US 8,133,989 B2 · Nucleic acid primer/construct compositions

Abstract

This invention provides novel processes for amplifying nucleic acid sequences of interest, including linear and non-linear amplification. In linear amplification, a single initial primer or nucleic acid construct is utilized to carry out the amplification process. In non-linear amplification, a first initial primer or nucleic acid construct is employed with a subsequent initial primer or nucleic acid construct. In other non-linear amplification processes provided by this invention, a first initial primer or nucleic acid construct is deployed with a second initial primer or nucleic acid construct to amplify the specific nucleic acid sequence of interest and its complement that are provided. A singular primer or a singular nucleic acid construct capable of non-linear amplification can also be used to carry out non-linear amplification in accordance with this invention. Post-termination labeling process for nucleic acid sequencing is also disclosed in this invention that is based upon the detection of tagged molecules that are covalently bound to chemically reactive groups provided for chain terminators. A process for producing nucleic acid sequences having decreased thermodynamic stability to complementary sequences is also provided and achieved by this invention. Unique nucleic acid polymers are also disclosed and provided in addition to other novel compositions, kits and the like.

Claims20

Dependent claims

A composition useful for the production of two or more copies of a hepatitis B virus (HBV) nucleic acid sequence comprising

(i) a first primer comprising at least two segments:
(A) a first segment (i) being substantially complementary to a first portion of said HBV nucleic acid sequence and (ii) capable of template dependent extension such that a first primer extension product is produced when said HBV nucleic acid sequence is used as a template;
and
(B) a second segment being (i) substantially non-identical and substantially non-complementary to said first segment, and (ii) substantially identical to a second portion of said hepatitis B virus (HBV) nucleic acid sequence, such that said second segment of said first primer is capable of hybridizing to said first primer extension product and
(ii) a second primer comprising two regions:
(A) a first region (1) being sufficiently complementary to a first portion of said first primer extension product to hybridize thereto, and (2) capable of template-dependent extension to produce a second primer extension product when said first primer extension product is used as a template;
and
(B) a second region being (1) substantially non-identical and substantially non-complementary to said first region, and (2) substantially identical to a second portion of said first primer extension product, such that said second region is capable of hybridizing to the complement of said second portion of said first primer extension product formed by extension of the first region with said first primer extension product as a template, thereby providing for binding of a second copy of said second primer to said first primer extension product under isothermal or limited cycling conditions.

5 more elements

A composition useful for the production of two or more copies of a hepatitis B virus (HBV) nucleic acid sequence comprising

(i) at least one first initial primer comprising two segments;
(A) a first segment (1) being sufficiently complementary to a first portion of said HBV nucleic acid sequence to hybridize thereto, and (2) capable of template dependent extension to produce a first primer extension product when said HBV nucleic acid sequence is used as a template, and
(B) a second segment being (1) substantially non-identical and substantially non-complementary to said first segment of said first initial primer, and (2) substantially identical to a second portion of said HBV nucleic acid sequence, such that said second segment of said first initial primer is capable of hybridizing to said first primer extension product, thereby providing for subsequent binding of a first segment of a second copy of said first initial primer to said first portion of said hepatitis B virus (HBV) nucleic acid sequence under isothermal or limited cycling conditions, such that a second copy of said first primer extension product is produced and displaces said first primer extension product;
and
(ii) at least one second initial primer comprising two regions:
(A) a first region (1) being sufficiently complementary to a first portion of said first primer extension product to hybridize thereto and (2) capable of template-dependent extension to produce a second primer extension product when said first extension product is used as a template, and
(B) a second region being (1) substantially nonidentical and substantially non- complementary to said first region, and (2) substantially identical to a second portion of said first primer extension product, such that said second region is capable of hybridizing to the complement of said second portion of said first primer extension product formed by extension of the first region with said first primer extension product as a template, thereby providing for binding of a second copy of said second initial primer to said first portion of said first primer extension product under isothermal or limited cycling conditions.

4 more elements

A composition useful for the production of two or more copies of a hepatitis B virus (HBV) nucleic acid sequence of interest comprising:

(i) at least one first initial primer comprising two segments;
(A) a first segment (1) being sufficiently complementary to a first portion of said HBV nucleic acid sequence to hybridize thereto, and (2) capable of template dependent extension to produce a first primer extension product, and
(B) a second segment being (1) substantially non-identical and substantially non-complementary to said first segment, and (2) substantially identical to a second portion of said specific nucleic acid sequence, such that said second segment of said first initial primer is capable of hybridizing to said first primer extension product, thereby providing for subsequent binding of a first segment of a second copy of said first initial primer to said first portion of said HBV nucleic acid sequence under isothermal or limited cycling conditions, such that a second copy of said first primer extension is produced and displaces said first primer extension product;
and
(ii) at least one second initial primer comprising two regions:
(A) a first region (1) being sufficiently complementary to a first portion of said first primer extension product to hybridize thereto and (2) capable of template-dependent extension to produce a second primer extension product and (B) a second region being (1) substantially nonidentical and substantially non-complementary to said first region, and (2) substantially identical to a second portion of said first primer extension product, such that said second region is capable of hybridizing to the complement of said second portion of said first primer extension product formed by extension of the first region with said first primer extension product as a template, thereby providing for binding of a second copy of a second initial primer to said first portion of said first extension product under isothermal or limited cycling conditions,
wherein the 5′ end of said second segment of said first initial primer is joined to the 5′ end of the second region of the second initial primer.

4 more elements

Dependencies are inferred from the claim text.

Nucleic acid primer/construct compositions

Life of this patent

Nucleic acid primer/construct compositions

Life of this patent

Abstract

Claims20