METHODS AND COMPOSITIONS FOR AMPLIFICATION OF RNA SEQUENCES

• US 20100167354A1
• Filed: 11/10/2009
• Published: 07/01/2010
• Est. Priority Date: 03/09/2001
• Status: Active Grant

• Current Assignee: NuGEN Technologies, Inc. (Tecan Group AG)
• Original Assignee: NuGEN Technologies, Inc. (Tecan Group AG)
• Inventors: Kurn, Nurith
• Granted Patent: US 8,071,311 B2
• Time in Patent Office: Days
• US Class Current: 435/91.200
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]

Abstract

The invention provides methods for isothermal amplification of RNA. The methods are particularly suitable for amplifying a plurality of RNA species in a sample. The methods employ a composite primer, a second primer and strand displacement to generate multiple copies of DNA products comprising sequences complementary to an RNA sequence of interest. In another aspect, the methods employ a single primer (which is a composite primer) and strand displacement to generate multiple copies of DNA products comprising sequences complementary to an RNA sequence of interest. In some embodiments, a transcription step is included to generate multiple copies of sense RNA of an RNA sequence of interest. The methods are useful for preparation of nucleic acid libraries and substrates for analysis of gene expression of cells in biological samples. The invention also provides compositions and kits for practicing the amplification methods, as well as methods which use the amplification products.

Claims

1-161. -161. (canceled)

162. A method for amplifying a target nucleic acid sequence or its complement to generate a plurality of single-stranded amplified products comprising performing linear, isothermal amplification on said target nucleic acid or its complement using a composite primer having a 3′

DNA portion and a 5′

RNA portion.

163. The method of claim 162 wherein the target nucleic acid comprises RNA and said target nucleic acid is selected from the group consisting of total RNA, tRNA, mRNA, rRNA, mitochondrial RNA, chloroplast RNA, DNA-RNA hybrids, viral RNA, cell free RNA, and mixtures thereof.

164. The method of claim 162 wherein the amplified products are further immobilized on a solid substrate.

165. The method of claim 164 wherein each amplified product further comprises a moiety that is capable of attachment to a solid substrate.

166. The method of claim 165 wherein each amplified product further comprises a modified base or a nucleotide analog that comprises a moiety that is capable of attachment to a solid substrate.

167. The method of claim 162 wherein the method further comprises a plurality of composite primers each comprising a 5′

RNA portion and a 3′

DNA portion wherein the 5′

RNA portion comprises a defined sequence and the 3′

DNA portion comprises a random sequence.

168. The method of claim 162 wherein the method further comprises a second composite primer having a 5′

RNA portion.

169. The method of claim 168 wherein the method further comprises a third composite primer having a 5′

RNA portion.

170. The method of claim 169 wherein the method further comprises a fourth composite primer having a 5′

RNA portion.

171. The method of claim 169 wherein the third composite primer is an amplification primer.

172. The method of claim 170 wherein the third and fourth composite primers are amplification primers.

173. The method of claim 168 wherein the sequence of the RNA portions of the first and second primers are the same.

174. The method of claim 173 wherein the third and fourth primers are the same.

175. The method of claim 168 wherein the sequence of the RNA portions of the first and second primers are not the same.

176. The method of claim 175 wherein the third and fourth primers are not the same

177. The method of claim 162 wherein the amplification products are further labeled.

178. The method of claim 177 wherein the label is selected from the group consisting of fluorescent dyes, radioisotopes, enzymes, steroids, colorimetric labels, ligands, and anti-ligands.

179. A composition comprising at least one RNA template and a plurality of random primers wherein the random primers comprise a 5′

RNA portion and a 3′

DNA portion.

180. The composition of claim 179, further comprising an RNA-dependent DNA polymerase.

181. The composition of claim 180 further comprising a DNA-dependent DNA polymerase.