# US 11,111,530 B2 · Methods for high level multiplexed polymerase chain reactions and homogeneous mass extension reactions

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- Number: US 11,111,530 B2
- Document ID: US11111530B2
- Status: Granted
- Kind: B2
- Application number: 15/863,644
- Filed: 2018-01-05
- Published: 2018-08-30
- Granted: 2021-09-07
- Earliest claimed: 2003-07-31
- Assignees: Agena Biosciences, Inc.
- Applicants: Agena Bioscience, Inc.
- Inventors: Martin Beaulieu; Dirk Johannes Van Den Boom
- Web page: https://patentgrep.com/patent/US11111530B2

## CPC

- C12Q 1/6858: Allele-specific amplification
- C12Q 1/6844: Nucleic acid amplification reactions
- C12Q 1/6872: involving mass spectrometry
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 2600/156: Polymorphic or mutational markers
- C12Q 2600/16: Primer sets for multiplex assays
- 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

Provided herein are optimized methods for performing multiplexed detection of a plurality of sequence variations. Also provided are methods for performing multiplexed amplification of target nucleic acid.

## Claims

### Claim 1

A method for performing multiplexed amplification of target nucleic acid, comprising:
(a) forming a mixture containing 7 or more pairs of primary and secondary primers and one or more target nucleic acids, wherein each primer pair amplifies a nucleic acid-target region of a target nucleic acid and a sequence tag is attached to one or both of the primary and secondary primers of one or more primer pairs; and
(b) amplifying a plurality of nucleic acid-target regions of (a) under amplification conditions, whereby at least 60% of the 7 or more nucleic acid-target regions attempted are amplified by the 7 or more amplification primer pairs to produce an amplified mixture of nucleic acid-target regions, wherein the amplification conditions comprise a multiplicity of thermocycles and amplification reactants comprising a polymerase, dNTPs and MgCl2, wherein the ratio of the concentration of MgCl2 to the concentration of each one of the dNTPs is less than or equal to 10:1 and only a single primer pair is used to amplify each particular nucleic acid target-region.

### Claim 2 (depends on 1)

The method of claim 1, wherein the concentration of each dNTP is between about 400-700 μM.

### Claim 3 (depends on 1)

The method of claim 1, wherein the free Mg2+ concentration is between 1.0-2.0 mM.

### Claim 4 (depends on 1)

The method of claim 1, wherein the polymerase is Taq polymerase.

### Claim 5 (depends on 1)

The method of claim 1, wherein the amplification reaction conditions comprise about 100 nM primer pairs, and between about 2.6 up to about 4.8 mM MgCl2.

### Claim 6 (depends on 1)

The method of claim 1, wherein a sequence tag is attached to both primers of one or more primer pairs.

### Claim 7 (depends on 1)

The method of claim 1, further comprising inactivating one or more amplification reactants, wherein the one or more amplification reactants includes a nucleotide and/or a primer.

### Claim 8 (depends on 7)

The method of claim 7, wherein the amplification reactant that is a nucleotide is inactivated by a phosphatase and/or the amplification reactant that is a primer is inactivated by a nuclease.

### Claim 9 (depends on 8)

The method of claim 8, wherein the nuclease is an exonuclease.

### Claim 10 (depends on 1)

The method of claim 1, wherein for each amplification primer pair, one primer is in lower concentration than the other primer.

### Claim 11 (depends on 1)

The method of claim 1, wherein the ratio of the concentration of MgCl2 to the concentration of each one of the dNTPs is less than or equal to 9:1.

### Claim 12 (depends on 1)

The method of claim 1, wherein the multiplicity of thermocycles is about 45.

### Claim 13 (depends on 1)

The method of claim 1, wherein the amplification conditions excludes the addition of one or more of BSA, glycerol, DMSO and urea.

### Claim 14 (depends on 4)

The method of claim 4, wherein the Taq polymerase is at a concentration of 0.03 units/μl.

### Claim 15 (depends on 1)

The method of claim 1, whereby at least 90% of the 7 or more nucleic acid-target regions attempted are amplified by the 7 or more amplification primer pairs.

### Claim 16 (depends on 1)

The method of claim 1, wherein the free Mg2+ concentration is between about 1.3 to about 1.7 mM.

### Claim 17 (depends on 1)

The method of claim 1, wherein the ratio of the concentration of MgCl2 to the concentration of each one of the dNTPs is less than or equal to 7:1.

### Claim 18 (depends on 1)

The method of claim 1, wherein the ratio of the concentration of MgCl2 to the concentration of each one of the dNTPs is less than or equal to 6:1.

### Claim 19 (depends on 1)

The method of claim 1, wherein the mixture of (a) contains 13 or more pairs of primary and secondary primers, whereby at least 60% of the 13 or more nucleic acid-target regions attempted are amplified by the 13 or more amplification primer pairs.

### Claim 20 (depends on 1)

The method of claim 1, wherein a sequence tag is attached to all primers.

### Claim 21 (depends on 1)

The method of claim 1, wherein the sequence tag is from 5 up to 20 nucleotides in length.

### Claim 22 (depends on 1)

The method of claim 1, wherein the sequence tag comprises the sequence of nucleotides of SEQ ID NO: 37.