Thermal Cycling Method

• US 20110097763A1
• Filed: 05/13/2009
• Published: 04/28/2011
• Est. Priority Date: 05/13/2008
• Status: Abandoned Application

• Current Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Original Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Inventors: Eckhardt, Allen E., Hua, Zhishan, Pamula, Vamsee K., Thwar, Prasanna, Srinivasan, Vijay, Pollack, Michael G.
• Application Number:
  US12/991,966
• Publication Number:
  US 20110097763A1
• Time in Patent Office: Days
• US Class Current: 435/91.2
• CPC Class Codes: B01L 2200/027 for microfluidic devices
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2300/0803 Disc shape
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0861 Configuration of multiple channels and/or chambers in a single devices
• 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 2300/088 Channel loops
• CPC Class Codes: B01L 2300/0883 Serpentine channels
• CPC Class Codes: B01L 2300/089 Virtual walls for guiding liquids
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2400/0427 Electrowetting
• CPC Class Codes: B01L 2400/043 magnetic forces
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B01L 3/502792 for moving individual droplets on a plate, e.g. by locally altering surface tension
• 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: B01L 7/525 with physical movement of samples between temperature zones
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

The invention relates to certain novel approaches to reducing or eliminating the movement of contaminants from one droplet to another on a droplet actuator via liquid filler fluid. In one application, droplet actuators are used to conduct genetic analysis using polymerase chain reaction (PCR) techniques. The invention addresses the need for improved methods of performing PCR on a droplet actuator that provide for optimum amplification and detection of a sample target.

Claims

1-104. -104. (canceled)

105. A method of thermal cycling a droplet, the method comprising:

(a) providing a droplet at least partially surrounded by a liquid filler fluid, wherein the droplet;

(i) potentially comprises a target nucleic acid; and

(ii) comprises reagents sufficient to cause amplification in the presence of the target nucleic acid, the reagents comprising a fluorophore that does not significantly partition into the filler fluid during the execution of a thermal cycling protocol; and

(b) adjusting the temperature of the droplet according to a thermal cycling protocol to induce amplification in the presence of the target nucleic acid.

106. The method of claim 105 wherein the fluorophore comprises a polar fluorophore.

107. The method of claim 105 wherein the fluorophore is substantially impermeable to cell membranes.

108. The method of claim 105 wherein the fluorophore is completely impermeable to cell membranes.

109. The method of claim 105 wherein the fluorophore comprises the EVAGREEN®

fluorophore.

110. The method of claim 105 wherein the fluorophore comprises the TO-PRO1 fluorophore.

111. The method of claim 105 wherein the filler fluid consists essentially of silicone oil, optionally doped with one or more additives.

112. The method of claim 105 wherein the filler fluid consists essentially of a 10 to 20-carbon oil, optionally doped with one or more additives.

113. The method claim 105 wherein the filler fluid consists essentially of a 15 to 20-carbon oil, optionally doped with one or more additives.

114. The method of claim 105 wherein the filler fluid consists essentially of hexadecane oil, optionally doped with one or more additives.

115. The method of claim 105 wherein the filler fluid consists essentially of degassed oil, optionally doped with one or more additives.

116. The method of claim 105 wherein providing a droplet comprises providing a droplet in a droplet operations gap of a droplet actuator.

117. The method of claim 116 wherein adjusting the temperature of the droplet according to a thermal cycling protocol comprises heating and/or cooling the droplet in the droplet operations gap of a droplet actuator.

118. The method of claim 116 wherein adjusting the temperature of the droplet according to a thermal cycling protocol comprises transporting the droplet between thermal zones in the droplet operations gap using electrode mediated droplet operations.

119-317. -317. (canceled)