WO2014025500A2

WO2014025500A2 - Thermostable enzyme-based extractions on an integrated microfluidic chip for biological analysis

Info

Publication number: WO2014025500A2
Application number: PCT/US2013/050628
Authority: WO
Inventors: James P. Landers; Paul Kinnon; Brian Root; James D. SAUL
Original assignee: Microlab Horizon Llc
Priority date: 2012-07-16
Filing date: 2013-07-16
Publication date: 2014-02-13
Also published as: WO2014025500A3

Abstract

liquid extraction methods are used to recover nucleic acid samples in a fully-automated microfiuidic chip tor biological analysis. A sample is introduced to a rnicrofiuidic chip through a sample acceptor that can be isolated from the surroundings, but. fluidly coupled to a first domain containing a pre-loaded liquid extraction solution. The liquid extraction solution uses one or more thermostable proteinases to extract DNA from the biological sample by submerging an introduced sample and returning with an extracted DNA solution to the first domain, A fraction of the resultant extracted DNA solution is directed through a side channel toward the second domain, where it mixes with PGR reagents and a fraction of pre-PCR solution is thermally cycled in a PGR reaction chamber. The amplified DMA solution is then mixed with a separations solution for separation by capillary electrophoresis and detection by a DNA Analyzer, After the first extraction step, the sample solution is diluted at every processing step.

Description

Priority C laim

This application relies on and claims the benefit of the filing date of U.S. provisional patent application number 61 /672, 122, filed 16 July 2012, the entire disclosure of which is hereby incorporated herein by reference.

Background

The contents of a ceil of any type contains materials that can be analyzed for identification of the organism by type, condition, individuality, or prediction of activity. For instance, DMA in a cell can be used to distinguish individuals by short tandem repeat (STR) analysis, identify genus or species of ah organism directly or indirectly by other unique DNA sequences, predict sensitivity to external factors like warfarin sensitivity, and predict behavior of the cell, tissue, or organism such as likelihood of turning cancerous such as BRAC testing for breast cancer. Detection of RNA in a cell can be used to measure expression of genes under ambient, stressed or in disease conditions for research and medical purposes,

Methods to access cellular contents for analysis encounter a variety of complications. First, the milieu of the cell itself often contains extraneous material that are inhibitory to extraction and subsequent analysis. This commonly includes mucus or blood contents for animal cells sampled from the animal, soil, paper, or cloth for animal cells used in forensic testing, soil for bacterial and fungus cells sampled from the environment, and many other extraneous materials. Then the cell itself must be lysed. Cells may include a celt wall or not; and may have any variety of outer coating from carbohydrates to lipids outside the cell wall or cell membrane. Once the cell is broken open, it will spill contents that include a multitude of protein, carbohydrate, lipid, small molecule and other nucleic acid matter that interferes with the substrate targeted for analysis. Each of these extraneous materials must be somehow stopped from interfering with the Intended analysis, whether by removal, destruction, or filtering.

Diagnostic or detection methods that involve the targeted analysis of cellular content may be directed at any type of cell, including but not limited to human, bacterial, fungal, or protozoan, or viral materials that present inside or outside a cell. They may be for identification, diagnosis of infectious or chromosomal disease, indication of risk factors, or detection of contaminants.

Microfluidic analysis of cellular materials has shown promise for automated, high-throughput analysis. Microfluidics reactions are scaled down from the standard lab protocols, so that far smaller amounts of samples and reagents can be used to achieve preparation and/or analysis of a target material. Preparative methods for microfluidic extraction typically involve solid-phase extraction, wherein a sample is put in contact with a silica matrix or beads to aid in the extraction of target samples from cells or viral particles. It may also include scaled-down Chelex extraction, or alkaline solution extraction. An extraction method In a microfluidic format is typically intended to prepare the sample for further analysis, also at the micro scale. Ideally, the preparation does not require intervention from a lab worker to ensure preparation results at the microscale are sufficient to facilitate subsequent analysis of the target material.

Chemical extraction was used by Ramesh at al (1] for the detection of Staphylococcus aureus, and by Tan et al |2] with human tissue for STR analysis. The methods are easily scaled down, but chemical methods are not tunable for activity the way enzymatic lysis is.

Microfluidic systems are useful for their automation, efficiency, and high-throughput. For analysis specifically of DNA and RNA by PCR, an Integrated system includes all steps from unprepared sample introduction to detection of amplified fragments, Such systems are described by scale and functionality in Easley et al (6] and landers et al US 20090170092 (3], both incorporated herein by reference.

For multiple biochemical processing steps at the microscale level, or < 100μΜ in a channel dimension, fewer steps are favorable for automation and economic needs. Increasing the numbers of materials and steps in a preparative process increases the error rates in performing the process, the expense of producing the apparatus and running the process, and time necessary for troubleshooting when errors occur.

One effort to use enzymatic lysis methods was done by Halt et af. [4] for interplanetary expeditions, where they used at least three enzymes and two detergents in a small-scale reaction, and discovered that it was effective for extraction. While that many components may be acceptable for limited scale, high-budget space exploration, it is entirely inappropriate for manufacturing systems for wider use in health care, forensic*, and other broad applications. They also end the investigation at extraction, failing to examine the consequences of such a complex extraction lysis solution.

Other descriptions of enzymatic extraction have failed to appreciably accelerate the rates of extraction to a useful time frame [5][6]. These studies, like many for the past two decades, have shown that proteases can be useful In extraction over long time frames of greater than one hour. The rapid analysis that is required for useful for modem applications like human identification and diagnostics need results in under 30 minutes, so that additional processing steps can be done In a similarly short time frame.

Enzymatic extraction methods are capable of being tuned to streamlined the extraction procedure for automation and reproducibility. The combination of microfluidic chips with enzymatic extraction offers an economically beneficial way to have fast, high-throughput diagnostics and analyses.

One method for extracting target material for analysis that requires few steps is using enzymes to extract material and counteract interfering species. As shown in Saul, Thermostable proteinases (US 7,547,510) [7], incorporated herein by reference, the Bacillus sp.EAl enzyme can be used in a closed system to extract DNA from an intact cell so that it can be used in PCR reactions or other methods of detecting sequences of DNA. It is also known to extract RNA from cells for analysis of expression profiles or otherwise identifying an organism. Additionally, select enzymes may be used for pre-extraction purification as shown in Saul et ai Purification Methods #3 (US 20070190552) [8J, incorporated herein by reference in its entirety.

Proteinases such as Bacillus EA1 and its functional equivalent offer substantial advantages over alternative extraction methods and other enzymes for the integrated microfluidic analysis of DNA. The thermal reCfUirements for achieving extraction provide inherent thermal processing steps that exclude non-desired reaction from occurring at the same time, For instance, by using the ideal extraction temperature at 75 degrees C ensures that target proteins to be digested by the protease are in a partially-unfolded state, thereby increasing digestion rates and overcoming 3 dimensional structures that might inhibit proteinase digestion. Furthermore, the high threshold for inactivation of the proteinase at above 90 degrees C, or preferably 95 C, ensures that a necessary population of pre-loaded proteinase will survive in the microfluidic chip through product transfer and manufacturing steps before the analysis is carried out. Finally, because the thermostable proteinase has substantially absent activity at 40 C or less, no reaction will proceed without the necessary externally-driven processing by a person running a heater or DNA Analyzer device.

The low (room) temperature inactivity of the proteinases described herein substantially distinguish it from known alternative proteinases in the art such as Proteinase K, which both has substantial reactivity below 40C, with an optimum temperature in nature of about 2SC {Ebeling 1974) [9]. Furthermore, proteinase K lacks significant activity above 65C except for short periods in the presence of substantial stabilizing agents. The proteinases described herein ail have peak activity above 65 C without additional additives (Daniel et al 1995) [10], Invention Summary

This invention involves the use of a class of thermostable enzymes for use in the extraction of ONA for analysis on a fully-integrated microfluidic chip, in this case a microfluidic chip is understood to comprise an acceptor for sample introduction that is fluidiy coupled to an extraction domain to extract the DNA, a PCR or amplification domain to perform the amplification of nucleic acids for further identification, and a separations/detection domain that wit! separate the amplified nucleic acid samples for detection by an optical system.

The integrated microfluidic chip is coupled to a DNA analyzer, an instrument that is specially designed to drive the reactions and fluid movement in the chip without human intervention. A DNA analyzer for the integrated chip example here comprises pneumatic pumping functions, contact heating functions such as resistive heating to drive extraction, thermal cycling functions such as lasers for focused, rapid thermal cycling to effect PCR, etectrokinetlc drive functions to transfer sample solutions to a separations channel and perform electrophoretlc separation, optical excitation and detection functions to identify a plurality of fluorescent signals that can be used to identify a DNA signature, and a computer to drive all the functions, with or without human intervention. The DNA analyzer is able to perform these functions with more than one channel in parallel ^0f In sequence on the same chip. Such as device is described in Bell et al. {US 2011/0229898)(11J, which is incorporated herein by reference in its entirety.

The Enzymes used for liquid extraction are proteinase enzymes characterized as having a functional domain comprising an amino acid sequence of WXQXDNQFXASYDA (SEQ ID NO:l) where X at positions 2, 4, and 9 comprises any hydrophobic residue, Including and without limitation, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. Exemplary functional domains comprise an amino acid sequence of WADVDNQFFASVDA (SEQ ID NO:2) OR WADADNQFFASYDA (SEQ ID ND:3).

Although the class of proteinase enzymes comprising this functional domain may vary at the primary amino acid sequence level considerably outside of this functional domain, enzymes possessing SEQ ID NO:l have substantially the same proteinase activity, Exemplary enzyme sequences comprising SEQ ID NO:l are disclosed herein.

functional Equivalents of Bacillus EA1

The thermostable properties of Bacillus EA1 and homologues and functional equivalents are all useful for automated, microscale extractions. Many proteinases can be substituted for Bacillus EA1 in similar enzymatic extractions based on similar structure and function.

Proteinase Enzyme;

EAl proteinase is related to many other proteinases by its functional domains. The following points are the relevant observations about the amino acid sequence and its functional characteristics of the enzyme that make it ideal for nucleic acid extraction.

Bacillus sp. strain EAi is a bacterium for which no formal species name has been assigned. On this basis, It is likely that other organisms have been discovered, or are yet to be discovered, that are closely related strains of the same organism. The genus name Bacillus and Beobacillus san be used interchangeably with these organisms. It would be expected that enzymes produced by closely related bacteria would be functional equivalents of those from Bacillus sp. strain EAi. Closely related organisms are recognisable by their identical or very similar 16S rRNA gene sequences. Examples of such organisms are B. kaustiphilus, B. thermofeovarans and 8. caldofyticus. EAl proteinase is a member of the Peptidase M4 (Thermolysin) family and is presently the most stable, naturally occurring enzyme of this group. Although it is widely accepted that the thermal characteristics of an enzyme cannot be easily predicted from primary amino sequence alone, the thermolysin family of proteinases is one of the most studied enzymes for elucidating this relationship and a large body of information has gathered over the iast thirty years, This information has allowed correlation of amino acids within specific domains of the protein (from both naturally occurring and artificial enzymes) with thermal stability, A number of mutational studies have been performed and many sequence variants have been studied in order to elucidate the impact of certain amino acid sites on parameters such as thermal stability and temperature optima. Because of codon redundancy, it is understood that genes can use different codons to produce proteins that are identical to EAl proteinase, it is also understood that the N- terminal region of the peptide is a pre-pro region that is removed during activation and any sequence differences in this region are irrelevant to the functional characteristics of the mature enzyme, In EAl proteinase the N-terrninal pre-pro region is 227 amino acids long. The mature peptide is 319 amino acids. These numbers may be different in other related peptides. Related enzymes can be produced from native organisms, cloned genes, mutated genes either by site-directed or random mutagenesis or by de novo synthesis of genes or proteins

Some previously identified proteinases have identical amino acid sequences. Since the original discovery of EAl proteinase, a second organism has been characterized which produces a proteinase identical to EA1. The sequence as deposited in GenBank is currently provisional and Incomplete, but we infer that the organism, Geobacllluskaustophilus has a neutral proteinase gene which is approximately 99% similar to that of Bacillus sp. EA1 but this gene produces a protein that is identical to EAl proteinase [12]. It is a given that any enzyme with an identical amino sequence to EAl proteinase would have identical properties. And so for functional purposes the enzyme from GeobacfUuskaustophiius enzyme is EAl proteinase isolated from an alternative source. Likewise, we would consider other proteinases from related organisms with the same amino acid sequence to be identical to EAl proteinase, with equivalent functionality.

Some previously identified proteinases have similar amino acid sequencesThe key factors relating to the success of EAl proteinase in DNA extraction are the following: (a) Thermostability above 65°C. The enhanced thermal stability of EAl proteinase means that the substrate, proteins, are partially or wholly denatured (relaxed) making them more susceptible to hydrolysis. Proteinases that work at lower temperatures often require chaooropic agents and/or surfactants to achieve a simiiar level of

denaturation. Although surfactants may be used with EAl proteinase to enhance activity, these can be milder and less likely to impact on downstream applications such as the PCR. (b) Although the above characteristic must be true to achieve the enhanced activity, the proteinase must not be so stable that it cannot be removed by a heat-kill step in a closed-tube reaction, (c) The proteinase must function in solutions that are compatible with the PCR. For this reason, a neutral proteinase Is preferred, (d) Many of the thermolysin (from Bacittus thermoproteolyticus) family come close to sharing all of these

characteristics with EAl proteinase, (e) The thermolysin family of peptidases are very closely related in terms of sequence similarity. Despite this similarity, different members have different thermal characteristics and so one or a few amino acid changes can bring about large changes thermal stability. For this reason, these enzymes have been ideal for studying the impact of amino acid changes on thermal stability and a large body of information is available, (f) Alternatively, random mutagenesis could be used to create variants of less stable members of this family to create variants of EAl proteinase that are functionary similar, (h) It should also be realized that a large proportion of the amino acids in any enzyme can be changed without altering its functional characteristics. Such inconsequential variants of EAl proteinase could be created by mutating EAl proteinase or a related proteinase or be deliberately inserted Into a synthetic peptide or gene to mask the functional identity with EAl.

Key sites in the proteinase relate to the heat stability of the proteinase. These sites, and the residues that are critical, are identified in the sequence alignment below. Veltman et al (1998) [l3]described the sites on the thermoiysin family of proteinases that are responsible for binding calcium and thereby conferring thermal stability. These sites are very highly conserved in this family.

Saul et al {1995)[l4] describes a similar proteinase to EAl proteinase that has been isolated from

Bacilluscaldolyticus YP-T. This proteinase differs by only a glycine at position 61 rather than valine. Despite this single change, EAl proteinase (V61) is significantly more stable than YP-T proteinase (061).

This difference is described by Saul et al 1995) [ 14) who stated: It has been shown that the rate of thermal inactivatfon in thermolysin-like proteinases is determined by partial denaturation which renders the protein susceptible to autolysis.

There is evidence that a region proximal to ⁽361 to V61 substitution is the first to become susceptible to autolysis and so is the most important (Eijsink et al. 1995) [15]. This site has been studied by Eijsink using Thermoiysin (where the site is numbered as position 58}. Eijsink determined that substituting the Glycine with Alanine, significantly increased thermal stability [15],

Saul et al, (1995) considered that the changes G→ A or G→ V resulted in an exposed region of the protein to become more hydrophobic.THlS IS THE KEY SITE THAT DEFINES EAl*equivalent thermostable proteases.

Eijsink et al. (1995) [15] conducted a broad study using mutagenesis and demonstrated that mutations within the region 56 - 69 generated the most thermostable variants of thermoiysin. Veltman et al (1997) [13) confirmed that the region of bases 55 to 69 was crucial for the thermal stability of the protein.

Mansfield et al (1997) [16] further confirmed that the region of bases 55 to 69 was crucial for the thermal stability of the protein by stabilising it with a S-S bond between amino acid 8 and 59.

THIS IS THE KEY REGION THAT DEFINES EAl-equivaient thermostable proteases.

Our insight into these studies and additional experiments show that the G61 ->A61 or G61 -> V61 or G61 -> another hydrophobic amino acid is critical in defining EA1-equivalent thermostable proteases in terms of functionality with respect to nucleic acid extraction in a closed-system.

To mimic the thermal stability of EAl proteinase for use in a DNA extraction kit one can mutate amino acids 56 - 69 for enhancing a thermofysin-like proteinase to resemble the thermal characteristics of EAl. The approaches for achieving these changes would be to:

a j mutate site 61 of similar proteinases to an alternative amino acid (possibly a more hydrophobic amino acid);

b) search for a naturally occurring enzyme which has an alternative amino acid at position 61;

c) screen for more stable variants of thermoiysin like enzymes within region 55 to 69;

d) mutate the region 55 - 69 for more stable variants.

In all cases* an enzyme that is functionally equivalent to EAl proteinase could be generated. Embodiments of Functionally Equivalent proteins of Bacillus EA1 for Extraction

Applying the insights described above, proteins that fit the functional equivalency requirement include the following thermostable protease embodiments;

1. In one embodiment said thermophilic protease is selected from the group comprising EAl, Ak1, NprS, NprT, BT1, YP-T, aqualysin I, NprM, Caidolysin, Caldolase, Rt41A, RT4A2, and variants thereof, preferably said thermophilic protease is EAl or Ak1 or a variant thereof.

2. adding to the composition said one or more mesophilic enzyme and at least one thermophilic protease; and maintaining said composition at a first temperature or temperature range at which said one or more mesophilic enzyme has enzymatic activity, but at which said at least one thermophilic protease has minimal or no proteolytic activity; and maintaining said composition at a second temperature or temperature range at which said at (east one thermophilic protease has proteolytic activity; and maintaining said composition at a temperature suitable for use; wherein said one or more mesothermic enzyme does not exhibit activity effective to remove celt walls.

3. Preferably, said one or more mesophilic enzyme is a nucleic acid-modifying enzyme, more

preferably said enzyme is selected from the group comprising kinases, polymerases, synthetases, llgases, methylases, transferases and recombinases.

4. A system preferably uses one of these embodiments in conjunction with an integrated DNA

analysis system as described in (US 20110223605 A1) [3], (US 20110229898 Al) [4], and (US 20110229897 Al} [5].

Figure 1.

A schematic of the integrated extraction process using liquid extraction with thermostable enzymes. The liquid extraction solution, containing a thermostable enzyme, is mixed into the sample input and extraction is performed. The solution is transferred back to domain 1 and a fraction of the solution is transferred to domain 2 for PCR reactions. Domain 2 comprises chambers for pre-loaded PCR reagents, channels connecting the chambers, a mixing chamber for the reagents and sample to be mixed, and a PCR reaction chamber to carry out thermal cycling. Domain 3 comprises channels and chambers containing separations reagents, the injection channels, and a separations channel loaded with sieving polymer.

Figure 2.a fully-manufactures integrated cartridge showing four DNA analysis systems in parallel on a chip that is about 5 inches fong by three inches wide. The Sample, or swab acceptors are shown being comprised of luer locks where the sample swabs with attach.

Figure 3.

A figure showing the thermal performance of an enzyme that is 87% similar to Baciifus EAl. Figure 4.

DNA yields from liquid extraction procedures and PCR reactions run in sequence on an integrated chip. Figure 5.

An STR profile from one of the donors in the liquid extraction and rapid PCR reaction carried out on an integrated microfluidic chip.

Figure 6.

101 is the sample acceptor where a sample or swab is coupled to the chip. 102 is the main chamber from domain 1, where the liquid extraction reagent is stored prior to sample introduction. Post sample introduction, the liquid extraction solution is pneumatically-driven into the sample chamber 101 to submerge the sample. The sample chamber 101 is then heated to between 65 -80 degrees C, preferably 75 degrees C to effect enzymatic extraction of DNA by the liquid extraction solution, which contains a thermophilic protease, The liquid now contains the sample biological material and its extracted DNA, and it is returned, by pneumatic means, to chamber 102. A portion of that sample is pneumatically directed into the side channel 103, and toward the PCR reagent chambers 104 and 105. The contents of the chambers 104 and 105 are driven to the mixing chamber 106 ahead of the sample solution, with minimal mixing done in the channels until all three solutions reach the mixing chamber. The mixed solutions are then driven to the PGR chamber, 107 (In a plane below the other chambers), where PCR thermal cycling is done by non-contact heating with a radiation source, such as a laser. The sample solution then contains amplified ONA fragments ready for separation and detection. The sample solution proceeds through additional channels until it is loaded into the separations channel by electrophoretic means, then separated by electrophoretic means in the channel 108, which is in the same plane than as the PCR channel on the chip.

Figure 7.

The bottom of a DNA analysis system, showing the opposite plane from Figure 6. 201 is the bottom of the sample acceptor. 202 is the Mixing chamber for PCR reagents with the sample solution, post-extraction. 203 is the PCR chamber where amplification takes place using non-contact thermal cycling. 204 is the cross-t channel where the sample solution is injected electrokinetically after mixing with the separations reagents, 205 is the separations channel where the amplified DNA fragments are separated and detected by the DNA Analyzer. 206 are loading ports for the pre-loading of reagents. 207 are vent ports that allow air to escape but not liquid, so that bubbles do not interfere with the reactions or fluid movement.

Figure 8.

A DNA Analysis instrument with integrated Chip inserted, 301 is the heating unit used to heat the sample acceptor, to effect extraction of DNA using a thermophilic enzyme.

In one example, a swab is used to collect cells from the cheek of a donor. The swab is placed into the sample acceptor 101 and isolated from the outside with a universal adapter like a luer lock. The Swab Is submerged In about 200 μl extraction solution containing a thermostable extraction enzyme such as Bacillus EAl. The solution is pumped repeatedly so that the solution is washed over the sample at least 2 times as the solution moves from domain 1 to the sample acceptor and back again until the extraction solution is held in the sample acceptor. The sample acceptor, while filled with the liquid extraction solution, is heated so that the solution reaches 75 degrees C and held for at least 3 minutes, The sample acceptor is then heated so that the solution is about 95 degrees C to for at least 1 minute to inactivate the thermostable enzyme. The resulting sample solution is directed back into the extraction chamber, or domain 1, 102. less than 5 μl of the sample solution is removed from the extraction chamber 102 and into the side channel 103, preferably containing about 5 ng of DNA. The sample solution is then mixed with a total of about 11 μl PCR reagents including a primer mix held in chamber 104 and a reaction mix held in chamber 105_> in the mixing chamber 106. About 1.5 μl of the sample solution, containing PCR reagents,, is moved into the PCR chamber 107, where it undergoes thermal cycling to amplify DNA fragments.

Subsequent to the ONA amplification by PGR using non-contact radiation means, the amplified ample solution is transferred to combine with ahout 17 ^ separations reagents including Hi-Di formamlde and a size standard such as LIZ (Life Technologies) and moved toward the separations channel. About 1% of the final sample solution is injected electrokinetically into the separations channel 108 for separations through a sieving polymer that is loaded into the separations channel and detection of hybridized fluorescent tags by a laser and CCD detection unit.

1. Extraction methods in lab with Bacillus sp. EA1 DNA

Reference sample procedure

1. Get saliva (buccal) sample with brush swab and cut off brush in to 0.7ml tube

2. Add 200ul of 2yGEM buffer (94ul buffer, 6ul enzyme) and vortex for 20 sec

3. Remove l00ul and pipette in to PCR tube

4. Thermocycle for 2min at 75C and 2min at 95C

2. Kapa thermostable enzyme compared to EA1 thermostable enzyme. Compare with our enzyme T stability at 75°C. Sigma F1TC protease assay kit

PROCEDURE

1. Supplement Buffer 3 to 1 mM CaCI2, 1 mM MgCI2

2. Dilute Kapa enzyme 2 μl + 230 μl 2x buffer

3. Add 980 μl 2x B3 buffer to Standard enzyme {20 μl) = 0.4 Units per 5 μl

4. Add 980 μl 2x B3 buffer to diluted Kapa (20 μl) * 0,4 Units per 5 μΐ

5. Dispense each into 8x tubes each containing 30 μl

6. Heat at 75° temperature for 0, A, 8, 15, 30, 60, 90 min

7. In triplicate assay with FITC Casein (Sigma)

8. Mix well - the FfTC is very viscous.

9. While chilled, add 15 μl of this solution to 5 μl of samples.

10. On ice pipette to mix

11. Load onto thermal cycler at 4*C and incubate at 70°C for 10 minutes. Add a 4°C cycle at the end to rapidly chill the tubes.

12. Add 100 μl of 0.6 M TCA (Trichloroacetic acid) and centrifuge at max speed for 10 min. 13, Transfer 50 μl of supernatant to a black microtitre dish and 220 μ1 of Assay Buffer (Tris).

14. Recorded the fluorescence intensity with excitation at 485 nm and monitoring the

emission wavelength of 535 nm. Measurements taken at 25*C. Sensitivity = 50.

(BioTek FU800),

3. Chip-based extraction experiment on an experimental bench to get a baseline DNA yield in the cartridge on the test bench. The materials necessary are a brush swab (Epicentre), prepGEM Enzyme kit (ZyGEM), 5mm Tris-HCL pH 8.3, Four-channel integrated cartridge as in Figure 2, a bench with appropriate interface capabilities for pneumatic drive in the cartridge, qPCRmastermix (Quanta), and STR primers.

Cartridge Procedure

1. Fill extraction domain with ZyGEM buffer; PSA over fill ports and hydrophobic membrane over air ports

2. Invert the tube in the tip of the syringe and pull the shaft of the swab slightly out of the cap so that the brush extends slightly in to the base of the syringe tip.

3. Connect the syringe barrel to the cartridge

4. See fluidic procedure below

5. Heat LE heater at 75C for 4min and 95C for 3 minutes

6. Remove 4 50ui fractions - fraction 1 is closest to the syringe - qPCR fractions 2 and 3

[B] Extraction with UE heater

1. Make up an extraction mix as before (94 uL IX buffer + 6 uLprepGEM)

2. Load through fill port as indicated

3. Seal fill ports with PSA.

4. Rinse mouth and swab cheek for 30 sec. Place swab in the syringe acceptor,

5. Attach syringe to the luer on cartridge.

Liquid Extraction heater was running off of an external PID controller and power supply for the heaters. Type-T thermocouple used for feedback was moved to the syringe holder that was being used for the test.

qPCR

1. Make up a qPCRmastermix

2. Aliquot 20 μl to a 96-well plate then add 5 μl standard/sample in triplicates.

3. Run 95C 3 min, [95C 15 s, 60C 45s] x 40 cycles

Test Bench Yields

4, Chip-based extraction and amplification combined in this experiment on the Liquid Extraetion/PCR test bench. Primers and water as loaded into the reagent reservoirs, along with

Extraction solution and dye, The fill ports were sealed with a PSA sticker and the ventports covered with a hydrophobic membrane.

The test bench was aligned along the ports and run to inject the sample into the cartridge, ft was then incubated at 76C for 4 min and 95C for 3 min. The pneumatic drive mixed the solution and made a 10 ul afiquot toward the PCR chamber.

PCR was run using 27 cycles of 72C-94C-60C after a hot start at 95G for 10 minutes.

After PCR, the pressure was released, vented, thenvalves were released. Sample was retrieved mixed with 20 μlHiDiFormamide and 0.5 μl UZ (life Technologies). After vortexing, spinning down, the sample was run on ABi 3100.

5. Sequence alignment for Enzymes

67.

68.

69 . EL.EA1 STSQEVNSVKQAPNAVGVY

70. Gb„kaustophllus_H TA426 STSQEWSVKQAFNAVOVY

71 . 6105512_p«p2 STSQEVASVKQAFDAVGEVK

72 . US6103512„pepl STSQEWSVKQAFWAVGVY

73 , S_Caldolyticus STS^EVNSW-QAFNAVSVY

74 , Thermolysin_Ob_V412}4C52 ST£¾EVKSVKQAPtIAVGVY

75 , Alicyclobacillus STSQEWSVKQAFHAVGVY

76 , B„thermoproteolytiCus STQQEVASVKQAFDAVGVK

77 .

78.

7$. Figure Legend

80. Alignment of the amino add sequence of a selection of thermoiystn tike proteinases. Key:

===== = Region known to be associated with thermal stability [3, 4, 5)

82. A = Amino acid changes known to confer thermal stability.

83. I = Site in EA1 proteinase where hydrophobic residues confer thermal stability [3].

84. + = Thermo-stabllizing calcium-binding site [2)

85. [] = Reference

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Claims

1. A method of performing DNA analysis, said method comprising:

i. providing an integrated DNA analysis cartridge comprising a microfluidic chip and one or more sample acceptors, wherein said sample acceptors are fluidly connected to a first domain of the microfluidic chip comprising a DNA extraction liquid, wherein said DNA extraction liquid comprises One or more thermophilic enzymes;

ii, introducing one or more samples potentially comprising DNA into the one or more sample acceptors of the Integrated DNA analysis cartridge;

iii. contacting the one or more samples with the DNA extraction liquid in the one or more sample acceptors under conditions effective to extract DNA from each sample; and

iv. analyzing the extracted DNA by directing a portion of the extracted DNA to fiuidiy coupled domains for PCR and electrophoretic separation steps.

2. the method of claim 1, wherein the portion of extracted DNA is less than 10% of the total final extraction solution.

3. The method of claim 1, wherein the portion of extracted DNA s comprises between 2 and 10 ng of DNA.

4. The method of claim 1, wherein the portion of extracted DNA comprises 5 ng of DNA.

5. The method of claim 1, wherein the one or more sample acceptors are fluidically coupled to

or more microstructures of the microfluidic chip.

6. The method of claim 1, wherein said one or more thermophilic enzymes is a proteinase.

7. The method of claim 1, wherein the one or more thermophilic enzymes has minimal activity at 40°C or below.

8. The method of claim 1, wherein the one of more thermophilic enzymes comprise a functional domain comprising an amino acid sequence of WXDXDNQFXASYDA (SEQ ID NO:l) wherein X at positions 2, 4, and 9 comprises a hydrophobic residue,

9. The method of claim 8, wherein X at positions 2, 4, and 9 of SEQ ID NO:l is selected from the group consisting of valine, alanine and phenylalanine.

10. The method of claim 8, wherein the one or more thermophilic enzymes comprise a functional domain comprising an amino sequence selected from the group consisting of SEQ ID NO: 2 or SEQ ID NO: 3,

11. The method of claim 1, wherein the one or more thermophilic enzymes comprise an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO; 6, and SEQ ID NO: 11

12. The method of claim 1, wherein the One or more thermophilic enzymes exhibits Bacillus EA1 enzyme activity,

13. The method of claim 1, wherein the one or more thermophilic enzymes is a thermolysin M4 protease.

14. The method of claim 1, wherein the one or more thermophilic enzymes Is chosen from the group consisting of EA1, Ak1, NprS, NprT, BT1, YP.T, aqualysin I, NprM, Caldolysin, Caldolase, Rt41A, RT4A2, and variants thereof, preferably said thermophilic protease is EA1 or Akl or a variant thereof.

15. The method of claim 1, wherein in the DNA extraction liquid further comprises one or more mesophilic enzymes, said method further comprising maintaining said composition at a first temperature or temperature range at which said one or more mesophilic enzyme has enzymatic activity, but at which said at least one thermophilic protease has minimal or no proteolytic activity; maintaining said composition at a second temperature or temperature range at which said at least one thermophilic protease has proteolytic activity; and maintaining said composition at a temperature suitable for use; wherein said one or more mesothermic enzyme does not exhibit activity effective to remove cell walls.

16. The method of claim 15, wherein , said one or more mesophilic enzymes is a nucleic arid- modifying enzyme selected from the group consisting of kinases, polymerases, synthetases, ligases, methylases, transferases and recombinases.

17. The method of claim 1, wherein the DNA extraction liquid further comprises a mesophilic enzyme in solution with the thermophilic enzyme.

18. The method of claim 1, wherein the conditions effective to extract DNA from each sample

comprises at least:

i. Submerging the sample in the sample acceptor with substantially all of the DNA extraction liquid held in the first domain

ii. Heating the sample acceptor to achieve a temperature of between 65-80 degrees

C in the DNA extraction liquid

iii. Holding said temperature for more than 3 minutes to effect DNA extraction from the sample

iv, Increasing the temperature to greater than 90 degrees C to effect autocataiysis of the thermophilic enzyme

v. Redirecting the extraction liquid to the first domain for further processing

19. The method of claim 1, wherein the one or more microstructures of the microfiudic chip comprise reaction reservoirs, reagent carrier reservoirs, separation channels, inlets, or any combination thereof 20. The method of claim 1 further comprising:

i. pumping the extracted DNA after said contacting into a reaction reservoir on the microfiudic chip prior to said analyzing.

21. The method of claim 20, wherein said pumping is carried out using a hydrodynamic

pressure/vacuum system

22. The method of claim 20 wherein said reaction reservoir is a polymerase chain reaction (PCR) reservoir said method further comprising;

i. introducing PCR reagents from a reagent carrier on the microfluidic chip into the PCR reservoir and ii. subjecting the extracted and purified DNA and PCR reagents to one or more

polymerase chain reaction cycles.

23, The method of claim 1, wherein DNA from more than one sample is simultaneously extracted in more than one sample acceptor of the integrated DNA analysis cartridge.

24. The method of claim 1, wherein the sample comprises a portion of a FTA card, a portion of a

cigarette, a piece of fabric, or biological tissue.

25, The method of claim 1, wherein the sample is on a swab.

26. The method of claim I, wherein said incubating is carried out for about 6 minutes to effect DNA extraction.

27. The method of claim 1, wherein the integrated DNA analysis cartridge further comprises a thermal module to control the temperature of the DNA extraction liquid during said contacting.

28. The method of claim 27, wherein the thermal module comprises a resistance heater that is

temperatlre-regulated by a controller module.

29. The method of claim 28, wherein the resistance heater is positioned so as to contact the one or more sample acceptors.

30. An integrated DNA analysis cartridge comprising:

a microfluidic chip comprising one or more reservoirs connected by one or more channels, including a reagent carrier reservoir comprising a DNA extraction liquid, wherein said DNA extraction liquid comprises one or more thermophilic proteinase enzymes and one or more sample acceptors suitable for extracting DNA from a sample, wherein said sample acceptors are fluidically coopled to the microfluidic chip.

31. The cartridge of claim 30, wherein the one or more thermophilic enzymes has minimal activity at 40°C or below.

32. The cartridge of claim 30, wherein the one or more thermophilic enzymes comprise a functional domain comprising an amino acid sequence of WXDXDNQFXASYDA (SEQ 1D NO:l) wherein X at positions 2, 4, and 9 comprises a hydrophobic residue.

33. The cartridge of claim 30, wherein X at positions 2, 4, and 9 of SEQ ID NO:1 is selected from the group consisting of valine, alanine and phenylalanine.

34. The cartridge of claim 30, wherein the one or more thermophilic enzymes comprise a functional domain comprising an amino sequence selected from the group consisting of SEQ iD NO: 2 or SEQ ID NO: 3.

35. The cartridge of claim 30, wherein the one or more thermophilic enzymes comprise an amino add sequence selected from the group consisting of SEQ ID NQ:4, SEQ ID NO:5, SEQ ID NO: 6, and SEQ ID NO: 11

36. The cartridge of claim 30, wherein the one or more thermophilic enzymes comprises Baci!Ius EAi enzyme activity.

37 The cartridge of claim 30, wherein the one or more thermophilic enzymes comprises Bacillus EAI

38. The cartridge of claim 30, wherein the DNA extraction liquid comprises one or more mesophKic enzymes.

39. The cartridge of claim 30 further comprising:

1. a thermal control domain that controls the temperature of the DNA extraction liquid in the sample acceptors.

40. The cartridge of claim 30 wherein the microfludic chip further comprises one or more reagent carrier reservoirs, separation channels, inlets, electrode reservoirs or any combination thereof.