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. A kit comprising:
a population of polynucleotide primers, wherein the population of polynucleotide primers is configured to generate from a whole human genome or whole human transcriptome a library comprising a representative amplifiable copy of the whole human genome or whole human transcriptome, wherein the primers of the population:
(a) comprise a variable region and a constant region, wherein the constant region is positioned 5′ to the variable region; and
(b) have sequences that are 70% or more comprised of two non-complementary and non-self-complementary nucleotides within the variable and constant regions,
wherein the population of primers all comprise the same constant region,
wherein the variable region is 4 to 20 nucleotides in length and is degenerate among the plurality of primers,
wherein the kit comprises an amount of primers of the population effective to generate the library from the whole genome or whole transcriptome; and
a polymerase.
2. The kit according to claim 1 , wherein the primers of the population have sequences that are 75% or more comprised of two non-complementary and non-self-complementary nucleotides.
3. The kit according to claim 1 , wherein the primers of the population have sequences that are 80% or more comprised of two non-complementary and non-self-complementary nucleotides.
4. The kit according to claim 1 , wherein the primers of the population have sequences that are 85% or more comprised of two non-complementary and non-self-complementary nucleotides.
5. The kit according to claim 1 , wherein the primers of the population have sequences that are 90% or more comprised of two non-complementary and non-self-complementary nucleotides.
6. The kit according to claim 1 , wherein the primers of the population have sequences that are 95% or more comprised of two non-complementary and non-self-complementary nucleotides.
7. The kit according to claim 1 , wherein the primers of the population have sequences that are 97% or more comprised of two non-complementary and non-self-complementary nucleotides.
8. The kit according to claim 1 , wherein the primers of the population have sequences that are 100% comprised of two non-complementary and non-self-complementary nucleotides.
9. The kit according to claim 1 , wherein the non-complementary and non-self-complementary variable and constant regions consist of:
(i) guanines and adenines;
(ii) cytosines and thymidines;
(iii) adenines and cytosines; or
(iv) guanines and thymidines.
10. The kit according to claim 1 , wherein the constant region comprises 6 to 100 nucleotides.
11. The kit according to claim 1 , wherein the kit further comprises a second polymerase.
12. The kit according to claim 1 , wherein the polymerase is an isothermally amplifying polymerase.
13. The kit according to claim 1 , wherein the polymerase is a strand displacing polymerase.
14. The kit according to claim 1 , wherein the kit further comprises a polymerase processivity-enhancing compound.
15. The kit according to claim 14 , wherein the polymerase processivity enhancing compound is a single-stranded DNA binding protein or a helicase.
16. The kit according to claim 1 , wherein the kit further comprises an agent that facilitates polymerization through GC-rich DNA and/or RNA.
17. The kit according to claim 1 , wherein the constant region and the variable region each comprise at least 70% non-complementary nucleotides, wherein the at least 70% non-complementary nucleotides of both the constant region and the variable region are: guanines, adenines, or combinations thereof; cytosines, thymidines/uridines, or combinations thereof; adenines, cytosines, or combinations thereof; or guanines, thymidines/uridines, or combinations thereof.
18. The kit according to claim 17 , wherein the constant region and the variable region each comprise at least 75% non-complementary nucleotides.
19. The kit according to claim 18 , wherein the constant region and the variable region each comprise at least 80% non-complementary nucleotides.
20. The kit according to claim 19 , wherein the constant region and the variable region each comprise at least 85% non-complementary nucleotides.
21. The kit according to claim 20 , wherein the constant region and the variable region each comprise at least 95% non-complementary nucleotides.
22. The kit according to claim 1 , wherein the constant region and the variable region of each primer are adjacent.
23. The kit according to claim 1 , wherein each primer comprises 1 to 3 random bases at the distal 3′ end.
24. The kit according to claim 1 , wherein the primers of the population comprise a labeled nucleotide.
25. The kit according to claim 1 , wherein the variable region comprises a degenerate nucleotide.
26. The kit according to claim 1 , wherein the primers of the population range in length from 10 nucleotides to 120 nucleotides.
27. The kit according to claim 1 , wherein the polymerase is a non-naturally-occurring polymerase.
28. The kit according to claim 1 , wherein the polymerase is selected from a 9° Nm polymerase, a Klenow exo-fragment of DNA Polymerase I, a mutant form of T7 phage DNA polymerase that lack 3′ to 5′ endonuclease activity, a modified Pyrococcus furiosus DNA polymerase,-, a exo-Bst large fragment polymerase, a modified Thermococcus litoralis DNA polymerase lacking 3′-5′ endonuclease activity, a phage T5 exo-DNA polymerase, a chemically modified Taq DNA polymerase requiring thermal activation, or a thermosequenase polymerase, or a genetically modified Taq polymerase comprising an N-terminal deletion.
29. The kit of claim 1 , wherein the library comprises at least 80% of the human genome.
30. The kit of claim 1 , wherein library comprises at least 90% of the human genome.
31. The kit of claim 1 , wherein the library comprises at least 99% of the human genome.
32. A kit comprising:
a population of polynucleotide primers ranging in length from 10 to 120 nucleotides, wherein the population of polynucleotide primers is configured to generate from a whole human genome or whole human transcriptome a library comprising a representative amplifiable copy of the whole human genome or whole human transcriptome, wherein the primers of the population:
(a) comprise a variable region and a constant region that is comprised of:
(i) guanines and adenines;
(ii) cytosines and thymidines;
(iii) adenines and cytosines; or
(iv) guanines and thymidines,
wherein the constant region comprises 6 to 100 nucleotides and is positioned 5′ to the variable region; and
(b) have sequences that are 75% or more comprised of two non-complementary and non-self-complementary nucleotides within the variable and constant regions,
wherein the population of primers all comprise the same constant region,
wherein the variable region is 4 to 20 nucleotides in length and is degenerate among the plurality of primers,
wherein the kit comprises an amount of primers of the population effective to generate the library from the whole genome or whole transcriptome; and
a non-naturally occurring polymerase.