Nucleic acid encoding reactions

• US 9,074,204 B2
• Filed: 05/21/2012
• Issued: 07/07/2015
• Est. Priority Date: 05/20/2011
• Status: Active Grant

• Current Assignee: Fluidigm Corporation
• Original Assignee: Fluidigm Corporation
• Inventors: Anderson, Megan, Chen, Peilin, Fowler, Brian, Jones, Robert C., Kaper, Fiona, Lebofsky, Ronald, May, Andrew
• Primary Examiner(s): Babic, Christopher M
• Assistant Examiner(s): PRIEST, AARON A
• Application Number:
  US13/476,911
• Publication Number:
  US 20130005585A1
• Time in Patent Office: 1,142 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 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/65 using markers enzymes used as markers C12N15/52
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation

Abstract

Described herein are methods useful for incorporating one or more adaptors and/or nucleotide tag(s) and/or barcode nucleotide sequence(s) one, or typically more, target nucleotide sequences. In particular embodiments, nucleic acid fragments having adaptors, e.g., suitable for use in high-throughput DNA sequencing are generated. In other embodiments, information about a reaction mixture is encoded into a reaction product. Also described herein are methods and kits useful for amplifying one or more target nucleic acids in preparation for applications such as bidirectional nucleic acid sequencing. In particular embodiments, methods of the invention entail additionally carrying out bidirectional DNA sequencing. Also described herein are methods for encoding and detecting and/or quantifying alleles by primer extension.

Claims

1. A method for amplifying a target nucleic acid, the method comprising:

amplifying a target nucleic acid using;

a set of inner primers, wherein the set comprises;

an inner, forward primer comprising a target-specific portion and a first primer binding site;

an inner, reverse primer comprising a target-specific portion and a second primer binding site, wherein the first and second primer binding sites are different;

a first set of outer primers, wherein the set comprises;

a first outer, forward primer comprising a portion specific for the first primer binding site; and

a first outer, reverse primer comprising a barcode nucleotide sequence and a portion specific for the second primer binding site;

a second set of outer primers, wherein the set comprises;

a second outer, forward primer comprising a barcode nucleotide sequence and a portion specific for the first primer binding site; and

a second outer, reverse primer comprising a portion specific for the second primer binding site;

to produce two target amplicons, wherein;

a first target amplicon comprises 5′

-first primer binding site-target nucleotide sequence-second primer binding site-barcode nucleotide sequence-3′

; and

a second target amplicon comprises 5′

-barcode nucleotide sequence-first primer binding site-target nucleotide sequence-second primer binding site-3′

;

wherein the method comprises amplifying a plurality of target nucleic acids and produces target amplicons having common first and common second primer binding sites, wherein at least some target amplicons have different barcode nucleotide sequences.

2. The method of claim 1, wherein the barcode nucleotide sequence in each target amplicon produced from a given target nucleic acid is the same, and wherein each target amplicon comprises only one barcode nucleotide sequence.

3. The method of claim 1, wherein the first and second primer binding sites are binding sites for DNA sequencing primers.

4. The method of claim 1, wherein the outer primers each additionally comprise an additional nucleotide sequence, wherein:

the first outer, forward primer comprises a first additional nucleotide sequence, and the first outer, reverse primer comprises a second additional nucleotide sequence; and

the second outer, forward primer comprises the second additional nucleotide sequence, and the second outer, reverse primer comprises the first additional nucleotide sequence; and

the first and second additional nucleotide sequences are different; and

said amplifying produces two target amplicons, wherein;

a first target amplicon comprises 5′

-first additional nucleotide sequence-first primer binding site-target nucleotide sequence-second primer binding site-barcode nucleotide sequence-second additional nucleotide sequence-3′

; and

a second target amplicon comprises 5′

-second additional nucleotide sequence-barcode nucleotide sequence-first primer binding site-target nucleotide sequence-second primer binding site-first additional nucleotide sequence 3′

.

5. The method of claim 4, wherein the first or second additional nucleotide sequence comprises a primer binding site.

6. The method of claim 5, wherein the first and second additional nucleotide sequences each comprise a primer binding site.

7. The method of claim 4, wherein the first set of outer primers comprises PE1-CS1 (SEQ ID NO:

9) and PE2-BC-CS2 (SEQ ID NO;

10), and the second set of outer primers comprises PE1-CS2 (SEQ ID NO;

11) and PE2-BC-CS1 (SEQ ID NO;

12).

8. The method of claim 1, wherein said amplifying is carried out in a single amplification reaction.

9. The method of claim 1, wherein said amplifying comprises employing the inner primers in a first amplification reaction and employing the outer primers in a second amplification reaction, wherein said second amplification reaction is separate from the first.

10. The method of claim 9, wherein the second amplification reaction comprises two separate amplification reactions, wherein one employs the first set of outer primers and the other employs the second set of outer primers.

11. The method of claim 10, wherein the target amplicons produced in the two separate second amplification reactions are pooled.

12. The method of claim 1, wherein the plurality of target nucleic acids is selected from the group consisting of genomic DNA, cDNA, fragmented DNA, DNA reverse-transcribed from RNA, a DNA library, and nucleic acids extracted or amplified from a cell, a bodily fluid or a tissue sample.

13. The method of claim 12, wherein the plurality of target nucleic acids is amplified from a formalin-fixed, paraffin-embedded tissue sample.

14. The method of claim 1, wherein the method additionally comprises sequencing the target amplicons.

15. The method of claim 1, wherein the method comprises amplifying the target amplicons using primers that bind to the first and second additional nucleotide sequences to produce templates for DNA sequencing.

16. The method of claim 15, wherein one or both primers that bind to the first and second additional nucleotide sequences are immobilized on a substrate.

17. The method of claim 1, wherein the amplification is carried out by isothermal nucleic acid amplification.

18. The method of claim 15, wherein the method comprises performing DNA sequencing using the templates and target sequencing primers that bind to the first and second primer binding sites, wherein said target sequencing primers prime sequencing of the target nucleotide sequence(s).

19. The method of claim 18, wherein the target sequencing primers are present in substantially equal amounts so as to produce both 5′

and 3′

DNA sequence information from each target nucleotide sequence.

20. The method of claim 15, wherein the method comprises performing DNA sequencing using the templates and barcode sequencing primers that bind to the first and second primer binding sites, wherein said barcode sequencing primers prime sequencing of the barcode nucleotide sequences(s).

21. The method of claim 20, wherein the barcode sequencing primers are present in substantially equal amounts so as to produce barcode sequences from each of the first and second target amplicons.

22. The method of claim 18, wherein the method comprises performing DNA sequencing using the templates and barcode sequencing primers that are reverse complements of the target sequencing primers.

23. The method of claim 22, wherein the target sequencing primers and the barcode sequencing primers comprise CS1 (SEQ ID NO:

13), CS2 (SEQ ID NO;

14), CS1rc (SEQ ID NO;

15), and CS2rc (SEQ ID NO;

16).

24. The method of claim 1, wherein the barcode nucleotide sequence is selected so as to avoid substantial annealing to the target nucleic acids.

25. The method claim 1, wherein the barcode nucleotide sequence identifies a particular sample.

26. The method of claim 15, wherein for at least 50 percent of the sequences determined from DNA sequencing, each determined sequence is present at greater than 50 percent of the average number of copies of that sequence and less than 2-fold said average number of copies.

27. The method of claim 15, wherein for at least 70 percent of the sequences determined from DNA sequencing, each determined sequence is present at greater than 50 percent of the average number of copies of that sequence and less than 2-fold said average number of copies.

28. The method of claim 15, wherein for at least 90 percent of the sequences determined from DNA sequencing, each determined sequence is present at greater than 50 percent of the average number of copies of that sequence and less than 2-fold said average number of copies of sequences.

29. The method of claim 1, wherein the average length of the target amplicons is less than 200 bases.

30. The method of claim 1, wherein the first amplification reaction is carried out in a volume in the range of about 1 picoliter to about 50 nanoliters.

31. The method of claim 1, wherein the first amplification reaction is carried out in a volume in the range of about 5 picoliters to about 25 nanoliters.

32. The method of claim 1, wherein the first amplification reaction(s) is/are formed in, or distributed into, separate compartments of a microfluidic device prior to amplification.

33. The method of claim 32, wherein the microfluidic device is fabricated, at least in part, from an elastomeric material.

34. The method of claim 1, wherein the first amplification reaction(s) is/are carried out in (a) fluid droplet(s).

35. The method of claim 34, wherein a plurality of first amplification reactions are carried out in fluid droplets in an emulsion.