Amplification and analysis of whole genome and whole transcriptome libraries generated by a DNA polymerization process

• US 20070054311A1
• Filed: 11/06/2006
• Published: 03/08/2007
• Est. Priority Date: 03/07/2003
• Status: Abandoned Application

• Current Assignee: Emmanuel Kamberov, Eric Bruening, Irina Sleptsova, Jonathon Pinter, Takao Kurihara, Tong Sun, Vladimir Makarov
• Original Assignee: Emmanuel Kamberov, Eric Bruening, Irina Sleptsova, Jonathon Pinter, Takao Kurihara, Tong Sun, Vladimir Makarov
• Inventors: Makarov, Vladimir, Kurihara, Takao, Kamberov, Emmanuel, Bruening, Eric, Sleptsova, Irina, Sun, Tong, Pinter, Jonathon
• Application Number:
  US11/593,625
• Publication Number:
  US 20070054311A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/15 incorporating a consensus or conserved sequence
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms

Abstract

The present invention regards a variety of methods and compositions for whole genome amplification and whole transcriptome amplification. In a particular aspect of the present invention, there is a method of amplifying a genome comprising a library generation step followed by a library amplification step. In specific embodiments, the library generating step utilizes specific primer mixtures and a DNA polymerase, wherein the specific primer mixtures are designed to eliminate ability to self-hybridize and/or hybridize to other primers within a mixture but efficiently and frequently prime nucleic acid templates.

Claims

1-183. -183. (canceled)

184. A plurality of polynucleotides, wherein the polynucleotides in the plurality comprise nucleic acid sequence that is substantially non-self-complementary and substantially non-complementary to other polynucleotides in the plurality.

185. The plurality of claim 184, wherein said nucleic acid sequence is further defined as rendering the polynucleotides substantially incapable of at least one of the following:

self-hybridization;

self-priming;

hybridization to another polynucleotide in the plurality;

or

initiation of a polymerization reaction in the plurality.

186. The plurality of claim 184, wherein the polynucleotides are further defined as having a 5′

to 3′

orientation and comprising a constant region that is 5′

to a variable region.

187. The plurality of claim 186, wherein the constant region is for subsequent amplification.

188. The plurality of claim 186, wherein the variable region is for random annealing, random priming, or both.

189. The plurality of claim 186, wherein the constant and variable regions are each comprised of two non-complementary nucleotides.

190. The plurality of claim 186, wherein the constant and variable regions are each comprised of guanines, adenines, or both.

191. The plurality of claim 186, wherein the constant and variable regions are each comprised of cytosines, thymidines, or both.

192. The plurality of claim 186, wherein the constant and variable regions are each comprised of adenines, cytosines, or both.

193. The plurality of claim 186, wherein the constant and variable regions are each comprised of guanines, thymidines, or both.

194. The plurality of claim 186, wherein said constant region comprises about 6 to about 100 nucleotides.

195. The plurality of claim 186, wherein said second region comprises about 4 nucleotides to about 20 nucleotides.

196. The plurality of claim 186, wherein the polynucleotide is further comprised of 0 to about 3 random bases at its distal 3′

end.

197. The plurality of claim 186, wherein the first region and the second region are each comprised of guanines and thymidines and wherein the polynucleotide comprises 0, 1, 2, or 3 random bases at its 3′

end.

198. The plurality of claim 184, wherein the nucleic acid sequence is comprised of base or backbone analogs.

199. The plurality of claim 184, wherein the concentration of each polynucleotide in the plurality of primers is adjusted for optimal priming of a given nucleic acid.

200. The plurality of claim 199, wherein the concentration of all polynucleotides in the plurality is equimolar.

201. The plurality of claim 199, wherein the given nucleic acid comprises part or all of a genome.

202. The plurality of claim 199, wherein the given nucleic acid comprises part or all of a transcriptome.

203-205. -205. (canceled)

206. A kit comprising a plurality of polynucleotides, wherein the polynucleotides comprise nucleic acid sequence that is substantially non-self-complementary and substantially non-complementary to other polynucleotides in the plurality, said plurality dispersed in a suitable container.

207. The kit of claim 206, further comprising a polymerase.

208. The kit of claim 207, wherein said polymerase is a strand-displacing polymerase.

209. The kit of claim 208, wherein said strand-displacing polymerase is □

29 Polymerase, Bst Polymerase, Vent Polymerase, 9o Nm Polymerase, Klenow fragment of DNA Polymerase I, MMLV Reverse Transcriptase, Tth DNA polymerase, AMV Reverse Transcriptase, human HIV Reverse Transcriptase, a mutant form of T7 phage DNA polymerase that lacks 3′

-5′

exonuclease activity, or a mixture thereof.

210-242. -242. (canceled)