| 主权项 |
1. A method of synthesizing a polynucleic acid wherein said method comprises the steps of
a) providing a target template which comprises at least a first and a second reciprocal primer binding region; b) providing a first primer comprising a first and a second segment, wherein the first segment is substantially complementary to the first reciprocal primer binding region on the template and the second segment comprises a sequence that is substantially complementary to another region in the first primer or a region in the amplicon generated from the first segment of the first primer such that the second segment is able to form a loop, wherein, when the first primer comprises a second segment that is substantially complementary to a region in the amplicon generated from the first segment of the first primer, the first reciprocal primer binding region also encompasses a region on the template which is substantially identical to the second segment of the first primer; c) providing a second primer comprising a first and optionally a second segment, wherein the first segment is substantially complementary to the second reciprocal primer binding region on the template and the optional second segment comprises a sequence that it substantially complementary to another region in the second primer or a region in the amplicon generated from the first segment of the second primer such that the second region is able to form a loop, wherein, when the second primer comprises a second segment that is substantially complementary to a region in the amplicon generated from the first segment of the second primer, the second reciprocal primer binding region also encompasses a region on the template which is substantially identical to the second segment of the second primer; d) providing at least one stem primer which is capable of binding to the region between the first and second reciprocal primer binding regions, wherein the at least one stem primer is (i) a simple primer, which is a primer that is complementary to a primer binding site on a polynucleic acid and which contains fewer than 5 nucleotides 3′ or 5′ of the primer region which is substantially complementary to the primer binding site; (ii) a loop-forming primer, which is a primer that comprises a first and a second segment, wherein the first segment is substantially complementary to a primer binding region on the template and the second segment comprises a sequence that is substantially complementary to a region in the amplicon generated from the first segment of the first primer such that the second segment is able to form a loop; (iii) a hairpin primer, which is a primer comprising a first and a second segment, wherein the first segment is substantially complementary to a primer binding region on a template and the second segment comprises a sequence that is substantially complementary to another region in the primer; (iv) a loop-providing primer; which is a hairpin primer in which the inverted repeats are separated by a linker region; or (v) a chimeric primer; e) providing the necessary reagents and conditions to perform synthesis of the polynucleic acid; f) performing synthesis of the polynucleic acid. |
