim 35 or claim 36 wherein said modifying groups are attached to terminal nucleotides.

### Claim 38 (depends on 35, 36, 37)

A mixture of two modified double-stranded oligonucleotides according to any of claims 35-37.

### Claim 39 (depends on 38)

The mixture of claim 38 wherein the two double-stranded oligonucleotides comprise three strands.

### Claim 40 (depends on 1)

The amplification reaction mixture of claim 1, wherein the DNA polymerase is thermostable.