MULTI-PRIMER AMPLIFICATION METHOD FOR BARCODING OF TARGET NUCLEIC ACIDS

• US 20100273219A1
• Filed: 04/02/2010
• Published: 10/28/2010
• Est. Priority Date: 04/02/2009
• Status: Active Grant

• Current Assignee: Fluidigm Corporation
• Original Assignee: Fluidigm Corporation
• Inventors: Wang, Jun, Kaper, Fiona, Chen, Peilin, Anderson, Megan, May, Andrew
• Granted Patent: US 8,691,509 B2
• Time in Patent Office: Days
• US Class Current: 435/91.500
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/087 Multiple sequential chambers
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/0655 pinch valves
• CPC Class Codes: B01L 3/50273 characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• CPC Class Codes: B01L 3/502738 characterised by integrated valves microvalves F16K99/0001
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• 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 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2527/143 Concentration of primer or probe
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2549/119 using nested primers
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

In certain embodiments, the present invention provides amplification methods in which nucleotide tag(s) and, optionally, a barcode nucleotide sequence are added to target nucleotide sequences. In other embodiments, the present invention provides a microfluidic device that includes a plurality of first input lines and a plurality of second input lines. The microfluidic device also includes a plurality of sets of first chambers and a plurality of sets of second chambers. Each set of first chambers is in fluid communication with one of the plurality of first input lines. Each set of second chambers is in fluid communication with one of the plurality of second input lines. The microfluidic device further includes a plurality of first pump elements in fluid communication with a first portion of the plurality of second input lines and a plurality of second pump elements in fluid communication with a second portion of the plurality of second input lines.

Claims

1. A method for amplifying a plurality of target nucleic acids in a plurality of samples, the method comprising:

preparing an amplification mixture for each target nucleic acid, said amplification mixture comprising;

a forward primer comprising a target-specific portion;

a reverse primer comprising a target-specific portion, wherein the forward primer additionally comprises a first nucleotide tag and/or the reverse primer additionally comprises a second nucleotide tag; and

at least one barcode primer comprising a barcode nucleotide sequence and a first and/or second nucleotide tag-specific portion, wherein the barcode primer is in excess of the forward and/or reverse primer(s);

subjecting each amplification mixture to amplification to produce a plurality of target amplicons comprising tagged target nucleotide sequences, each comprising first and/or second nucleotide tags flanking the target nucleotide sequence, and at least one barcode nucleotide sequence at the 5′

or 3′

end of the target amplicon.

2. The method of claim 1, wherein the forward primer additionally comprises a first nucleotide tag.

3. The method of claim 1, wherein the reverse primer additionally comprises a second nucleotide tag.

4. The method of claim 1, wherein the concentration of the barcode primer in the amplification mixtures is at least 4-fold the concentration of the forward and/or reverse primer(s).

5. The method of claim 4, wherein the concentration of the barcode primer in the amplification mixtures is at least 50-fold the concentration of the forward and/or reverse primer(s).

6. The method of claim 1, wherein the first and/or second nucleotide tags and/or the barcode nucleotide sequence are selected so as to avoid substantial annealing to the target nucleic acids.

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

8. The method of claim 1, wherein the barcode primer comprises a barcode nucleotide sequence and a first nucleotide tag-specific portion.

9. The method of claim 8, wherein a plurality of forward primers comprise the same first nucleotide tag.

10. The method of claim 9, wherein the all forward primers used to amplify target sequences in each sample comprise the same first nucleotide tag.

11. The method of claim 7, wherein the forward and reverse primers for each target are initially combined separately from the sample, and each barcode primer is initially combined with its corresponding sample.

12. The method of claim 11, wherein T targets are amplified in S samples, T and S being integers greater than one, the method additionally comprising preparing S×

T amplification mixtures wherein the initially combined forward and reverse primers are added to the initially combined samples and barcode primers.

13. The method of claim 1, wherein the amplification is carried out for at least 3 cycles to introduce the first and second nucleotide tags and the barcode nucleotide sequence.

14. The method of claim 13, wherein the amplification is carried out for between 5 and 50 cycles.

15. The method of claim 1, wherein the amplification is carried out for a sufficient number of cycles to normalize target amplicon copy number across targets and across samples.

16. The method of claim 1, wherein at least 50 percent of the target amplicons are present at greater than 50 percent of the average number of copies of target amplicons and less than 2-fold the average number of copies of target amplicons.

18. The method of claim 16, wherein at least 70 percent of the target amplicons are present at greater than 50 percent of the average number of copies of target amplicons and less than 2-fold the average number of copies of target amplicons.

30. The method of claim 1, additionally comprising recovering the target amplicons from the amplification mixtures.

31. The method of claim 30, wherein the target amplicons are recovered in a volume and/or copy number that varies less than about 50% among the recovered target amplicons.

56. The method of claim 1, wherein the method is performed to prepare target nucleic acids for sequencing.

17. A method for amplifying a plurality of target nucleic acids in a plurality of samples, the method comprising:

preparing an amplification mixture for each target nucleic acid, said amplification mixture comprising;

a forward primer comprising a target-specific sequence; and

a reverse primer comprising a target-specific sequence;

subjecting each amplification mixture to amplification to produce a plurality of target nucleotide sequences;

tagging the target nucleotide sequences to produce a plurality of target amplicons, each comprising first and/or second nucleotide tags flanking the target nucleotide sequence;

wherein at least 50 percent of the target amplicons are present at greater than 50 percent of the average number of copies of target amplicons and less than 2-fold the average number of copies of target amplicons.

19. The method of claim 17, wherein the average length of the target amplicons is at least 25 bases.

20. The method of claim 17, wherein the average length of the target amplicons is at least 50 bases.

21. The method of claim 17, wherein the average length of the target amplicons is at least 100 bases.

22. The method of claim 17, wherein the average length of the target amplicons is at least 200 bases.

23. The method of claim 17, wherein the average length of the target amplicons is at least 1 kilobase.

24. The method of claim 17, wherein at least 90 percent of the target amplicons are present at greater than 50 percent of the average number of copies of target amplicons and less than 2-fold the average number of copies of target amplicons.

25-29. -29. (canceled)

32-55. -55. (canceled)

57. A microfluidic device comprising:

a plurality of first input lines;

a plurality of second input lines;

a plurality of sets of first chambers, wherein each set of first chambers is in fluid communication with one of the plurality of first input lines;

a plurality of sets of second chambers, wherein each set of second chambers is in fluid communication with one of the plurality of second input lines;

a plurality of first pump elements in fluid communication with a first portion of the plurality of second input lines; and

a plurality of second pump elements in fluid communication with a second portion of the plurality of second input lines.

58-65. -65. (canceled)

66. A method of operating a microfluidic device having an assay chamber, a sample chamber, and a harvesting port, the method comprising:

closing a fluid line between the assay chamber and the sample chamber;

flowing a sample into the sample chamber via a sample input line;

flowing an assay into the assay chamber via an assay input line;

opening the fluid line between the assay chamber and the sample chamber;

combining at least a portion of the sample and at least a portion of the assay to form a mixture;

reacting the mixture to form a reaction product;

closing the fluid line between the assay chamber and the sample chamber;

flowing a harvesting reagent from the harvesting port to the sample chamber; and

removing the reaction product from the microfluidic device.

67-81. -81. (canceled)

82. A method of preparing reaction products, the method comprising:

providing M samples;

providing N assays;

mixing the M samples and N assays to form M×

N pairwise combinations, each of the M×

N pairwise combinations being contained in a closed volume;

forming M×

N reaction products from the M×

N pairwise combinations; and

recovering the M×

N reaction products.

83-95. -95. (canceled)