Method and apparatus for analysis and sorting of polynucleotides based on size

Patent metadata

Field	Value
Publication number	US-9383337-B2
Application number	US-201313741249-A
Country	US
Kind code	B2
Filing date	Jan 14, 2013
Priority date	Sep 25, 1996
Publication date	Jul 5, 2016
Grant date	Jul 5, 2016

Abstract

The invention relates to a microfabricated device and methods of using the device for analyzing and sorting polynucleotide molecules by size.

First claim

We claim:

1. A method of analyzing an individual polynucleotide single-stranded DNA molecule in a solution using a microfluidic device, the method comprising :
transporting the individual molecule through a detection region having a volume of between 1 femtoliter and 1 picoliter that is positioned in a microfluidic channel of the device without hydrodynamically focusing the solution with a sheath fluid, and without previous size separation of polynucleotides in the solution; and
determining the size of the molecule as it passes through the detection region.

2. The method of claim 1 wherein the size of the individual molecule passing through the detection region is calculated and stored in a data storage unit.

3. The method of claim 1 wherein the channel is microfabricated in a silicon substrate.

4. The method of claim 1 , wherein the size of the individual molecule is determined by measuring a reporter attached to the DNA molecule as it passes through the detection region.

5. The method of claim 4 , wherein the reporter is a fluorescent label.

6. The method of claim 1 , wherein the individual molecule is transported through the detection region separate and apart from other polynucleotides that are initially present in the solution.

7. The method of claim 1 , further comprising diverting the individual molecule into a branch channel downstream of the detection region if it is determined to have a selected size.

8. The method of claim 1 , wherein the microfluidic channel is between about 1 and 10 micrometers in width and about 1 and 10 micrometers in depth.

9. A method of analyzing a plurality of single-stranded DNA molecules using a microfluidic device, the method comprising:
flowing the solution by hydrostatic pressure through a microfluidic channel in the device such that the molecules pass one-by-one through a detection region located within the channel without hydrodynamically focusing the solution with a sheath fluid; and
determining the size of individual molecules in the plurality as they pass through the detection region.

10. The method of claim 9 , wherein the concentration of the DNA molecules in the solution has been adjusted so that the probability that the detection region will contain two or more molecules at the same time (P x2 ) is no more than 0.1.

11. The method of claim 9 , wherein the molecules are attached with a fluorescent label, and the size of each molecules is determined by measuring fluorescence as it passes through the detection region.

12. The method of claim 9 , further comprising diverting molecules from the plurality into a branch channel downstream of the detection region when they are determined to have a size within a selected size range.

13. The method of claim 9 , wherein the detection region is between 1 femtoliter and 1 picoliter in volume.

14. The method of claim 9 , wherein the microfluidic channel is between about 1 and 10 micrometers in width and about 1 and 10 micrometers in depth.

Assignees

• California Inst Of Techn

Inventors

• Quake Stephen R
• Volksmuth Wayne D

Classifications

• B01L2200/0647

  Handling flowable solids, e.g. microscopic beads, cells, particles · CPC title

• B01L2400/0487

  fluid pressure, pneumatics · CPC title

• B01L2400/0421

  electrophoretic flow · CPC title

• B01L2400/0415

  electrical forces, e.g. electrokinetic · CPC title

• B01L2300/0654

  Lenses; Optical fibres · CPC title