Integrated sample preparation systems and stabilized enzyme mixtures

• US 8,961,899 B2
• Filed: 06/24/2013
• Issued: 02/24/2015
• Est. Priority Date: 05/06/2010
• Status: Active Grant

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: Ibis Biosciences Incorporated
• Inventors: Eshoo, Mark, Picuri, John
• Primary Examiner(s): Mui, Christine T
• Application Number:
  US13/925,355
• Publication Number:
  US 20130274147A1
• Time in Patent Office: 610 Days
• Field of Search: 422/502, 422/501, 422/500, 422/50, 506/40, 506/33
• US Class Current: 422/502
• 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 2200/16 Reagents, handling or storing thereof
• CPC Class Codes: B01L 2300/0672 Integrated piercing tool
• CPC Class Codes: B01L 2300/0681 Filter
• 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 2300/0887 Laminated structure
• CPC Class Codes: B01L 2400/0481 squeezing of channels or chambers
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/0655 pinch valves
• CPC Class Codes: B01L 2400/0683 mechanically breaking a wall or membrane within a channel or chamber
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: B01L 3/502753 characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation separation in general B01D; microapparatus for analysis using electrophoresis G01N27/44791; sample preparation G01N1/28
• 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 9/527 for microfluidic devices, e.g. used for lab-on-a-chip
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• 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 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation

Abstract

The present invention provides integrated sample preparation systems and stabilized enzyme mixtures. In particular, the present invention provides microfluidic cards configured for processing a sample and generating DNA libraries that are suitable for use in sequencing methods (e.g., next generation sequencing methods) or other suitable nucleic acid analysis methods. The present invention also provides stabilized enzyme mixtures containing an enzyme (e.g., an enzyme used in whole genome amplification), BSA, and a sugar. Such enzyme mixtures may be lyophilized and stored at room temperature without significant loss of enzyme activity for months.

Claims

1. A microfluidic card, comprising:

a) a loading port configured for introduction of a biological sample;

b) a nucleic acid extraction sub-circuit wherein said nucleic acid extraction sub-circuit comprises;

i) a nucleic acid extraction component configured to bind nucleic acids present in said biological sample;

ii) a wash buffer;

iii) an elution buffer; and

iv) a component configured for moving said elution buffer over said nucleic acid extraction component to generate a mixture of extracted nucleic acids; and

c) an amplification sub-circuit, wherein said amplification sub-circuit comprises a stabilized enzyme mixture, wherein said enzyme mixture comprises at least one amplification-related enzyme useful for performing amplification on said extracted nucleic acid to generate amplified nucleic acid,

d) a fragmentation sub-circuit, wherein said fragmentation sub-circuit comprises;

i) a reagent mixture configured for digesting nucleic acid to generate fragmented nucleic acid; and

/or

ii) a fragmentation component configured for mechanically fragmenting nucleic acid to generate fragmented nucleic acid; and

iii) at least one type of enzyme configured for polishing the ends of said fragmented nucleic acid; and

e) a linker sub-circuit, wherein said linker ligation sub-circuit comprises;

i) nucleic acid linkers configured for use in sequencing methods;

ii) a ligation enzyme mixture configured for ligating said nucleic acid linkers to nucleic acid to generate a nucleic acid sequencing library; and

iii) at least one type of enzyme configured for polishing the ends of said nucleic acid sequencing library; and

f) a purification sub-circuit, wherein said purification sub-circuit comprises a nucleic acid purification component.

2. The microfluidic card of claim 1, further comprising:

g) a lysis sub-circuit operably linked to said loading port, wherein said lysis sub-circuit comprises;

i) a lysis chamber; and

ii) a lysis buffer wherein said lysis sub-circuit is configured to lyse said biological sample with said lysis buffer to generate a lysed sample.

3. The microfluidic card of claim 1, wherein said at least one amplification-related enzyme comprises an enzyme useful for performing whole genome amplification (WGA), PCR, or TMA.

4. The microfluidic card of claim 1, wherein said at least one amplification-related enzyme is selected from the group consisting of:

Phi-29 polymerase, E. coli DNA polymerase I, inorganic pyrophosphatase, or any combination thereof.

5. The microfluidic card of claim 1, wherein said stabilized enzyme mixture further comprises:

i) BSA, ii) a sugar, and iii) at least one additional component selected from the group consisting of;

an inorganic salt, a divalent metal cation, a buffering agent, an emulsifier, and a reducing agent.

6. The microfluidic card of claim 1, wherein said stabilized enzyme mixture further comprises:

i) BSA, ii) a sugar, iii) an inorganic salt, iv) a divalent metal cation, v) a buffering agent, vi) an emulsifier, vii) and a reducing agent.

7. The microfluidic card of claim 1, further comprising an outlet port configured to allow a user to withdraw at least a portion of said nucleic acid sequencing library.

8. The microfluidic card of claim 5, wherein said BSA is present at a concentration of 0.05%-3.0%, said sugar is present at a concentration of 5-35%, said inorganic salt is present at a concentration of 1 mM-25 mM, said divalent metal cation is present at a concentration of 1 mM-30 mM, said buffering agent is present at a concentration of 10 mM-100 mM, said emulsifier is present at a concentration of 0.01%-0.15%, and said reducing agent is present at a concentration of 1 mM-10 mM in said stabilized enzyme mixture.

9. The microfluidic card of claim 2, further comprising a waste chamber operably linked to one or more of said lysis sub-circuit, said nucleic acid extraction sub-circuit, said amplification sub-circuit, said fragmentation sub-circuit, and/or said linker ligation sub-circuit and said purification sub-circuit.

10. The microfluidic card of claim 1, further comprising a processing instrument configured for passing buffers and samples.

11. The microfluidic card of claim 1, further comprising one or more sealed packages comprising one or more of said lysis buffer, said wash buffer, said elution buffer, said stabilized enzyme mixture, said reagent mixture configured for digesting said amplified nucleic acid, and said ligation enzyme mixture.

12. The microfluidic card of claim 2, wherein in said lysis sub-circuit, said nucleic acid extraction sub-circuit, said amplification sub-circuit, said fragmentation sub-circuit, said linker ligation sub-circuit, and said purification sub-circuit are operably linked in sequence.

13. The microfluidic card of claim 2, wherein said lysis buffer, said wash buffer, and said elution buffer are a single buffer.

14. A system comprising an automated nucleic acid sequencer and a microfluidic card of claim 7, wherein said outlet port of said microfluidic card is in operable fluid communication with said nucleic acid sequencer.

15. The microfluidic card of claim 1, wherein said linker is a nucleic acid adaptor.