# US 5,686,271 A · Apparatus for performing magnetic cycle reaction

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- Number: US 5,686,271 A
- Document ID: US5686271A
- Status: Granted
- Kind: A
- Application number: 08/353,573
- Filed: 1994-12-09
- Granted: 1997-11-11
- Earliest claimed: 1994-06-09
- Assignees: Gamera Bioscience Corporation
- Inventors: Alec Mian; Stephen G. Kieffer-Higgins
- Web page: https://patentgrep.com/patent/US5686271A

## CPC

- B03C 1/035: Open gradient magnetic separators, i.e. separators in which the gap is unobstructed, characterised by the configuration of the gap
- B03C 1/28: Magnetic plugs and dipsticks
- C12Q 1/6834: Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q 1/6846: {Common amplification features}
- C12Q 1/686: Polymerase chain reaction [PCR]
- G01N 35/0098: {involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation (magnetic particles used in immunoassays G01N33/54326; magnetic separation in general B03C)}
- Y10T 436/206664: Ozone or peroxide
- Y10T 436/207497: Molecular oxygen
- B03C: MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS (separating isotopes B01D59/00; combinations of magnetic or electrostatic separation with separation of solids by other means B03B, B07B)
- 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)
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

The invention provides an apparatus for performing a process for amplification of specific nucleic acid sequences based upon the separation of nucleic acid strands by an electromagnetic field. This means of separation allows the use of mesophilic polymerases in the amplification process, thereby increasing the speed and fidelity of the amplification process, as well as the size of target nucleic acid that can be amplified.

## Claims

### Claim 1

An apparatus for performing a magnetic cycle reaction, the apparatus comprising in combination:
(a) a temperature-controlled sample block comprising a plate containing an array of a multiplicity of sample tube wells, wherein a portion of a sample tube fits within the sample tube well, and heating and cooling means in thermal contact with the sample block for controlling the temperature thereof;
(b) a first magnetic element, wherein the first magnetic element is located above the sample block and in magnetic proximity thereof, wherein the first magnetic element comprises a multiplicity of coils, wherein the coils comprise a multiplicity of rams of an electricity-conducting wire around a soft iron core, and wherein the flow of electric current through the wire comprising the coil creates a magnetic field surrounding the first magnetic element which extends to the sample block;
(c) a second magnetic dement, wherein the second magnetic element is located beneath the sample block and in proximity thereof, wherein the second magnetic element comprises a multiplicity of coils, wherein the coils comprise a multiplicity of turns of an electricity-conducting wire around a soft iron core, and wherein the flow of electric current through the wire comprising the coil creates a magnetic field surrounding the second magnetic element which extends to the sample block;
(d) a controller comprising a microprocessor, wherein the controller is operatively linked to the temperature-controlled sample block and each of the first and second magnetic elements, wherein the controller is also operatively linked to a user interface and the controller controls electric current flow through each of the magnetic elements and the temperature of the temperature-controlled sample block; and
(e) a power supply that is operatively linked to the controller and supplies electrical power to the apparatus.

### Claim 2 (depends on 1)

A method for performing a magnetic cycle reaction to amplify a specific DNA fragment using the apparatus of claim 1, the method comprising:
(a) incorporating into a nucleic acid strand complementary to the target nucleic acid a primer attached to a solid phase (solid phase primer) to create a solid phase strand bound to the target nucleic acid;
(b) separating the solid phase strand and the target nucleic acid;
(c) incorporating into a nucleic acid strand complementary to the solid phase strand a primer attached to a magnetic particle (magnetic primer) to yield a duplex having one solid phase strand and one magnetic strand;
(d) separating the solid phase strand from the magnetic strand by applying an electromagnetic field of sufficient strength to dissociate the duplex, wherein said electromagnetic field is generated by the first magnetic element of the apparatus of claim 1;
(e) allowing magnetic primers complementary to the solid phase strand to anneal to the solid phase strand by applying an electromagnetic field, wherein said electromagnetic field is generated by the second magnetic element of the apparatus of claim 1, and allowing solid phase primers complementary to the magnetic strand to anneal to the magnetic strand;
(f) extending the annealed solid phase and magnetic primers with a suitable DNA polymerase; and
(g) repeating steps (d) through (f) as many times as necessary to obtain a desired quantity of amplified DNA.

### Claim 3 (depends on 1)

A method of amplifying a specific single stranded target nucleic acid using the apparatus of claim 1, the method comprising the steps of:
(a) incorporating into a nucleic acid strand complementary to the target nucleic acid a magnetic primer to create a magnetic strand bound to the target nucleic acid by extending an annealed magnetic primer with a polymerase;
(b) separating the magnetic strand and the target nucleic acid;
(c) incorporating into a nucleic acid strand complementary to the magnetic strand a solid phase primer to yield a duplex having one solid phase strand and one magnetic strand by extending an annealed solid phase primer with a polymerase;
(d) separating the solid phase strand from the magnetic strand by applying an electromagnetic field of sufficient strength to dissociate the duplex, by generating a magnetic field in the first magnetic element of the apparatus of claim 1;
(e) allowing magnetic primers complementary to the solid phase strand to anneal to the solid phase strand by generating a magnetic field in the second magnetic element of the apparatus of claim 1, and allowing solid phase primers complementary to the magnetic strand to anneal to the magnetic strand;
(f) extending the annealed solid phase and magnetic primers with a suitable DNA polymerase; and
(g) repeating steps (d) through (f) as many times as necessary to obtain a desired quantity of amplified DNA.

### Claim 4 (depends on 1)

A method of amplifying a specific double stranded target nucleic acid using the apparatus of claim 1, the method comprising the steps of:
(a) separating the strands of the target nucleic acid to yield a first strand and a second strand;
(b) incorporating a solid phase primer into a strand complementary to the first strand by extending an annealed solid phase primer with a polymerase, to yield a first heteroduplex having the first strand and a solid phase strand, and incorporating a magnetic primer into a strand complementary to the second strand by extending an annealed magnetic primer with a polymerase, to yield a second heteroduplex having the second strand and a magnetic strand;
(c) separating the strands of the first heteroduplex and of the second heteroduplex;
(d) allowing magnetic primers complementary to the solid phase strand to anneal to the solid phase strand and allowing solid phase primers complementary to the magnetic strand to anneal to the magnetic strand;
(f) extending the annealed magnetic and solid phase primers with a DNA polymerase; and
(g) separating the solid phase strand from the magnetic strand by applying an electromagnetic field of sufficient strength to dissociate the duplex, by generating a magnetic field in the first magnetic element of the apparatus of claim 1;
(h) allowing magnetic primers complementary to the solid phase strand to anneal to the solid phase strand by generating a magnetic field in the second magnetic element of the apparatus of claim 1, and allowing solid phase primers complementary to the magnetic strand to anneal to the magnetic strand;
(I) extending the annealed magnetic and solid phase primers with a DNA polymerase; and
(j) repeating steps (g) through (I) as many times as necessary to obtain a desired quantity of amplified DNA.