Self-primed amplification system

A mixture of amplification primers composed of first and second primers for copying a target nucleic acid,

• (a) said first primer comprising two domains of nucleic acid sequences in the following order from 3′ to 5′:
• i. a sense, target-specific binding domain;
• ii. a non-target, antisense primer binding domain;
• (b) said second primer comprising two domains of nucleic acid sequences in the following order from 3′ to 5′:
• i. a non-target sense primer binding domain;
• ii. an antisense, target-specific binding domain wherein the sequence of the domain complements a sense sequence of the target nucleic acid which lies upstream of the target nucleic acid sequence complementary to the sense, target-specific binding sequence.

A mixture of nucleic acid primers for copying a target nucleic acid comprising a primer mixture selected from the group consisting of:

• (a) a primer comprising two domains of nucleic acid sequences in the following order from 3′ to 5′:
• i. a sense, target-specific binding domain;
• ii. a non-target, antisense primer binding domain;
• and
• a primer having 3′ sequence identity to the sequence of the non-target, antisense primer binding domain;
• (b) a primer comprising two domains of nucleic acid sequences in the following order from 3′ to 5′:
• i. a non-target sense primer binding domain;
• ii. an antisense, target-specific binding domain wherein the sequence of the domain complements a sense sequence of the target nucleic acid which lies upstream of the target nucleic acid sequence complementary to the sense, target-specific binding sequence;
• and
• a primer complementary to the non-target sense primer binding domain;
• and
• (c) a combination of the above four primers.

A method of copying a target nucleic acid said method comprising:

• (a) combining in an aqueous solution:
• a target nucleic acid, a nucleic acid polymerase, a molar excess of dNTP, a non-target antisense-primer, a non-target sense-primer, and self-priming nucleic acid polymerase (SNAP) primers for copying a target nucleic acid, the primers comprising four domains of nucleic acid sequences in the following order from 3′ to 5′:
• i. a sense, target-specific binding domain;
• ii. a non-target, antisense primer binding domain;
• iii. a non-target sense primer binding domain;
• iv. an antisense, target-specific binding domain, wherein the sequence of the domain complements a sense sequence of the target nucleic acid which lies upstream of the target nucleic acid sequence complementary to the sense, target-specific binding sequence;
• wherein domains i and ii are joined together to form a first primer and domains iii and iv are joined together to form a second primer or wherein i, ii, iii, and iv are all joined together to form a single primer;
• (b) allowing the SNAP primers to bind to the target nucleic acid and to the non-target sense primer, to permit the nucleic acid polymerase to extend the primers to create:
• i. a nascent sense primer having a non-target primer sequence and a target-specific binding sequence having identity to a sense sequence of the target nucleic acid which lies upstream of the target nucleic acid sequence complementary to the sense, target-specific binding sequence of the SNAP primer;
• and
• ii. a long SNAP extension (antisense) product;
• (c) denaturing the nucleic acid strands;
• (d) allowing the nascent sense primer to bind to the long, SNAP extension (antisense) product to permit the nucleic acid polymerase to extend the nascent sense primer to yield a long, nascent primer extension (sense) product having a non-target antisense primer binding sequence;
• (e) denaturing the nucleic acid strands;
• (f) allowing the non-target antisense primer to bind to the long, nascent primer-extension (sense) product to permit the nucleic acid polymerase to extend the non-target antisense primer to yield a short, antisense primer extension (antisense) product terminating in a non-target sense primer binding sequence;
• (g) denaturing the nucleic acid strands;
• (h) allowing the non-target sense primer to bind to the short primer extension (antisense) product to yield a short non-target primer extension (sense) product terminating in a non-target antisense primer binding sequence;
• and
• (i) repetitively allowing the non-target primers to bind to the short extension products in a series of polymerase extension and denaturation cycles to amplify the target nucleic acid.

A method for copying at least 2 different target nucleic acids comprising

• (a) combining in an aqueous solution:
• at least 2 different target nucleic acids, nucleic acid polymerase, a molar excess of dNTPs, non-target antisense primers, non-target sense-primers, and self-priming nucleic acid polymerase (SNAP) primers for copying at least 2 different target nucleic acids, the primers comprising four domains of nucleic acid sequences in the following order from 3′ to 5′:
• i. a sense, target-specific binding domain;
• ii. a non-target, antisense primer binding domain;
• iii. a non-target sense primer binding domain;
• iv. an antisense, target-specific binding domain, wherein the sequence of the domain complements a sense sequence of the target nucleic acid which lies upstream of the target nucleic acid sequence complementary to the sense, target-specific binding sequence;
• wherein domains i and ii are joined together to form a first primer and domains iii and iv are joined together to form a second primer, or wherein i, ii, iii, and iv are all joined together to form a single primer;
• (b) allowing the SNAP primers to hybridize to said at least 2 different target nucleic acids and to the non-target sense primers, to permit the nucleic acid polymerase to extend the primers to create:
• i. nascent sense primers having a non-target primer sequence and a target-specific binding sequence having identity to a sense sequence of the target nucleic acid which lies upstream of the target nucleic acid sequence complementary to the sense, target-specific binding sequence of the SNAP primer;
• and
• ii. long SNAP extension (antisense) products;
• (c) denaturing the nucleic acid strands;
• (d) allowing the nascent sense primers to bind to the long, SNAP extension (antisense) products to permit the nucleic acid polymerase to extend the nascent sense primers to yield long, nascent primer extension (sense) products having a non-target antisense primer binding sequence;
• (e) denaturing the nucleic acid strands;
• (f) allowing the non-target antisense primers to bind to the long, nascent primer extension (sense) products to permit the nucleic acid polymerase to extend the non-target antisense primers to yield a short, antisense primer extension (antisense) products terminating in a non-target sense primer binding sequence;
• (g) denaturing the nucleic acid strands;
• (h) allowing the non-target sense primer to bind to the short primer extension(antisense) product to yield a short non-target primer extension (sense) product terminating in a non-target antisense primer binding sequence;
• and
• (i) repetitively allowing the non-target primers to bind to the short extension products in a series of nucleic acid polymerase extension and denaturation cycles to amplify the target nucleic acid.

A kit comprising:

• a container having a self-priming nucleic acid polymerase (SNAP) primer for copying a target nucleic acid comprising four domains of nucleic acid sequences in the following order from 3′ to 5′:
• i. a sense, target-specific binding domain;
• ii. a non-target, antisense primer binding domain;
• iii. a non-target sense primer binding domain;
• iv. an antisense, target-specific binding domain, wherein the sequence of the domain complements a sense sequence of the target nucleic acid which lies upstream of the target nucleic acid sequence complementary to the sense, target-specific binding sequence;
• wherein domains i and ii are joined together to form a first primer and domains iii and iv are joined together to form a second primer or wherein i, ii, iii, and iv are all joined together to form a single primer.