AMPLIFICATION AND ANALYSIS OF WHOLE GENOME AND WHOLE TRANSCRIPTOME LIBRARIES GENERATED BY A DNA POLYMERIZATION PROCESS

• US 20160355879A1
• Filed: 07/22/2016
• Published: 12/08/2016
• Est. Priority Date: 03/07/2003
• Status: Active Grant

• Current Assignee: Takara Bio Usa, Inc. (Ningbo Junsheng Investment Group Co., Ltd.)
• Original Assignee: Rubicon Genomics, Inc
• Inventors: PINTER, Jonathon H., KAMBEROV, Emmanuel, SUN, Tong, BRUENING, Eric, SLEPTSOVA, Irina, KURIHARA, Takao, MAKAROV, Vladimir L.
• Granted Patent: US 10,837,049 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• 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 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 have a nucleotide sequence that is substantially non-self-complementary and substantially non-complementary to other primers in the population, wherein the primers are composed of a constant region and a variable region, the constant region being positioned 5′

to the variable region, wherein primers in the population comprise non-complementary and non-self-complementary nucleotides selected from guanines and adenines;

cytosines and thymidines/uridines;

adenines and cytosines;

or guanines and thymidines/uridines in a relative proportion effective to render 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; 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.

2. The method of claim 1, wherein said nucleic acid molecules comprise single stranded nucleic acid molecules.

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

4. The method of claim 1, wherein said nucleic acid molecules are double stranded nucleic acid molecules.

5. The method of claim 4, wherein said double stranded nucleic acid molecules are DNA, RNA, or DNA-RNA chimeras.

6. The method of claim 1, wherein said nucleic acid molecules are a mixture of single stranded and double stranded nucleic acid molecules.

7. The method of claim 6, wherein said single stranded molecules are RNA and said double stranded molecules are DNA.

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

9. The method of claim 8, wherein said amplifying step is carried out using a polymerase chain reaction.

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

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

16. The method of claim 1, wherein the primers of the population further comprise 0 to 3 random bases at its distal 3′

end.

17. The method of claim 1, wherein the constant region and the variable region are each comprised of guanines and thymidines and wherein the primers of the population further comprise 0, 1, 2, or 3 random bases at their 3′

ends.

31. The method of claim 8, 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.

38. The method of claim 8, 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.

48. The method of claim 1, wherein the polymerase is a thermostable polymerase.

50. The method of claim 1, wherein the nucleic acid molecule/primer mixture is subjected to polymerase chain reaction having polymerization steps comprising two or more DNA denaturation steps.

51. The method of claim 50, wherein step c) is further defined as comprising:

(1) a first series of polymerase chain reaction steps comprising subjecting the nucleic acid molecule/primer mixture to a thermostable polymerase in the presence of the primers under conditions having two or more cycling DNA denaturation steps; and

(2) a second series of polymerase chain reaction steps using primers that comprise the constant region sequence.

52. The method of claim 1, wherein said nucleic acid sequence is further defined as rendering the polynucleotides 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.

53. The method of claim 1, wherein at least 75% of a sequence of a primer in the population is comprised of two non-complementary and non-self-complementary nucleotides.

54. The method of claim 53, wherein at least 80% of a sequence of a primer in the population is comprised of two non-complementary and non-self-complementary nucleotides.

55. The method of claim 54, wherein at least 85% of a sequence of a primer in the population is comprised of two non-complementary and non-self-complementary nucleotides.

56. The method of claim 55, wherein at least 90% of a sequence of a primer in the population is comprised of two non-complementary and non-self-complementary nucleotides.

57. The method of claim 56, wherein at least 95% of a sequence of a primer in the population is comprised of two non-complementary and non-self-complementary nucleotides.

58. The method of claim 57, wherein at least 97% of a sequence of a primer in the population is comprised of two non-complementary and non-self-complementary nucleotides.

59. The population of claim 58, wherein 100% of a sequence of a primer in the population is comprised of two non-complementary and non-self-complementary nucleotides.

10-13. -13. (canceled)

18-30. -30. (canceled)

32-37. -37. (canceled)

39-47. -47. (canceled)

49. (canceled)