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US 2025/0002984 A1Published application
Methods for amplification of nucleic acids with endonuclease-mediated shifting equilibrium (em-seq)
AssigneeDNAe Diagnostics Limited
InventorKamil Andrzej LIPINSKI
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Abstract
The embodiments and improvements relate to the design of molecular biology assays based on isothermal amplification of nucleic acids with Strand Displacement Amplification (SDA). The embodiments describe a novel method for SDA termed Endonuclease-Mediated Shifting Equilibrium Amplification (EM-SEq), which improves exponential kinetics and specificity of the reaction and enables amplification on solid surfaces.
Claims19
A method for the sequencing a double stranded nucleic acid molecule, comprising the steps of:
- A. carrying out an isothermal clonal amplification step comprising:
- a. modifying ends of the double stranded nucleic acid molecule such that at least one end comprises a region of sequence which is single stranded under the conditions of the isothermal process;
- b. copying the nucleic acid molecule having modified ends using at least one amplification primer which is immobilized on a solid support, wherein the at least one amplification primer hybridizes to at least one of the single stranded ends, and wherein the at least one immobilized amplification primer comprises:
- (i) a first 3′ region which is configured to hybridize to a single stranded end of the modified nucleic acid molecule;
- (ii) a second region which is an endonuclease recognition site;
- and
- (iii) a third 5′ region having a Tm which is higher than the temperature of the isothermal process and higher than the Tm of the first region;
- and wherein at least a portion of the 5′ region extends beyond the 3′ end of the modified template population of nucleic acid molecules such that extension of the 3′ end of the modified template provides a complete recognition site for the endonuclease, and a double stranded 5′ region;
- and extension of the 3′ end of the at least one amplification primers by strand displacement provides a copy the template;
- c. using the complete recognition site for the endonuclease to nick the extended strand, thereby releasing a free 3′-OH group within the one or more amplification primers;
- and
- d. extending the freed 3′-OH group by strand displacement to re-copy the modified nucleic acid;
- wherein steps b and c are performed isothermally;
- B. denaturing the double stranded amplicon to provide an single stranded template immobilized on the solid support;
- and
- C. hybridizing a sequencing primer to the single stranded template and sequencing the single stranded template using next generation sequencing.
13 more elements
A method for the sequencing a double stranded nucleic acid molecule in a sample using an ISFET sensor comprising an array of wells, each well comprising at least one immobilized amplification primer, the method comprising:
- A. carrying out an isothermal clonal amplification process comprising:
- a. modifying ends of the double stranded nucleic acid molecule such that at least one end comprises a region of sequence which is single stranded under the conditions of the isothermal process;
- b. contacting the sample with the ISFET array such that the concentration of molecules in the sample gives rise to an average occupancy of less than one molecule per well;
- c. copying the nucleic acid molecule having modified ends using at least one of the immobilized primers in a well of the ISFET sensor, wherein the at least one amplification primer hybridizes to at least one of the single stranded ends, and wherein the at least one immobilized amplification primer comprises:
- (i) a first 3′ region which is configured to hybridize to a single stranded end of the modified nucleic acid molecule;
- (ii) a second region which is an endonuclease recognition site;
- and
- (iii) a third 5′ region having a Tm which is higher than the temperature of the isothermal process and higher than the Tm of the first region;
- and wherein at least a portion of the 5′ region extends beyond the 3′ end of the modified template population of nucleic acid molecules such that extension of the 3′ end of the modified template provides a complete recognition site for the endonuclease, and a double stranded 5′ region;
- and extension of the 3′ end of the at least one amplification primers by strand displacement provides a copy the template;
- d. using the complete recognition site for the endonuclease to nick the extended strand, thereby releasing a free 3′-OH group within the one or more amplification primers;
- and
- e. extending the freed 3′-OH group by strand displacement to re-copy the modified nucleic acid;
- wherein steps b and c are performed isothermally;
- B. treating the amplified nucleic acid molecules to remove nicks by strand extension
- C. denaturing the double stranded amplicon to provide single stranded copies of a first strand of the templates immobilized on the solid support;
- and
- D. hybridizing a first sequencing primer to the single stranded template and sequencing the single stranded template to obtain a first read.
15 more elements
A method for isothermal amplification of a double stranded nucleic acid molecule, comprising:
- a. modifying ends of the double stranded nucleic acid molecule such that at least one end comprises a region of sequence which is single stranded under the conditions of the isothermal process;
- b. copying the nucleic acid molecule having modified ends using at least one amplification primer, wherein the at least one amplification primer hybridizes to at least one of the single stranded ends, and wherein the at least one immobilized amplification primer comprises:
- (i) a first 3′ region which is configured to hybridize to a single stranded end of the modified nucleic acid molecule;
- (ii) a second region which is an endonuclease recognition site;
- and
- (iii) a third 5′ region having a Tm which is higher than the temperature of the isothermal process and higher than the Tm of the first region;
- and wherein at least a portion of the 5′ region extends beyond the 3′ end of the modified template population of nucleic acid molecules such that extension of the 3′ end of the modified nucleic acid molecule provides a complete recognition site for the endonuclease, and a double stranded 5′ region;
- and extension of the 3′ end of the at least one amplification primer by strand displacement provides a copy the template;
- c. using the complete recognition site for the endonuclease to nick the extended strand, thereby releasing a free 3′-OH group within the one or more amplification primers;
- and
- d. extending the freed 3′-OH group by strand displacement to re-copy the modified nucleic acid;
- wherein steps b and c are performed isothermally.
9 more elements
Dependencies are inferred from the claim text.