Multi-primer amplification method for tagging of target nucleic acids

Patent metadata

Field	Value
Publication number	US-9677119-B2
Application number	US-201414180262-A
Country	US
Kind code	B2
Filing date	Feb 13, 2014
Priority date	Apr 2, 2009
Publication date	Jun 13, 2017
Grant date	Jun 13, 2017

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.

First claim

What is claimed is:

1. A method for amplifying more than 100 target nucleic acids, the method comprising:
preparing more than 100 amplification mixtures using a microfluidic device, each amplification mixture having a fixed volume between 1 picoliter and 500 nanoliters;
wherein one amplification mixture is prepared for each target nucleic acid, each 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;
using at least two additional primers to tag the target nucleotide sequences to produce a plurality of target amplicons, each comprising first and/or second nucleotide tags at the end(s) of the amplicon; and
performing automated sequencing on the plurality of target amplicons to yield sequencing data showing that
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.

2. The method of claim 1 , 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.

3. The method of claim 1 , wherein the average length of the target amplicons is at least 25 bases.

4. The method of claim 1 , wherein the average length of the target amplicons is at least 50 bases.

5. The method of claim 1 , wherein the average length of the target amplicons is at least 100 bases.

6. The method of claim 1 , wherein the average length of the target amplicons is at least 200 bases.

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

8. The method of claim 1 , 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.

9. The method of claim 1 , wherein the volume of the amplification mixtures is in the range of about 5 picoliters to about 25 nanoliters.

10. The method of claim 1 , wherein the amplification mixtures are formed in separate compartments of a microfluidic device prior to amplification.

11. The method of claim 1 , wherein the microfluidic device permits recovery of pools of reaction products, and the variability, with respect to volume and/or copy number, of each pool recovered from the device is less than 10 percent.

12. The method of claim 11 , wherein the variability is less than 5%.

13. The method of claim 1 , wherein each target amplicon is capable of being bound by DNA sequencing primers.

14. The method of claim 1 , wherein the method is performed in determining the copy numbers of the target nucleic acids in each sample.

15. The method of claim 1 , wherein the method is performed in determining the genotypes at loci corresponding to the target nucleic acids.

16. The method of claim 1 , wherein the method is performed in determining the expression levels of the target nucleic acids.

Assignees

• Fluidigm Corp

Inventors

• May Andrew
• Chen Peilin
• Wang Jun
• Kaper Fiona
• Anderson Megan

Classifications

• B01L2300/0816

  Cards, e.g. flat sample carriers usually with flow in two horizontal directions · CPC title

• B01L2400/0487

  fluid pressure, pneumatics · CPC title

• B01L3/502738 Primary

  characterised by integrated valves (throttle valves in microfluidic sample containers B01L3/502746) · CPC title

• B01L2300/0867

  Multiple inlets and one sample wells, e.g. mixing, dilution · CPC title

• B01L2300/087

  Multiple sequential chambers · CPC title