# US 11,299,777 B2 · Isothermal amplification components and processes

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- Number: US 11,299,777 B2
- Document ID: US11299777B2
- Status: Granted
- Kind: B2
- Application number: 16/089,063
- Filed: 2017-03-06
- Published: 2020-08-13
- Granted: 2022-04-12
- Earliest claimed: 2016-04-04
- Assignees: Nat Diagnostics, Inc.
- Applicants: Nat Diagnostics, Inc.
- Inventors: Andrew P. Miller; Honghua Zhang
- Web page: https://patentgrep.com/patent/US11299777B2

## CPC

- C12Q 1/6844: Nucleic acid amplification reactions
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 1/6816: characterised by the detection means (C12Q1/6804 takes precedence)
- C12Q 1/689: for bacteria
- G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)}
- C12Q 2521/101: DNA polymerase
- C12Q 2521/107: RNA dependent DNA polymerase,(i.e. reverse transcriptase)
- C12Q 2527/101: Temperature
- C12Q 2527/113: Time
- C12Q 2531/113: PCR
- C12Q 2561/113: Real time assay
- C12Q 2600/16: Primer sets for multiplex assays
- G01N 2021/6432: {Quenching}
- 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
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The technology relates in part to methods and compositions for isothermal amplification of nucleic acids.

## Claims

### Claim 1

A method for detecting a target nucleic acid sequence in a sample, the method comprising:
(a) amplifying a target nucleic acid sequence comprising a first strand and a second strand complementary to each other in an isothermal amplification condition, wherein the amplifying comprises contacting a non-denatured nucleic acid comprising the target nucleic acid sequence with:
i) a first primer and a second primer, wherein the first primer is capable of hybridizing to a sequence of the first strand of the target nucleic acid sequence, and the second primer is capable to hybridizing to a sequence of the second strand of the target nucleic acid sequence; and
ii) an enzyme having a hyperthermophile polymerase activity, thereby generating a nucleic acid amplification product at detectable levels within 10 minutes, wherein the nucleic acid amplification product comprises:
(1) the sequence of the first primer, and the reverse complement thereof,
(2) the sequence of the second primer, and the reverse complement thereof, and
(3) a spacer sequence flanked by (1) the sequence of the first primer and the reverse complement thereof and (2) the sequence of the second primer and the reverse complement thereof, wherein the spacer sequence is 1 to 10 bases long; and
wherein the amplifying does not comprise using any enzyme other than the enzyme having a hyperthermophile polymerase activity, and the amplifying does not comprise denaturing the non-denatured nucleic acid; and
(b) detecting the nucleic acid amplification product.

### Claim 2 (depends on 1)

The method of claim 1, wherein step (b) further comprises determining the amount of the non-denatured nucleic acid that comprises the target nucleic acid sequence in the sample.

### Claim 3 (depends on 1)

The method of claim 1, wherein the non-denatured nucleic acid is a genomic nucleic acid, a plasmid nucleic acid, a mitochondrial nucleic acid, a cellular nucleic acid, or an extracellular nucleic acid.

### Claim 4 (depends on 1)

The method of claim 1, wherein the non-denatured nucleic acid is a bacterial nucleic acid or a viral nucleic acid.

### Claim 5 (depends on 1)

The method of claim 1, wherein the target nucleic acid sequence is a bacterial nucleic acid sequence or a viral nucleic acid sequence.

### Claim 6 (depends on 1)

The method of claim 1, wherein the non-denatured nucleic acid is a double-stranded DNA.

### Claim 7 (depends on 1)

The method of claim 1, wherein the non-denatured nucleic acid is a product of reverse transcription reaction.

### Claim 8 (depends on 7)

The method of claim 7, wherein the non-denatured nucleic acid is a product of reverse transcription reaction generated from a cellular RNA, a mRNA, a microRNA, a bacterial RNA, or a viral RNA.

### Claim 9 (depends on 1)

The method of claim 1, further comprising generating the non-denatured nucleic acid by a reverse transcription reaction before step (a).

### Claim 10 (depends on 1)

The method of claim 1, wherein the enzyme having a hyperthermophile polymerase activity has a reverse transcriptase activity.

### Claim 11 (depends on 1)

The method of claim 1, wherein the enzyme having a hyperthermophile polymerase activity has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:8 or a functional fragment thereof.

### Claim 12 (depends on 1)

The method of claim 1, wherein the enzyme having a hyperthermophile polymerase activity is a polymerase comprising the amino acid sequence of SEQ ID NO: 8.

### Claim 13 (depends on 1)

The method of claim 1, wherein the enzyme having a hyperthermophile polymerase activity has 10% or less exonuclease activity compared to an unmodified hyperthermophile polymerase.

### Claim 14 (depends on 1)

The method of claim 1, wherein the sample comprises nucleic acids from prokaryotes or eukaryotes.

### Claim 15 (depends on 14)

The method of claim 14, wherein the sample comprises nucleic acids from a virus or a bacterium.

### Claim 16 (depends on 1)

The method of claim 1, wherein the method does not comprise contacting the non-denatured nucleic acid with a single-stranded DNA binding protein prior to or during step (a).

### Claim 17 (depends on 1)

The method of claim 1, wherein the amplifying the target nucleic acid sequence is performed at a constant temperature between 55 degrees Celsius and 75 degrees Celsius.

### Claim 18 (depends on 17)

The method of claim 17, wherein the amplifying the target nucleic acid sequence is performed at a constant temperature of 65 degrees Celsius.

### Claim 19 (depends on 1)

The method of claim 1, wherein the first primer, the second primer, or both is 8 to 16 bases long.

### Claim 20 (depends on 1)

The method of claim 1, wherein the nucleic acid amplification product is 20 to 40 bases long.

### Claim 21 (depends on 1)

The method of claim 1, wherein the spacer sequence comprises a portion of the target nucleic acid sequence.

### Claim 22 (depends on 21)

The method of claim 21, wherein the spacer sequence is 1 to 5 bases long.

### Claim 23 (depends on 1)

The method of claim 1, further comprising contacting the nucleic acid amplification product with a signal-generating oligonucleotide capable of hybridizing to the amplification product, wherein the single-generating oligonucleotide comprises a fluorophore, a quencher, or both.

### Claim 24 (depends on 1)

The method of claim 1, wherein the detecting the nucleic acid amplification product comprises detecting a fluorescent signal.

### Claim 25 (depends on 24)

The method of claim 24, wherein the fluorescent signal is from a molecular beacon.

### Claim 26 (depends on 1)

The method of claim 1, wherein the method is performed in a single reaction vessel.

### Claim 27 (depends on 1)

The method of claim 1, wherein the first primer, the second primer, or both comprise one or more of DNA bases, modified DNA bases, or a combination thereof.