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

• US 10,837,049 B2
• Filed: 07/22/2016
• Issued: 11/17/2020
• Est. Priority Date: 03/07/2003
• Status: Active Grant

• Current Assignee: Takara Bio Usa, Inc. (Ningbo Junsheng Investment Group Co., Ltd.)
• Original Assignee: Takara Bio Usa, Inc. (Ningbo Junsheng Investment Group Co., Ltd.)
• Inventors: Kamberov, Emmanuel, Sun, Tong, Bruening, Eric, Pinter, Jonathon H., Sleptsova, Irina, Kurihara, Takao, Makarov, Vladimir L.
• Primary Examiner(s): Kim, Young J
• Application Number:
  US15/216,783
• Publication Number:
  US 20160355879A1
• Time in Patent Office: 1,579 Days
• Field of Search: None
• 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 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• 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

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. A method of preparing a plurality of nucleic acid molecules having a known constant region at each end, the method comprising:

a) obtaining a sample comprising nucleic acid molecules;

b) subjecting said nucleic acid molecules to a population of primers to form a nucleic acid molecule/primer mixture, wherein all the primers of the population have a nucleotide sequence that is substantially non-self-complementary and substantially non-complementary to other primers in the population, wherein all the primers comprise a constant region of at least 6 nucleotides in length, the constant region consisting of all nucleotides of a contiguous known sequence that is constant among all the primers of the population, and a variable region at the distal 3′

end of at least 4 nucleotides in length, wherein the variable region begins with a degenerate nucleotide, the constant region being positioned 5′

to the variable region, wherein the constant region and the variable region each comprise greater than 50% non-complementary nucleotides, wherein the greater than 50% 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, rendering the population of primers 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; and

c) subjecting said nucleic acid molecule/primer mixture to a thermostable polymerase under conditions to generate the plurality of molecules including the known constant region at each end.

2. The method of claim 1, wherein said nucleic acid molecules comprise single stranded nucleic acid molecules that comprise DNA, RNA, or DNA-RNA chimeras.

3. The method of claim 1, wherein said nucleic acid molecules are double stranded nucleic acid molecules that comprise DNA, RNA, or DNA-RNA chimeras.

4. The method of claim 1, wherein the method further comprises a step d) of amplifying the plurality of the molecules to produce amplified molecules.

5. The method of claim 4, wherein said amplifying step is carried out using a polymerase chain reaction.

6. The method of claim 4, wherein said method further comprises the steps of:

modifying the amplified molecules to incorporate modified nucleotide bases, thereby producing labeled molecules, said amplified molecules further defined as single stranded DNA, double stranded DNA, or a mixture thereof;

generating single stranded molecules from the labeled molecules, said single stranded molecules capable of hybridizing to complementary sequences arrayed in known locations on a substrate; and

analyzing at least one hybridization signal.

7. The method of claim 4, wherein a tag is incorporated on the ends of the amplified molecules and wherein said constant region is penultimate to the tags on each end of the amplified molecules.

8. The method of claim 1, wherein said constant region is 6 to 100 nucleotides in length.

9. The method of claim 1, wherein said variable region is 4 nucleotides to 20 nucleotides in length.

10. The method of claim 1, wherein said nucleic acid sequence is further defined as rendering the population of primers substantially incapable of the following:

self-hybridization;

self-priming;

hybridization to another polynucleotide in the plurality; and

initiation of a polymerization reaction in the plurality.

11. The method of claim 1, wherein the constant region and the variable region of each primer are adjacent.

12. The method of claim 1, wherein the constant region is at the distal 5′

end of each primer.

13. The method of claim 12, wherein the constant region and the variable region of each primer are adjacent.

14. The method of claim 1, wherein the variable region comprises at least 5 degenerate nucleotides.

15. The method of claim 1, wherein the method further comprises amplifying the plurality of molecules including the known constant region at each end with primers that comprise the constant region.

16. A method of preparing a plurality of nucleic acid molecules having a known constant region at each end, the method comprising:

a) obtaining a sample comprising nucleic acid molecules;

b) subjecting said nucleic acid molecules to a population of primers to form a nucleic acid molecule/primer mixture, wherein all the primers of the population have a nucleotide sequence that is substantially non-self-complementary and substantially non-complementary to other primers in the population, wherein all the primers comprise a constant region at the distal 5′

end of at least 6 nucleotides in length, the constant region consisting of all nucleotides of a contiguous known sequence that is constant among all the primers of the population, 1 to 3 random bases at the distal 3′

end, and a variable region of at least 4 nucleotides in length, wherein the variable region begins with a degenerate nucleotide, the variable region being positioned adjacent to the random base(s), wherein the constant region and the variable region each comprise greater than 50% non-complementary nucleotides, wherein the greater than 50% 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, rendering the population of primers 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; and

c) subjecting said nucleic acid molecule/primer mixture to a thermostable polymerase under conditions to generate the plurality of molecules including the known constant region at each end.

17. The method of claim 16, wherein the constant region and the variable region are each comprised of guanines and thymidines/uridines.

18. The method of claim 16, wherein the constant region and the variable region of each primer are adjacent.

19. A method of preparing a plurality of nucleic acid molecules having a known constant region at each end, the method comprising:

a) obtaining a sample comprising nucleic acid molecules;

b) subjecting said nucleic acid molecules to a population of primers to form a nucleic acid molecule/primer mixture, wherein the primers of the population have a nucleotide sequence that is substantially non-self-complementary and substantially non-complementary to other primers in the population, wherein the primers comprise a constant region at the distal 5′

end of at least 6 nucleotides in length, the constant region consisting of all nucleotides of a contiguous known sequence that is constant among all the primers of the population, and a variable region of at least 4 nucleotides in length, the constant region being positioned 5′

to the variable region, wherein the constant region and the variable region each comprise at least 80% non-complementary nucleotides, wherein the at least 75% 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, rendering the population of primers 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; and

c) subjecting said nucleic acid molecule/primer mixture to a polymerase under conditions to generate the plurality of molecules including the known constant region at each end.

20. The method of claim 19, wherein at least 85% of the constant region and the variable region are comprised of the non-complementary nucleotides.

21. The method of claim 20, wherein at least 90% of the constant region and the variable region are comprised of the non-complementary nucleotides.

22. The method of claim 21, wherein at least 95% of the constant region and the variable region are comprised of the non-complementary nucleotides.

23. The method of claim 22, wherein at least 97% of the constant region and the variable region are comprised of the non-complementary nucleotides.

24. The method of claim 23, wherein 100% of the constant region and the variable region are comprised of the non-complementary nucleotides.

25. The method of claim 19, wherein the constant region and the variable region of each primer are adjacent.

26. The method of claim 19, wherein each primer comprises 1 to 3 random bases at the distal 3′

end.

27. A method of preparing a plurality of nucleic acid molecules having a known constant region at each end, the method comprising:

a) obtaining a sample comprising nucleic acid molecules;

b) subjecting said nucleic acid molecules to a population of primers to form a nucleic acid molecule/primer mixture, wherein the primers of the population have a nucleotide sequence that is substantially non-self-complementary and substantially non-complementary to other primers in the population, wherein the primers comprise a constant region and a variable region, the constant region being positioned 5′

to the variable region, wherein the constant region and the variable region each comprise greater than 50% non-complementary nucleotides, wherein the greater than 50% 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, rendering the population of primers 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; and

c) subjecting said nucleic acid molecule/primer mixture to a polymerase under conditions to generate the plurality of molecules including the known constant region at each end, wherein each primer of the population is comprised of at least 70% of the non-complementary nucleotides.

28. The method of claim 27, wherein the constant region is at least 6 nucleotides in length and the variable region is at least 4 nucleotides in length.

29. The method of claim 28, wherein the constant region is at the distal 5′

end of the primers, the variable region is at the distal 3′

end of the primers, the primers comprise 1 to 3 random bases at the distal 3′

end, the constant region and the variable region of each primer are adjacent, or a combination thereof.

30. A method of preparing a plurality of nucleic acid molecules having known constant region at each end, comprising:

a) obtaining a sample comprising nucleic acid molecules;

b) subjecting said nucleic acid molecules to a population of primers to form a nucleic acid molecule/primer mixture, wherein the primers of the population are non-self-complementary and non-complementary to other primers in the population, and comprise in a 5′

to 3′

orientation a constant region and a variable region, wherein the constant region sequence has a known sequence that is constant among the primers of the population and the variable region sequence is degenerate among the primers of the population, and further wherein the sequence of the primers comprises at least 70% of two types of non-complementary nucleotides selected from the group consisting of adenines and guanines;

adenines and cytosines;

guanines and thymidines; and

cytosines and thymidines, such that the primers of the population will not cross-hybridize or self-hybridize under the conditions employed in step c); and

c) subjecting said nucleic acid molecule/primer mixture to a polymerase under conditions to generate the plurality of molecules including the known constant region at each end.

31. A method of preparing a plurality of nucleic acid molecules having known constant region at each end, comprising:

a) obtaining a sample comprising nucleic acid molecules;

b) subjecting said nucleic acid molecules to a population of primers to form a nucleic acid molecule/primer mixture, wherein all the primers of the population are non-self-complementary and non-complementary to other primers in the population, and comprise in a 5′

to 3′

orientation a constant region and a variable region, wherein the constant region sequence consists of all nucleotides of a contiguous known sequence that is constant among all the primers of the population and the variable region sequence is degenerate among all the primers of the population, wherein the variable region begins with a degenerate nucleotide, and further wherein the sequence of the constant region comprises at least 70% of two types of non-complementary nucleotides selected from the group consisting of adenines and guanines;

adenines and cytosines;

guanines and thymidines; and

cytosines and thymidines, and the sequence of the variable region comprises greater than 50% non-complementary nucleotides selected from the group consisting of adenines and guanines;

adenines and cytosines;

guanines and thymidines; and

cytosines and thymidines, such that all the primers of the population will not cross-hybridize or self-hybridize under the conditions employed in step c); and

c) subjecting said nucleic acid molecule/primer mixture to a thermostable polymerase under conditions to generate the plurality of molecules including the known constant region at each end.

32. The method of claim 31, wherein the constant region is at least 6 nucleotides in length and the variable region is at least 4 nucleotides in length.

33. The method of claim 31, wherein the sequence of the variable region comprises at least 70% of two types of non-complementary nucleotides selected from the group consisting of adenines and guanines;

adenines and cytosines;

guanines and thymidines; and

cytosines and thymidines.