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US 2005/0089902 A1Published application
Methods and compositions for siRNA expression
AssigneeThe Scripps Research Institute
InventorsLianxing Zheng, Sheng Ding, Peter Schultz
Life of this patent
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Abstract
One-step methods for generating siRNA expression cassettes are provided. Expression cassettes useful for siRNA are also provided.
Claims42
A method for producing two complementary strands of a tripartite DNA comprising a 5′ amplification primer binding site and a 3′ amplification primer binding site with an intervening sequence between the two sites, the method comprising,
- providing a polymerase extension reaction mixture comprising the following reagents:
- a) an initial double-stranded DNA comprising
- a left end comprising a 5′ amplification primer binding site;
- and
- a right end complementary to a 3′ end of an intervening linking primer;
- b) the intervening linking primer comprising:
- a 5′ end complementary to a left end of a terminal DNA;
- a 3′ end complementary to the right end of the initial DNA;
- and
- an intervening sequence between the 5′ end and the 3′ end;
- c) the terminal double-stranded DNA comprising
- a right end that is a 3′ amplification primer binding site;
- and
- a left end complementary to the 5′ end of the intervening linking primer;
- d) DNA polymerase and components sufficient for a polymerase chain reaction;
- reacting the reagents of the mixture in at least two thermocycles, wherein
- the first cycle comprises
- a melting temperature to denature double-stranded DNA;
- an initial annealing temperature to anneal the intervening linking primer to a strand of the initial double-stranded DNA;
- and
- an extension temperature for the polymerase to extend a 3′ end of the strand of the initial double-stranded DNA using the intervening linking primer as a template to produce an intermediate product comprising the strand of the initial double stranded DNA and a complement of the intervening linking primer;
- and
- the second cycle comprises
- a melting temperature to denature double-stranded DNA;
- an annealing temperature to anneal the intermediate product to a strand of the terminal double-stranded DNA;
- and
- an extension temperature for the polymerase
- to extend the 3′ end of the intermediate product using the strand of the terminal double-stranded DNA as a template to produce a single stranded tripartite DNA comprising the strand of the initial double stranded DNA, the complement of the intervening linking primer, and the complement of the strand of the terminal double stranded DNA sequences;
- and
- to extend the 3′ end of the strand of the terminal double stranded DNA using the intermediate product as a template to produce the complement of the single-stranded tripartite DNA.
27 more elements
The method of claim 4, wherein the initial DNA and terminal DNA each further comprise a pol III terminator.
A kit for generating a siRNA polynucleotide, the kit comprising,
- a) an initial double-stranded DNA comprising
- a left end comprising a 5′ amplification primer binding site;
- and
- a right end complementary to a 3′ end of an intervening linking primer;
- b) the intervening linking primer comprising:
- a 5′ end complementary to a left end of a terminal DNA;
- and
- a 3′ end complementary to the right end of the initial DNA;
- an intervening sequence between the 5′ end and the 3′ end;
- c) the terminal double-stranded DNA comprising
- a right end that is a 3′ amplification primer binding site;
- and
- a left end complementary to the 5′ end of the intervening linking primer;
- d) a 5′ amplification primer;
- and
- e) a 3′ amplification primer.
13 more elements
The kit of claim 23, wherein the initial DNA and terminal DNA each further comprise a pol III terminator.
A mixture comprising,
- a) an initial double-stranded DNA comprising
- a left end comprising a 5′ amplification primer binding site;
- and
- a right end complementary to a 3′ end of an intervening linking primer;
- b) the intervening linking primer comprising:
- a 5′ end complementary to a left end of a terminal DNA;
- and
- a 3′ end complementary to the right end of the initial DNA;
- an intervening sequence between the 5′ end and the 3′ end;
- c) the terminal double-stranded DNA comprising
- a right end that is a 3′ amplification primer binding site;
- and
- a left end complementary to the 5′ end of the intervening linking primer;
- d) a 5′ amplification primer;
- and
- e) a 3′ amplification primer.
13 more elements
Dependencies are inferred from the claim text.