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

• US 8,206,913 B1
• Filed: 03/03/2010
• Issued: 06/26/2012
• 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: Kamberov, Emmanuel, Sun, Tong, Bruening, Eric, Pinter, Jonathon H., Sleptsova, Irina, Kurihara, Takao, Makarov, Vladimir L.
• Primary Examiner(s): Kim, Young J
• Application Number:
  US12/716,681
• Time in Patent Office: 846 Days
• Field of Search: None
• US Class Current: 435/6.1
• 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 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 a plurality of primers of the population and the variable region sequence is degenerate among said plurality of primers of the population, and further wherein the sequence of the constant and variable regions consists essentially of only 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.

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

3. The method of claim 2, wherein said amplifying step is carried out using a polymerase chain reaction.

4. The method of claim 2, 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.

5. The method of claim 2, 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.

6. The method of claim 5, wherein the tag is a homopolymeric sequence that comprises poly cytosine (poly C) or poly guanine (poly G).

7. The method of claim 6, wherein the incorporation of the homopolymeric poly C or poly G is carried out through the use of terminal deoxynucleotidyl transferase.

8. The method of claim 6, wherein the incorporation of the homopolymeric poly G or poly C comprises ligation of an adaptor comprising the homopolymeric poly G or poly C to the ends of the amplified molecules.

9. The method of claim 6, wherein the incorporation of the homopolymeric poly C or poly G comprises replicating the amplified molecules with DNA polymerase, said replicating utilizing a primer comprising in a 5′

to 3′

orientation;

the homopolymeric poly C or poly G; and

the constant region.

10. The method of claim 6, wherein the amplified molecules comprising the homopolymeric poly C or poly G are further amplified using a first primer complementary to a desired sequence in the nucleic acid molecule and a second primer complementary to the poly C or poly G.

11. The method of claim 6, wherein the amplified molecules comprising the homopolymeric sequence poly C or poly G are further amplified using a mixture of primers complementary to different desired sequences in the nucleic acid molecule and a separate poly C or poly G primer.

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

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

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

end.

15. 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.

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

17. 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.

18. The method of claim 1, 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.