Amplification and analysis of whole genome and whole transcriptome libraries generated by a DNA polymerization process
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.
1 .- 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 . (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 . (canceled)