| 摘要 |
The present teachings provide methods, compositions, and kits for performing primer extension reactions. In some embodiments, a reverse transcription reaction is performed on a target polynucleotide with a hot start primer comprising a blunt-ended self-complementary stem, and a loop, and extension products form at high temperatures but reduce extension product formation at low temperatures. |
| 主权项 |
1. A method of reducing primer extension products at a low temperature and allowing primer extension at a high temperature comprising;
forming a reaction mixture at the low temperature below about 27° C., wherein the reaction mixture comprises a target polynucleotide, a reverse transcriptase, and a hot start primer, wherein the hot start primer comprises a loop and a self-complementary stem, wherein the self-complementary stem comprises a target-specific region, but said loop does not, wherein the target-specific region of the self-complementary stem is hybridized with a quencher region in the self-complementary stem when at the low temperature, and wherein the self-complementary stem is unable to hybridize with the target polynucleotide when at the low temperature; elevating the temperature of the reaction mixture to a high temperature between 35° C.-60° C., wherein the target-specific region of the self-complementary stem is unhybridized with the quencher region in the self-complementary stem at the high temperature, and wherein the target-specific region hybridizes to the target polynucleotide; extending the target-specific region of the self-complementary stem with the reverse transcriptase to form a primer extension product; and, generating a primer extension product at the high temperature but not at the low temperature. |
