Digital analyte analysis
View Patent ↗The invention generally relates to droplet based digital PCR and methods for analyzing a target nucleic acid using the same. In certain embodiments, methods of the invention involve forming sample droplets containing, on average, a single target nucleic acid, amplifying the target in the droplets, excluding droplets containing amplicon from the target and amplicon from a variant of the target, and analyzing target amplicons.
1. A method for detecting a plurality of targets in a biological sample, said method comprising the steps of:
a) forming one or more microdroplets each comprising a single nucleic acid template and a plurality of primer pairs each pair specific for a different target site on the template, wherein only a single primer pair binds to the nucleic acid template in one microdroplet;
b) amplifying the nucleic acid template in the one or more microdroplets; and
c) analyzing products from the amplifying step.
2. The method of claim 1 , wherein said forming steps comprises:
a) providing a first sample fluid comprising a single nucleic acid template;
b) providing a second sample fluid comprising a plurality of primers each pair specific for a different target site on the template; and
c) merging the first and second sample fluids to form a droplet comprising the single nucleic acid template and a plurality of primer pairs.
3. The method of claim 2 , wherein the first and second sample fluids are merged in the presence of an electric field.
4. The method of claim 2 , wherein a first sample droplet formed from the first sample liquid is contacted with the second sample fluid.
5. The method of claim 2 , wherein said second sample fluid further comprises reagents for conducting a polymerase chain reaction.
6. The method of claim 1 , wherein the single nucleic acid template is a DNA or an RNA.
7. The method of claim 1 , wherein said analyzing step comprises detecting the presence or absence of the plurality of targets.
8. The method according to claim 7 , wherein detecting is by sequencing.
9. The method according to claim 8 , wherein sequencing is sequencing by synthesis.
10. The method according to claim 8 , wherein prior to sequencing, the products of the amplifying step are released from the droplets.
11. The method according to claim 10 , wherein prior to the releasing step, an adaptor is attached to the products of the amplifying step.
12. The method according to claim 10 , wherein prior to the releasing step, a barcode sequence is attached to the products of the amplifying step.
13. The method according to claim 10 , wherein prior to sequencing, the released produced are attached to a surface.
14. The method according to claim 13 , wherein the surface is a surface of a flow cell.
15. The method of claim 4 , wherein the first sample droplet is contacted with a second sample fluid stream.
16. The method of claim 2 , wherein a second sample droplet formed from the second sample liquid is contacted with the first sample fluid.
17. The method of claim 16 , wherein the second sample droplet is contacted with a first sample fluid stream.