US 9,315,863 B2 · Sequence-specific methods for homogeneous, real-time detection of lamp products

Abstract

Presented herein are methods and compositions for generating sequence-specific, secondary amplification products during Loop-mediated Isothermal Amplification (LAMP). Conventional LAMP produces a preponderance of high molecular weight DNA structures concatenated into self-complementary hairpins, which are not amenable to detection by routine probe-based hybridization methods, making multiplex detection of two or more targets or sequence variants in closed-tube formats extremely difficult. Provided herein, for example, are methods for generating secondary LAMP products bearing a fragment of the original target sequence embedded within low-molecular weight products that are devoid of competitive hairpin structures, the lack of which enhances probe-based detection of target sequences. These secondary products can, for example, be produced in real-time, during the LAMP process, and can provide the option of detecting multiple target sequences within a single tube using, e.g., a homogenous, real-time fluorescence format.

Claims29

Dependent claims

A method for generating a nucleic acid signal extension product during a loop-mediated isothermal amplification reaction, comprising:

hybridizing portions of a first nucleic acid signal primer, a first nucleic acid amplification primer, and a first nucleic acid displacement primer to complementary portions of a nucleic acid target sequence,
wherein the first nucleic acid signal primer hybridizes to the target sequence downstream of the first nucleic acid amplification primer;
wherein the first nucleic acid amplification primer comprises i) a 3′ terminal portion complementary to a portion of the nucleic acid target sequence, and ii) a 5′ terminal portion that does not hybridize but is complementary to a portion of the target sequence;
and
wherein the first nucleic acid displacement primer hybridizes to the target sequence upstream of the first nucleic acid amplification primer;
extending the hybridized first nucleic acid signal primer on the nucleic acid target sequence to produce a first signal extension product;
extending the hybridized first nucleic acid amplification primer on the nucleic acid target sequence wherein such extension displaces the first signal extension product from the nucleic acid target sequence and produces a first amplification extension product;
extending the hybridized first nucleic acid displacement primer, wherein such extension displaces the first amplification extension product from the nucleic acid target sequence and produces a first displacement extension product;
and
detecting the first signal extension product.

7 more elements

A method for generating nucleic acid signal extension products during a loop-mediated isothermal amplification reaction, comprising:

a. hybridizing a first nucleic acid signal primer to a nucleic acid target sequence region, wherein the target sequence comprises at least one self-complementary hairpin structure generated during a loop-mediated isothermal amplification reaction;
b. extending the hybridized first nucleic acid signal primer on the nucleic acid target sequence to produce a first signal extension product, wherein said first signal extension product comprises a hairpin structure;
c. hybridizing portions of a second nucleic acid amplification primer, as second nucleic acid amplification primer and second nucleic acid displacement primer to complementary portions of the first signal extension product, and extending the second hybridized nucleic acid signal primer on the first signal extension product to produce a second signal extension product;
d. extending the hybridized second nucleic acid amplification primer on the first signal extension product wherein such extension displaces the second signal extension product from the first signal extension product and produces a second amplification extension product;
e. extending the second nucleic acid displacement primer, wherein such extension displaces a second amplification extension product from the first signal extension product and producing a second displacement extension product;
and
f. detecting the second signal extension product.

4 more elements

A method for concurrently generating nucleic acid signal extension products during a loop-mediated isothermal amplification reaction, comprising:

a. hybridizing portion of a first signal primer to a first single stranded target sequence;
b. hybridizing a portion of a first nucleic acid amplification primer to the first single stranded target sequence upstream of the hybridized portion of the first signal primer,
wherein the first amplification primer comprises i) a 3′ terminal portion complementary to a portion of the first single stranded target nucleic acid sequence, and ii) a 5′ terminal portion that does not hybridize but is complementary to a portion of the first single stranded target sequence;
c. hybridizing a first displacement primer to the first single stranded target sequence upstream of the first amplification primer;
d. extending the hybridized first signal primer on the first single stranded target sequence to produce a first signal extension product;
e. extending the hybridized first amplification primer such that it displaces the first signal extension product from the target sequence;
f. extending the hybridized first displacement primer such that it displaces the first amplification extension product from the target sequence;
g. hybridizing a portion of a second signal primer to the displaced first signal extension product;
h. hybridizing a portion of a second amplification primer to the first signal extension product upstream of the hybridized second signal primer, wherein the second amplification primer comprises:
i) a 3′ terminal portion complementary to a portion of the first signal extension product, and ii) a 5′ terminal portion that does not hybridize but is complementary to a portion of the first signal extension product;
i. hybridizing a second displacement primer to the first signal extension product upstream of the second amplification primer;
j. extending the hybridized second signal primer on the first signal extension product to produce a second signal extension product;
k. extending the hybridized second amplification primer on the first signal extension product such that it displaces the second signal extension product from the first signal extension product;
l. extending the hybridized second displacement primer such that it displaces the second amplification extension product from the first signal extension product;
and
m. detecting the first and second signal extension products.

13 more elements

Dependencies are inferred from the claim text.

Sequence-specific methods for homogeneous, real-time detection of lamp products

Life of this patent

Sequence-specific methods for homogeneous, real-time detection of lamp products

Life of this patent

Abstract

Claims29