TWI539001B - Methods for rapid multiplexed amplification of target nucleic acids - Google Patents

Description

Method for rapid multiplex amplification of target nucleic acids

The present invention relates generally to methods for rapidly amplifying one or more loci in a nucleic acid sample, as well as thermal cyclers and systems suitable for performing such methods.

The present application claims US Provisional Application No. 60/921,802, filed on Apr. 4, 2007; U.S. Provisional Application No. 60/964,502, filed on August 13, 2007, and U.S. Provisional Application, filed on Feb. 12, 2008 The priority of the filing date of 35 USC § 119(e) of the application Serial No. 61/028, 073, the entire disclosure of each of which is hereby incorporated by reference. The present application is also incorporated by reference in its entirety into the two U.S. patent applications filed on the same date; the first is entitled "INTEGRATED NUCLEIC ACID ANALYSIS", attorney number 07-801-US; and second The title is "PLASTIC MICROFLUIDIC SEPARATION AND DETECTION PLATFORMS", attorney case number 07-865-US.

Polymerase chain reaction (PCR) is an enzymatic reaction that facilitates rapid exponential amplification of nucleic acid sequences in vitro. In forensic science, PCR can be used to identify individuals based on amplification of a small region of the human genome containing a repetitive DNA called a short tandem repeat (STR). The unit length of a given STR repeat is in the range between 2-10 base pairs, and the STR is usually in a non-coding and flanking sequence, but sometimes in the coding region (Edwards et al ., Am . J) .Hum.Genet. 1991, 49, 746-756). Hundreds of thousands of STR loci exist in the human genome, appearing on average every 6-10 kb (Beckman and Weber, Genomics 1992, 12, 627-631) and appear to be highly polymorphic (Edwards et al ., Trans. Assoc. Am.). Physicians 1989, 102, 185-194). STR analysis has become a major tool in a comprehensive set of forensic science with a growing set of applications including paternity testing, human identification in major disasters, and routine infant typing.

Although several commercially available STR kits have been developed for the synthesis of PCR products with high specificity, there are still important areas that can improve current STR technology. Most importantly, the average time to perform multiplex PCR using a commercial STR typing test kit is approximately 2.14 hours; the time consuming and labor intensive nature of such assays has resulted in a backlog of forensic laboratory work. Although the advent of automated instruments that process multiple samples simultaneously can help alleviate significant bottlenecks in the yield of typing, an increased number of samples to be analyzed will require further accelerated processing. In addition, there is a need to increase the sensitivity of STR assays and to improve detection of amplified products (Gill, Croat. Med . J. 2001, 42, 229-32). Currently available STR kits contain 9 to 16 loci and research in this area is ongoing to increase the number of detectable loci. Four or more loci can be used for certain applications of STR analysis in this field.

PCR can also be applied over a wide range of clinical settings. Their eg, PCR can be used to diagnose such a group A streptococcal (Group A Streptococci), methicillin-resistant Staphylococcus aureus (of S. aureus) and vancomycin-resistant enterococci (Enterococci) due to the Bacterial infections, and PCR is generally more sensitive than culture-based diagnostic techniques. Fungal infections can be similarly diagnosed. PCR can be used to diagnose respiratory viruses (such as respiratory syncytial virus, adenovirus and influenza virus and parainfluenza virus), genitourinary virus (such as herpes simplex virus and typing detection of human papilloma virus), meningitis (for example) Herpes simplex virus, Epstein-Barr virus, varicella-zoster virus and enterovirus, and hepatitis (eg hepatitis B and C). PCR is also applicable to pre-implantation genetic diagnosis, including assessment of aneuploidy and diagnosis of genetic diseases. From oncology to rheumatology and from hematology to gastroenterology, it is difficult to find medical fields that are not affected by PCR.

PCR has also been used in a variety of non-clinical settings, including veterinary identification (similar to human STR typing), veterinary diagnosis, assessment of food safety, detection of agricultural pathogens, and pharmacogenomics. The increased use of applications involves the identification of biological weapons agents in clinical and environmental samples. Immediate PCR is used in applications that are essentially identical to the PCR itself, ie, close relatives that allow PCR to quantify the amount of product present in the reaction after each amplification cycle (see Espy et al , Clinical MicrobiOlogy Reviews 2006, 19, 1656). -256).

Most commercially available thermal cycling instruments are limited in that they are opposite to the temperature of the PCR solution, which receives temperature feedback directly from the module temperature and controls the module temperature. Therefore, the critical solution temperature distribution for the success of PCR may vary widely from the desired distribution.

In addition, most of the literature on increasing PCR speed and sensitivity has focused on each amplification of a specific locus ("single-check") and as required for forensic STR typing, clinical diagnosis, and non-clinical applications. Simultaneous expansion of multiple loci ("multiple assays") only reported limited success. For example, a 160 nL chamber coupled to an integrated heater has been shown to amplify and separate the four STRs contained in the Y-STR assay within 80 minutes with the detection limit of 20 copies of the template DNA. (Liu et al ., Anal. Chem. 2007, 79, 1881-1889). Increased PCR sensitivity due to reduced PCR reaction volume has also been reported for the PowerPlex ^® 16 System, although no attempt has been made to increase the rate of reaction (Schmidt et al , Int. J. Legal Med. 2006, 120, 42-48). However, neither of the reports provided a significantly shorter amplification time required in the art. Hopwood et al. ( International Congress Series 1288 (2006) 639-641) reported 100 minute amplification using a set of 11 STR primers. About clinical diagnostics, using nanometer chip system needs 97.5 minutes in the PCR amplification assays of seven species of respiratory virus panel (Takahashi et al., J.Clin.Microbiol 2008, doi: 10.1128 / JCM.01947-07).

Many PCR (and real-time PCR) applications, such as forensic human identification, clinical diagnostics, and biological weapon detection by STR typing, are time sensitive and optimal for many applications in multiple environments. In addition, available in limited samples (for example, from clinical or Many of these applications are used in environments where the pathogen is small, or a small number of human cells from forensic samples, and where the sensitivity of the reaction is critical.

Notably, Ibid . , Horsman et al. ( J. Forensic Sci. , 2007, 52, 784-799) on page 792 states that "PCR has become a common pursuit of analytical microchip researchers, as evidenced by the literature on the subject. However, There are still many ways to develop for forensic DNA analysis. A lot of work has not shown the use of commercially available forensic STR kits on a single device or further use of multiple STR amplifications. However, when fully developed, microchips PCR will undoubtedly save a lot of time and cost for the forensic community." Therefore, there is a need in the art for a rapid and sensitive method to successfully provide simultaneous amplification of multiple loci in a nucleic acid sample for a wide range of applications.

The apparatus, biochip, method and system of the present invention provide the ability to rapidly and controllably and reproducibly heat and cool a PCR solution based at least in part on the actual temperature of the solution via monitoring and control of the thermal cycler. The inventive apparatus, biochip, method and system disclosed herein provide monitoring and/or precise control of the reaction temperature of a solution within a biochip via a thermal sensor specifically incorporated into a commercial thermal cycler to avoid overheating or owing The ability to heat. The ability to rapidly heat and cool the reaction solution to such temperatures minimizes the ramp and settling time and allows the incubation time at the desired temperature to dominate the total step time. Additionally, the apparatus, biochips, methods, and systems of the present invention provided herein provide the ability to rapidly change and balance the temperature of the reaction solution, thereby greatly increasing the rate at which the amplification reaction can proceed.

Fast multiplex PCR amplification times as short as 17 minutes have been achieved using the apparatus, biochips, methods and systems of the present invention. Additional time reductions are possible based on the teachings of the present invention. In addition, the rapid PCR method of the present invention is effective over a wide dynamic range, is extremely sensitive and compatible with a variety of commercially available enzymes and reagents. For forensic applications, the apparatus, biochip, method and system of the present invention enable the generation of a complete map that satisfies the interpretation criteria of STR analysis The time required for the spectrum is significantly reduced.

In a first aspect, the present invention provides a thermal cycler comprising a temperature control element (TCE), wherein the first surface of the TCE is adapted to receive a sample chamber containing a solution and a sensing chamber containing a thermal sensor, wherein the thermal sensing The device provides feedback to the TCE to set or maintain the sample at the desired temperature. In a second aspect, the present invention provides a thermal cycler further comprising a second thermal sensor positioned to monitor the temperature of the first surface of the TCE.

In a second aspect, the invention provides a system comprising a biochip comprising one or more reaction chambers comprising a portion of the biochip having a volume, wherein each reaction chamber additionally comprises a microfluidic inlet channel and micro a fluid outlet channel, wherein each reaction chamber is less than 200 μm from the contact surface of the bio-wafer substrate; the system additionally includes a thermal cycler including a temperature control element (TCE), wherein the first surface of the TCE is adapted to receive the substrate containing the sample, And a thermal sensor positioned to measure the temperature of the sample within the substrate and providing feedback to the TCE to set or maintain the sample at a desired temperature, the thermal cycler being in thermal communication with the contact surface of the bio-wafer substrate.

In a third aspect, the present invention provides a system comprising a biochip comprising one or more reaction chambers, wherein each reaction chamber comprises a portion of the biochip having a volume, additionally comprising a microfluidic inlet channel and a microfluidic outlet channel, wherein each reaction chamber is less than 100 μm from the contact surface of the bio-wafer substrate; and a thermal cycler comprising a temperature control element (TCE), wherein the first surface of the TCE is adapted to receive the substrate containing the sample, and A thermal sensor positioned to measure the temperature of the sample within the substrate and provide feedback to the TCE to set or maintain the sample at a desired temperature, the thermal cycler being in thermal communication with the contact surface of the bio-wafer substrate.

In a fourth aspect, the invention provides a method for simultaneously amplifying a plurality of loci in a nucleic acid solution, comprising providing one or more reaction chambers, wherein each reaction chamber comprises (i) at least at least a nucleic acid solution of at least one copy of the target nucleic acid; (ii) one or more buffers; (iii) one or more salts; (iv) each corresponding to a plurality of loci to be amplified a primer set; (v) a nucleic acid polymerase and (vi) nucleotide; the temperature of the nucleic acid solution in each reaction chamber is sequentially denatured and bonded at a heating and cooling rate of about 4 to 150 ° C / sec ( A predetermined number of cycles of thermal cycling between the state and the extended state to obtain a plurality of amplified loci in each reaction chamber in about 97 minutes or less.

In a fifth aspect, the invention provides a method for simultaneously amplifying a plurality of loci in a nucleic acid solution, comprising providing one or more reaction chambers, wherein each reaction chamber comprises (i) at least a nucleic acid solution of at least one copy of the target nucleic acid; (ii) one or more buffers; (iii) one or more salts; (iv) a primer set corresponding to each of the plurality of loci to be amplified; v) a nucleic acid polymerase and (vi) nucleotide; the temperature of the nucleic acid solution in each reaction chamber is sequentially thermally cycled for a predetermined number of cycles at about 97 minutes at a heating and cooling rate of about 4 to 150 ° C/sec. A plurality of amplified loci are obtained in each reaction chamber in a shorter time.

In a sixth aspect, the invention provides a method for simultaneously amplifying 5 or more loci in a nucleic acid solution, comprising providing one or more reaction chambers, wherein each reaction chamber comprises (i) a nucleic acid solution of at least one replica of at least one target nucleic acid amplified; (ii) one or more buffers; (iii) one or more salts; (iv) corresponding to 5 or more loci to be amplified a primer set; (v) a nucleic acid polymerase and (vi) a nucleotide; in a heating and cooling rate of about 4 to 150 ° C / sec, the temperature of the nucleic acid solution in each reaction chamber is in a denatured state, a bonded state, and The predetermined number of cycles of thermal cycling between the expanded states is such that five or more amplified loci are obtained in each reaction chamber.

In a seventh aspect, the present invention provides an integrated bio-disc system comprising a bio-wafer comprising at least two microfluidically coupled reaction chambers, wherein the first reaction chamber is in thermal communication with a thermal cycler, the thermal cycler A temperature control element (TCE) is included, wherein the first surface of the TCE is adapted to receive a substrate containing the sample, and is positioned to measure the temperature of the sample within the substrate and provide feedback to the TCE to set or maintain the sample at a desired temperature An inductor, wherein a contact surface of the biochip is in thermal communication with the first surface of the thermal cycler; and A reaction chamber is fluidly connected and suitable for nucleic acid extraction, nucleic acid purification, pre-PCR nucleic acid clearance, post-PCR clearance, pre-sequencing removal, sequencing, post-sequencing removal, nucleic acid isolation, nucleic acid detection, reverse transcription, pre-reverse clearance a second reaction chamber after post-transcriptional clearance, nucleic acid ligation, nucleic acid hybridization or quantification, wherein the first reaction chamber is less than 200 [mu]m from the contact surface of the biochip.

Particular preferred embodiments of the present invention will become apparent from the following detailed description of the preferred embodiments.

Figure 1A is a photograph of an embodiment of a thermal cycler of the present invention.

1B is a photograph showing an embodiment of a 16-ribbon microfluidic biochip for the thermal cycler of FIG. 1A.

2A is a graph showing the module and the temperature profile of the reaction solution for one thermal cycle (total cycle time: 145.1 minutes) of the standard STR cycle scheme described herein.

Figure 2B is a graph showing the module and the temperature profile of the reaction solution for one thermal cycle (total cycle time: 19.56 minutes) of the fast cycle scheme described herein.

Fig. 3 is a graph showing the temperature distribution of a heat pump and a reaction solution of a thermal cycle of the thermal cycler of the present invention using rapid cycle conditions (total cycle time: 17.3 minutes).

4A is a graph showing STR patterns generated in a biowafer reaction using 0.5 ng of template DNA in accordance with the present invention.

Figure 4B is a graph showing the STR map generated in a tube reaction using 0.5 ng of template DNA in accordance with the present invention.

Figure 5A is a graph showing the effect of DNA template levels on signal intensity in a biowafer reaction.

Figure 5B is a graph showing the effect of DNA template levels on signal intensity in a tube reaction.

Figure 6A is a graph showing the effect of DNA template levels on the heterozygous peak height ratio (PHR) in a biowafer reaction.

Figure 6B is a graph showing the effect of DNA template levels on PHR in a tube reaction.

Figure 7A is a graph showing the effect of DNA template levels on non-template nucleotide addition (NTA) in a biowafer reaction.

Figure 7B is a graph showing the effect of DNA template levels on NTA in a tube reaction.

Figure 8A is a graph showing the effect of DNA template levels on a shadow in a biowafer reaction.

Figure 8B is a graph showing the effect of DNA template levels on shadow bands in a tube reaction.

FIG 9A shows the use of biochips is 1ng of template DNA primer COfiler ^TM to produce the group of (top) and the tube reactor graph (bottom) map.

9B shows the use of biochips 1ng of template DNA primer Identifiler ^TM to produce the group of (top) and the tube reactor graph (bottom) of the map of FIG.

Figure 10 is a graph showing a map of an example of a sequencing reaction as described in Example 5.

To achieve rapid multiplex nucleic acid amplification, such as PCR, the present invention provides thermal cycling apparatus, reaction vessels, and reaction conditions that can be used to amplify multiple loci within a target nucleic acid sample. As illustrated by the examples provided herein, the rapid thermal cycling process of the present invention can be carried out in a microfluidic biochip using the thermal cycler of the present invention and the methods described herein.

The methods provided by the present invention enable rapid multiplex amplification in applications other than the use of the biochips and thermal cyclers described herein. For example, especially contemplated (e.g. thermocycler module group and the Roche LightCycler ^TM) used in a conventional thermal cycler using thin-walled tubes and amplification other than PCR temperature cycling (e.g., PCR or isothermal rolling circle amplification) of the method.

The method, biochip and thermal cycler provided by the present invention are capable of amplifying a given amount of human genomic DNA (approximate amount of DNA in a single-nuclear human cell) containing at least 0.006 ng of a target nucleic acid locus in 100 minutes. Multiple loci in a nucleic acid solution. In other embodiments, less than 90 minutes, less than 80 minutes, less than 70 minutes, less than 60 minutes, less than 50 minutes, less than 40 minutes, less than 30 minutes, less than 20 minutes, less than 17.7 Amplification is performed in minutes, less than 15 minutes, less than 10 minutes, or less than 5 minutes.

In other embodiments, multiple loci derived from the genome of a bacterium, virus, fungus, animal or plant can be amplified starting from at least one copy of the target nucleic acid locus. For example, prior to the multiplex amplification reaction, the sample to be analyzed may comprise less than 1000 copies of the target nucleic acid, less than 400 copies, less than 200 copies, less than 100 copies, less than 50 copies, less than 30 copies, less than 10 A copy or at least 1 copy. In addition, if a target nucleic acid locus is present in more than one copy of the genome, amplification of less than a single genome equivalent of the DNA can be used. Typically, at least two loci and up to about 250 loci can be simultaneously amplified within each target nucleic acid of the sample according to the methods described herein. In addition, at least two loci and up to about 250 loci can be simultaneously amplified in a plurality of target nucleic acids according to the methods described herein.

The target nucleic acid used herein may be any nucleic acid such as a human nucleic acid, a bacterial nucleic acid or a viral nucleic acid. The target nucleic acid sample can be, for example, from one or more cells, tissues or body fluids (such as blood, urine, semen, lymph, cerebrospinal fluid or amniotic fluid) or other biological samples (such as tissue culture cells, buccal swabs, mouthwashes). Nucleic acid samples of feces, tissue sections, biopsy aspirate and archaeological samples such as bone or dried tissue. The target nucleic acid can be, for example, a DNA product that is reverse transcribed from DNA, RNA or RNA. The target sample can be derived from any source including, but not limited to, eukaryotes, plants, animals, vertebrates, fish, mammals, humans, non-humans, bacteria, microorganisms, viruses, biological sources, serum, plasma, blood, Urine, semen, lymph, cerebrospinal fluid, amniotic fluid, biological specimens, biopsies taken from needles, cancer, tumors, tissues, cells, cell lysates, crude cell lysates, tissue lysates, tissue culture cells, Oral swabs, mouthwashes, feces, dry tissues, forensic sources, autopsy bodies, archaeological sources, infections, nosocomial infections, sources of production, pharmaceutical preparations, biomolecular production, protein preparations, lipid preparations, carbohydrate preparations, Inanimate objects, air, soil, juice, metals, fossils, excavated materials and/or other materials and sources on or off Earth. Samples can also contain one source or different a mixture of materials of the source. For example, when the disclosed methods are used to amplify nucleic acids of such infected cells or tissues, the nucleic acid that infects the bacteria or virus can be amplified with the human nucleic acid. Types of suitable target samples include eukaryotic samples, plant samples, animal samples, vertebrate samples, fish samples, mammalian samples, human samples, non-human samples, bacterial samples, microbial samples, viral samples, biological samples, serum samples, Plasma samples, blood samples, urine samples, semen samples, lymph fluid samples, cerebrospinal fluid samples, amniotic fluid samples, biological sample samples, needle-absorbed biological samples, cancer samples, tumor samples, tissue samples, cell samples, Cell lysate sample, crude cell lysate sample, tissue lysate sample, tissue culture cell sample, buccal swab sample, mouthwash sample, stool sample, dried tissue sample, autopsy sample, archaeological sample, infection sample, hospital Infected samples, production samples, pharmaceutical preparation samples, biomolecule production samples, protein preparation samples, lipid preparation samples, carbohydrate preparation samples, inanimate object samples, air samples, soil samples, juice samples, metal samples, fossil samples, excavated materials Samples and / or other earth or ground Out-of-ball sample. Types of forensic samples include blood, dried blood, blood stains, buccal swabs, fingerprints, contact samples (such as staying on the glass lip, the inner edge of a baseball cap or the epithelial cells on the cigarette head), chewing gum, stomach contents, saliva , nail fragments, soil, sexually assaulted samples, hair, bone, skin and solid tissue. Types of environmental samples include unfiltered and filtered air and water, soil, surface swab samples, coatings and powders.

For example, the methods herein can provide amplified nucleic acid samples that are analyzed for information suitable for forensic interpretation, and specifically for compliance with forensic interpretation guidelines. These criteria include signal intensity, peak height balance in the locus, heterozygous peak height ratio (PHR), incomplete non-template nucleotide addition (NTA), and shadow (Scientific Working Group on DNA Analysis Methods, Short Tandem Repeat ( Scientific Working Group on DNA Analysis Methods, Short Tandem Repeat) STR) Interpretation Guidelines. Forensic Science Communications, 2000, 2(3)).

The phrase "fluid communication" as used herein refers to two fluid-containing chambers or other components or regions connected together such that fluid can flow between the two chambers, components or regions. move. Thus, the two chambers of "fluid communication" can be coupled together, for example, via a microfluidic channel between the two chambers such that fluid can flow freely between the two chambers. The microfluidic channels may optionally include one or more valves that may close or block to block and/or otherwise control fluid communication between the chambers.

As used herein, the term "poly (methyl methacrylate)" or "PMMA" means a synthetic polymer of methyl methacrylate, including (but not limited to) their trademark Plexiglas ^TM, Limacryl ^TM, R -Cast ^TM , Perspex ^TM , Plazcryl ^TM , Acrylex ^TM , ACrylite ^TM , ACrylplast ^TM , Altuglas ^TM , Polycast ^TM and Lucite ^TM , and those described in U.S. Patent Nos. 5,561,208, 5,462,995 and 5,334,424 Polymers, each of which is hereby incorporated by reference herein.

The term "polycarbonate" as used herein means a polyester of carbonic acid and a diol or a divalent phenol. Examples of such diols or divalent phenols are p-xylylene glycol, 2,2-bis(4-oxyphenyl)propane, bis(4-oxyphenyl)methane, 1,1-double ( 4-oxyphenyl)ethane, 1,1-bis(oxyphenyl)butane, 1,1-bis(oxyphenyl)cyclohexane, 2,2-bis(oxyphenyl) butane and mixtures thereof, including (but not limited to) their trademark ^{Calibre TM, Makrolon TM, Panlite TM} , Makroclear TM, Cyrolon TM, Lexan TM Tuffak ^TM and sellers.

The term "nucleic acid" as used herein is intended to include both single and double stranded DNA and RNA, as well as any and all alternative nucleic acids containing modified bases, sugars and backbones. Thus, it is to be understood that the term "nucleic acid" includes, but is not limited to, single or double stranded DNA or RNA (and may be in the form of a partial single or partial double strand), cDNA, aptamer, peptide nucleic acid ("PNA"). 2'-5' DNA (a synthetic material with a shortened skeleton that has a base spacing that matches the A configuration of DNA; 2'-5' DNA usually does not hybridize to DNA of Form B, but it is easy to RNA hybridization) and locked nucleic acids ("LNA"). Nucleic acid analogs include known analogs of natural nucleotides that have similar or improved binding to base pair properties, hybridization. The "similar" forms of purines and pyrimidines are well known in the art and include, but are not limited to, aziridine cytosine, 4-ethenylcytosine, 5-fluorouracil, 5-bromouracil, 5-carboxymethyl Aminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, inosine, N ⁶ -isopentenyl adenine, 1-methyladenine, 1-methyl pseudouracil , 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methyl Cytosine, N ⁶ -methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannose Beta-D-mannosylqueosine, 5-methoxyuracil, 2-methylthio-N-6-isopentenyl adenine, methyl uracil-5-oxyacetate, false Uracil, Q nucleoside, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxy Acetic acid and 2,6-diamino hydrazine. The DNA backbone analogs provided by the present invention include phosphodiester, phosphorothioate, dithiophosphate, methyl phosphonate, amino phosphate, alkyl phosphate, aminosulfonate, 3'-sulfur Acetal, methylene (methylimido), 3'-N-carbamate, morpholinyl carbamate and peptide nucleic acid (PNA), methyl phosphonate linkage or alternative phosphonate Ester and phosphodiester linkages (Strauss-Soukup, 1997, Biochemistry 36: 8692-8698) and benzyl phosphonate linkages as discussed in US 6,664,057; see also OLIGONUCLEOTIDES AND ANALOGUES, A PRACTICAL APPROACH, by F. Eckstein Edited by IRL Press at Oxford University Press (1991); Antisense Strategies , Annals of the New York Academy of Sciences, vol. 600, by Baserga and Denhardt (NYAS 1992); Milligan, 1993, J. Med. Chem. 36:1923 -1937; Antisense Research and Applications (1993, CRC Press). Nucleic acids herein can be synthesized from cells or synthesized according to any means known to those skilled in the art; for example, nucleic acids can be chemically synthesized or transcribed or reverse transcribed from cDNA or mRNA in other sources.

The term "through hole" as used herein means a through hole formed in a solid material to allow a fluid connection between the top and bottom surfaces of the material.

The term "locus" as used herein means one or more nuclei as defined herein. One or more specific locations on an acid (eg, one or more chromosomes).

The term "STR locus" as used herein means a nucleotide sequence consisting of two or more nucleotides located in a repeating pattern of a given locus of a target nucleic acid. The repeat pattern can range in length from 2 to 10 base pairs (bp) and is typically located in a non-coding intron region.

According to an aspect of the present invention, a thermal cycler having the ability to rapidly and controllably and reproducibly heat and cool a reaction solution is provided. An example of an embodiment of the thermal cycler of the present invention is shown in Figure 1A . The ability to rapidly heat and cool the temperature of the reaction solution minimizes the ramp and settling time, and allows the incubation time at the desired temperature to dominate the total step time, thereby enabling multiple cycle times to be minimized.

High heating and cooling rates can be achieved by using a temperature control element (TCE) either alone or in fluid communication with the heat sink. The TCE includes a heating and cooling component, a thermal sensor, a controller for receiving signals from the thermal sensor, and a power source. In a preferred embodiment, the first surface of the TCE can be adapted to receive a sample chamber containing a solution and an induction chamber containing another thermal sensor. In this configuration, the thermal sensor is positioned within the sensing chamber mounted to the TCE so that it simulates the conditions within the sample chamber. The sensing chamber is fabricated to have a stack of the same material as the sample chamber. A thermocouple mounted in the temperature sensor is embedded in the structure at a location similar to the sample in the sample chamber. The sensor reports the effective temperature of the solution in the sample chamber. Commercially available T- or K-type thermocouples (from Omega Engineering, Stamford, CT) are most suitable, but other types of thermocouples and thermal sensors can be used, including thermistors, semiconductors, and infrared. A thermal sensor in the induction chamber provides feedback to the TCE to set or maintain the sample at the desired temperature. In this way, the sample temperature can be indirectly measured and controlled without inserting the thermal sensor into the reaction chamber itself. Alternatively, the thermal sensor can be placed directly in the reaction chamber to set and maintain the sample temperature, eliminating the need for a sensing chamber. As will be appreciated by those skilled in the art, other types of sensors, such as pressure sensors, can be used in accordance with the teachings of the present invention.

By, for example, in a first surface for receiving a substrate (eg, a biochip below) Forming a recess, the first surface of the TCE can be adapted to accept a substantially flat substrate. Alternatively, the TCE may be adapted to accept one or more thin walled tubes defined as tubes having a wall diameter of an area less than 200 [mu]m thick. Preferably, the heat sink is a high efficiency heat sink such as, but not limited to, a fan cooling fin having a copper base and cooling fins. More preferably, the heat sink can be a fan cooled copper base and wings having a thermal resistance of about 0.4 ° C/W or less. A specific and non-limiting example of a high efficiency heat sink is E1U-N7BCC-03-GP (Coolermaster, Taiwan ROC).

The thermal cycler of the present invention can additionally include a thermal sensor positioned to monitor the first surface temperature of the TCE. Another thermal sensor can be added as needed to achieve further improvements in sample temperature control. The supplemental temperature that can be monitored includes a plurality of regions on the substrate and within the substrate, a plurality of regions on the heat sink and the heat sink, cooling air input and output, sample input and output, and their surrounding temperatures.

Good thermal communication is required between the TCE and the heat sink. When two mating faces are suitably prepared, direct physical contact is sufficient to provide sufficient heat transfer between the two components. The thermal interface material (TIM) between the TCE and the heat sink can be used to enhance the thermal coupling. Such TIMs include, but are not limited to, adhesives, greases, phase change materials (PCM), metallic thermal interface materials, ceramic thermal interface materials, soft metal alloys, indium, alumina nanolayer coatings, submicron films, Glycols, water, oils, antifreezes, epoxy compounds and others. Specific examples include Arctic Silver or Ceramique (Arctic Silver, Visalia, CA; compounds with thermal resistance <0.007 ° C - in ² /W), compressible heating springs HSD4 (Indium Corp, Utica, NY), HITHERM (GrafTech International Holdings) Inc., Lakewood, OH) or bond TCE directly to the surface of the heat sink. The thermal contact can be further enhanced by physically clamping the components together or by directly bonding the surface with an average force greater than 2 psi or greater than 5 psi or greater than 10 psi or greater than 30 psi or greater than 60 psi or greater than 100 psi or greater than 200 psi.

About thermocycler module, such as the Eppendorf Mastercycler ^TM ep gradient S thermocycler (via having a high thermal conductivity and lower thermal energy than the heat capacity of silver module), heat transfer between the substrate in contact with the TCE of the substrate may be by direct Increased by placing on TCE. Suitable TCEs include, but are not limited to, high heating and cooling capacity heat pumps and high power output Peltier devices; examples of Peltier devices are 9500/131/150B (Ferrotec, Bedford NH), XLT2393 (Marlow, Dallas TX). When used as part of the TCE of the thermal cycler of the present invention, the Peltier device is advantageously powered by an H-bridge. An example of an H-bridge device is 5R7-001 (Oven Industries).

When the Peltier device is used as part of the TCE of the thermal cycler of the present invention, it is advantageous to power the Peltier device by means of an H-bridge having pulse width modulation for heating and cooling. The temperature feedback from the thermal sensor that measures the temperature of the sample drives the TCE to set and maintain the desired sample temperature. Closed loop temperature control algorithms for controlling TCE include, but are not limited to, PID control and fuzzy logic control.

The thermal controllers include a control algorithm that provides the ability to quickly transition from one target temperature state to another. This transition can be divided into 3 different phases. In the first stage, there is a large difference between the actual temperature and the target temperature (for example, 1 to 20 ° C or higher). During this phase, the ramp occurs at or near the maximum rate of the TCE device. In the second phase, the transition phase, the actual temperature is close to the target temperature (less than about 1 to 20 ° C). In this case, the controller must reduce the power of the TCE to prevent exceeding the solution temperature and allow the target temperature to be quickly reached with minimal deviation and oscillation. In stage 3, the target temperature has been reached and the controller throttles the power of the thermal cycler to maintain the solution within a narrow range around the target temperature. Measuring the temperature with the sensor provides more accurate feedback of the actual temperature and also allows the temperature of the TCE surface temperature to be overdriven. Each of the above three stages can be further subdivided into multiple sub-stages to provide faster reaction times, more precise temperature control, increased stability, and increased tolerance to external changes.

In one example, the temperature of the substrate can be measured by placing a thin thermocouple in the channel of the substrate surface. In another example, the second thermal sensor can be housed in a housing that is substantially formed of the same material as the substrate used, which substantially holds the second thermal sensor At the same distance from the TCE and the reaction chamber on the substrate that is in contact with the TCE. The second thermal sensor can typically be separated from the substrate (ie, a separate inductor) and can be placed on the first surface of the TCE next to the substrate.

The heat sink may optionally include a variable speed cooling fan and/or a second heating element for controlling the temperature of the heat sink, wherein each additional element of the heat sink is in communication with the second control element. This makes it possible to adjust the cooling efficiency of the heat sink, in particular to keep the heat sink temperature substantially constant and independent of ambient temperature changes. The heater can also pre-adjust the heat sink to a substantially operating temperature.

To facilitate thermal coupling of the reaction solution in the substrate to the TCE, uniform thermal communication of the contact surface of the substrate with the first surface of the TCE can be provided by applying a force to the substrate to secure it to the TCE while the thermal cycler is operating. Preferably, the force is applied by temporarily holding the substrate to the first surface of the TCE and the member that can be easily removed after the thermal cycle is completed. For example, a wafer compression element (CCE) can be placed on the first surface of the TCE such that the substrate can be placed therebetween. The wafer compression element can then be engaged to hold the substrate in place during operation of the thermal cycler and released to allow the substrate to be removed. The proper integration of the CCE, TCE and heat sink allows the CCE to improve the thermal coupling between the two components and between all three.

The portion of the CCE that is in contact with the substrate can be formed from a low thermal mass insulating material including, but not limited to, a foam such as WF71 Rohcell foam (Inspec foams, Magnolia, AR). For the embodiments discussed herein, Rohacell is preferred. It has a specific heat capacity of 1.4-1.6 (J/gK) [or lower thermal mass] and a thermal conductivity of 0.0345 W/mK (or lower).

The biowafer compression element includes, but is not limited to, one or more grippers, springs, compressible foams, or compressed air bladders that are expandable to provide a force to hold the substrate on the first surface of the TCE. Preferably, the wafer compression element provides a substantially uniform force of from about 5 to about 250 psia to the surface of the substrate, and more preferably from about 20 to about 50 psia to hold the substrate to the first surface of the TCE. Notably, thermal communication between the contact surface of the bio-wafer substrate and the TCE can be provided in the absence of a thermal coupling solution such as a thermal grease or glycol, although Use these thermal coupling solutions as needed.

The biochip compression element provides force on the biochip-thermoelectric cooler-heat sink. This force is used to ensure good thermal contact between the biochip and the upper surface of the TCE and thus ensure heat transfer.

In one embodiment, the low thermal mass insulator is an air bladder and is used to provide low thermal mass and insulation properties. In another embodiment, the low thermal mass insulator is a foam liner. The clamping force can be applied to the foam pad by a pneumatic cylinder or a closed cell foam pad that is compressed or pressurized from the air bladder. In the latter case, the air bladder provides insulation and compression.

As noted above, the thermal cycler can have a temperature of from about 4 to 150 ° C per second, and preferably from about 8 to 150 ° C per second, and more preferably from about 10 to 150 ° C per second, on the first surface of the TCE surface. And / or cooling rate. The thermal cycler can also have a temperature of from about 4 to 150 ° C / sec and preferably from about 8 to 150 ° C / sec, and more preferably from about 10 - in the solution in the reaction chamber of the substrate in thermal communication with the first surface of the TCE. Heating or cooling rate of 150 ° C / sec. Additionally, the thermal cycler of the present invention can have a temperature stability of +/- 1.0 ° C, and preferably +/- 0.50 ° C and more preferably +/- 0.25 ° C.

Biochip

For purposes of illustration, an embodiment of a biochip (i.e., a substrate for a thermal cycler of the present invention) in accordance with another aspect of the present invention is shown in FIG. 1B , having 16 microfluidic systems, each containing and An inlet and an outlet in fluid communication with each of the reaction chambers formed in the biochip. However, the present disclosure is not intended to be limiting, and it will be readily appreciated by those skilled in the art that a biochip can contain an alternate number of microfluidic systems (below), including a biochip having one system and having two or more System biochip. The term "plurality" as used herein means two or more, four or more, eight or more, sixteen or more, 32 or more than 32, 48 or 48 or more, 64 or 64 or more, 96 or 96 or more, 128 or more, 2-16, 2-32, 2-48, 2-64, 2-96, 2-128, 8-128, 8-64 or 8-32 microfluidic channels.

The bio-wafer can comprise a substrate layer and a cover layer, wherein one or more portions of the microfluidic system comprising trenches and/or shaped recesses are patterned into the substrate layer. A series of through holes (i.e., through holes and/or inlets or outlets) may be formed in the cover layer to provide fluid access to the microfluidic channels and the reaction chamber, and may be located anywhere near the biochip. Alternatively, via holes may be formed in the substrate layer instead of the cap layer to achieve the same functionality. The upper surface of the substrate layer can be bonded to the lower surface of the cover layer to complete the microfluidic system. Becker and Gartner (Becker, 2000, Electrophoresis 21, 12-26 and Becker, 2008, Electrophoresis 390, 89) broadly review the techniques for making polymer-based microfluidic systems, which are hereby incorporated by reference in its entirety. . For example, unsaturated, partially unsaturated or saturated cyclic olefin copolymer "COC", unsaturated, partially unsaturated or saturated cyclic olefin polymer "COP", polymethyl methacrylate "PMMA", polycarbonate "PC"", polypropylene "PP", polyethylene "PE", polyetheretherketone "PEEK", poly(polydimethyloxane) "PDMA", polyamidene "PI" material to manufacture biochips. It is important to select a plastic having a glass transition temperature that is greater than the maximum temperature used in the amplification reaction. Any number of such methods and materials can be used to make the biochip described herein. In particular, biochips can be prepared by injection molding of plastic substrates such as COC or COP based polymers (currently marketed under the trademarks Topas ^{( TM)} , Zeonex ^{(TM )} , Zeonor ^(TM) and Apel ^(TM )). In this manufacturing method, an injection mold and a mold insert composed of a negative sheet to be formed are manufactured by processing and then surface polishing. The mold and the insert together enable the substrate layer to be fabricated and the resulting substrate to include channels, reaction chamber features, and vias. The substrate and the cover layer can be diffusion bonded by application of heat and pressure.

Alternatively, the biowafer can be prepared by hot stamping a thin thermoplastic film with a master of the negative to be fabricated. The master mold can be prepared by electroforming to replicate the device prepared in the solid substrate. The solid substrate can be a glass sheet that is patterned by standard photolithography and chemical etching known to those skilled in the art. The substrate and the cover layer are diffused by applying heat and pressure Hehe.

The substrate and cover layer of the biochip can be constructed from a variety of plastic substrates including, but not limited to, polyethylene, poly(acrylate) (eg, poly(methyl methacrylate)), poly(carbonate), And unsaturated, partially unsaturated or saturated cyclic olefin polymers (COP), or unsaturated, partially unsaturated or saturated cyclic olefin copolymers (COC). The thickness of the plastic substrate and cover layer used in the method of the present invention is kept thin to minimize its mass to thereby maximize heat transfer between the thermal cycler and the reaction solution contained in each reaction chamber during its use. The plastic substrate and the cover layer may each independently have a thickness of less than 2 mm, less than 1 mm, less than 750 μm, less than 650 μm, less than 500 μm, less than 400 μm, less than 300 μm, less than 200 μm or less than 100 μm; or the plastic substrate and the cover layer may each independently comprise A plastic having a thickness in the range of 25-2000 μm, 25-1000 μm, 25-750 μm, 25-650 μm, 25-500 μm, 25-400 μm, 25-300 μm, 25-200 μm or 25-100 μm. Preferably, at least one of the substrate and the cover layer has a thickness of less than about 200 [mu]m to maximize heat transfer to the reaction solution contained in the reaction chamber of the biochip. More preferably, the contact surface of the biochip in contact with the first surface of the TCE has a thickness of less than about 200 [mu]m.

Each reaction chamber may be formed to have a volume of, for example, less than 100 μL. Preferably, each reaction chamber has a volume of less than about 50 μL or less than about 40 μL or less than about 30 μL or less than about 25 μL or less than about 20 μL or less than about 15 μL or less than about 10 μL or less than about 5 μL or less than about 1 μL or less than about 0.1 μL. . Alternatively, each reaction chamber can be formed to have a volume ranging from about 0.1 μL to about 100 μL. Preferably, each reaction chamber has a volume ranging from about 0.1 μL to about 10 μL or from about 10 μL to about 50 μL. The reaction chamber is typically not coated with a polymer or decane coating. The reaction chamber can be designed to have inlet and outlet passages. Alternatively, a single channel can be used as an inlet and an outlet.

The biochip design of the present invention affects the advantages of microfluidics, including a high surface to volume ratio and reduced diffusion time for maximum heat transfer, as well as uniform heating and cooling. The use of microfluidic technology also provides advantages for fully integrated forensic analytical instruments. another In addition, based on the requirements of the desired application, it is tested and demonstrated that biofilms fabricated by diffusion bonding and bonding without the use of adhesives to bond various layers (e.g., COC layers) can withstand pressures of 100 to 1500 psi prior to failure. For example, the biowafer of the present invention is subjected to 450 psi, which is sufficient for the desired thermal cycling applications.

It should be noted that the particular embodiment of the biochip of the present invention set forth herein is substantially devoid of any heating elements integrated into the biochip for heating and/or cooling the reaction chamber. The thermal cycling of the reaction chamber on the biochip is provided externally, for example by the thermal cycler of the present invention. However, the heating element can be integrated into the biochip of the present invention.

In operation, a portion of the biochip can receive one or more reaction solutions via one or more inlets in fluid communication with one or more reaction chambers formed within the biochip, each reaction solution independently comprising one or more reagents ( For example for PCR) and/or nucleic acid samples. Simultaneous amplification of multiple samples can be performed by injecting each nucleic acid sample into a separate separation reaction chamber. A syringe for simultaneously injecting a plurality of samples into a plurality of samples or buffer wells can be provided with a biochip to enable simultaneous amplification of multiple samples. The injectors, for example, provide one of a plurality of samples to one of a plurality of reaction chambers. The syringe can introduce the sample into the channel according to any method known to those skilled in the art, such as by electrophoretic delivery, via needle or tube or channel pneumatic or liquid actuation that connects the sample to the reaction chamber.

Following amplification (and optionally the nucleic acid extraction and quantitative) enable amplification of nucleic acid products by (e.g. to Genebench-FX ^TM 100) or a plurality of outlet in fluid communication with the reaction chamber and to isolate fragments generated STR profiles.

The relatively low cost of plastic manufacturing allows the biochip of the present invention to be disposable, eliminating the labor required to reuse the biochip and substantially eliminating the possibility of contamination. Disposable items for single use are particularly advantageous for low copy number analysis where contamination (except for initial sampling) is not possible. Reusable plastic and glass biochips can be used where both pollution and labor are not a major consideration.

Integration method

The use of microfluidics enables the fabrication of features to perform more than one function on a single biochip. Such functions may include nucleic acid extraction, nucleic acid purification, pre-PCR nucleic acid clearance, post-PCR clearance, pre-sequencing clearance, sequencing, post-sequencing clearance, nucleic acid isolation, nucleic acid detection, reverse transcription, reverse transcription pre-clearing, reverse transcription. Post-clearing, nucleic acid ligation, nucleic acid hybridization, and quantification. Two or more of these functions may be microfluidically connected to enable continuous processing of the sample; this coupling is referred to as integration.

One form of microfluidic DNA extraction can be achieved by inserting a purification medium between the input and output channels. This purification medium can be based on cerium oxide fibers, and a chaotropic salt reagent is used to dissolve the biological sample, expose the DNA, and bind the DNA to the purification medium. The lysate is then transported through the purification medium via an input channel to bind the DNA. The bound DNA is washed by an ethanol based buffer to remove contaminants. This can be accomplished by flowing the wash reagent through the input channel through the purification membrane. The bound DNA is then eluted from the purified membrane by passing a suitable low salt buffer (see, for example, US 5,234,809 to Boom) through the input channel through the purification membrane and out of the output channel.

A DNA quantification method in microfluidic form is based on real-time PCR. In this quantitative method, a reaction chamber is fabricated between the input and output channels. The reaction chamber is coupled to the thermal cycler and the photoexcitation and detection system is coupled to the reaction chamber such that fluorescence from the reaction solution can be measured. The amount of DNA in the sample is related to the intensity of fluorescence from the reaction chamber per cycle (see, for example, Heid et al , Genome Research 1996, 6, 986-994).

For additional information on the integration of the microfluidic form, see U.S. Patent Application Serial No. 07-801-US, entitled "INTEGRATED NUCLEIC ACID ANALYSIS", which is hereby incorporated by reference in its entirety. The manner is incorporated herein. For additional information on the separation and detection of microfluidic forms, see U.S. Patent Application Serial No. 07-865-US, entitled "Plastic Microfluidic Separation and Detection Platforms", which is hereby incorporated by reference. Incorporated herein by reference in its entirety.

The microfluidic drive of the present invention is a means of transporting fluid within a reaction chamber that integrates a biochip. One type of microfluidic drive is achieved by incorporating a diaphragm pump that delivers a positive pressure and a negative pressure to the membrane by continuously applying a positive pressure to the membrane. Alternatively, a positive displacement pump can be connected to the input of the microfluidic chamber. The displacement of the pump forces the fluid through the microfluidic channel.

Integration can utilize microfluidic valves to gate fluid flow within the biochip. Valve control can be implemented in a passive or active configuration. The passive valve control structure includes a capillary valve that terminates fluid flow by using capillary pressure. The fluid can flow through the capillary valve control structure by applying a pressure large enough to overcome the capillary force. The active valve control structure includes a diaphragm valve that uses a flexible or semi-rigid structure at a point between the two channels. Pressure is applied to the membrane to close the passage. A vacuum is applied to the membrane to move it up the channel, allowing fluid to pass.

Amplification method

In another aspect, the invention provides a method of simultaneously amplifying a plurality of nucleic acid loci in one or more target nucleic acids via rapid polymerase chain reaction (PCR). The methods comprise providing one or more reaction solutions to one or more reaction chambers, wherein each reaction solution comprises (i) at least one copy of at least one target nucleic acid, wherein each target nucleic acid is identical or different and each target nucleic acid is independent Include a plurality of loci to be amplified; (ii) one or more buffers; (iii) one or more salts; (iv) a primer set corresponding to a plurality of loci to be amplified; (v) nucleic acid polymerization Enzymes and (vi) nucleotides. Each reaction solution, such as each target nucleic acid, can be the same or different, as desired, for example, by running multiple simultaneous analyses or simultaneously running multiple nucleic acid samples on the same nucleic acid sample.

Each reaction chamber may be contained in a biochip of the present invention as described above or in a thin-walled reaction tube. The thin walled reaction tube preferably has a wall thickness of less than about 200 [mu]m. Preferably, the thin walled reaction tube preferably has a wall thickness of less than about 100 [mu]m.

The primer for PCR amplification is an oligonucleotide sequence specifically designed to hybridize to the locus of the target DNA. These primers serve as a starting point for polymerase extension. To facilitate the analysis of amplified fragments, labeled primers can also be used in PCR reactions. Tag primer is the one that is coupled to the detectable part A nucleotide sequence; a non-limiting example of which is a fluorescent dye. When PCR is carried out with a fluorescently labeled primer, an amplicon having a fluorescent marker is produced. The method of performing rapid PCR is compatible with labeled and unlabeled primers, and rapid multiplex PCR has been demonstrated.

As noted above, the primer set can be any set of primers known to those skilled in the art for amplifying multiple loci with a target nucleic acid. For example, the primers suitable for amplifying one or more loci in a human nucleic acid sample are described in US 5,582,989; US 5,843,660; US 6,221,598; US 6,479,235; US 6,531,282 and US 7,008,771; No. 2003/0186272 and No. 2004/0137504, each of which is hereby incorporated by reference herein.

In addition, primers suitable for use in amplifying one or more loci of a viral nucleic acid sample are described in, for example, US 7,312,036; US 6,958,210; US 6,849, 407; US 6, 790, 952 and US Pat. in.

Examples of primers suitable for amplifying one or more loci in a bacterial nucleic acid sample are described in US 7,326,779, US 7,205,111, US 7,074,599, US 7,074,598, US 6,664,080, and US 5,994,066 This is incorporated herein by reference.

Salts and buffers include those familiar to those skilled in the art, including those in which MgCl ₂ and Tris-HCl and KCl, respectively. Buffers may contain surfactants such as Tween, dimethyl hydrazine (DMSO), glycerol, bovine serum albumin (BSA), and polyethylene glycol (PEG), among others, those skilled in the art. A familiar additive. Nucleotides are typically deoxynucleoside triphosphates such as deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP) and deoxythymidine triphosphate ( dTTP), which is also added to the synthesis mixture in sufficient amounts for amplification of the target nucleic acid.

The solution can optionally be heated to and maintained at a first temperature suitable for thermal initiation activation of the nucleic acid polymerase for a first period of time. Typically, the first period of time is less than about 90 seconds. The first temperature can range from about 95 to about 99 °C. A device that can be activated in 60 seconds or less Polymerases with a hot initiation mechanism include those that use antibody-mediated thermal initiation and aptamer-mediated thermal initiation mechanisms. Alternatively, it is not necessary to use a hot starting polymerase in the present invention.

Subsequently, the temperature of the reaction solution is sequentially cycled between the denatured state, the bonded state, and the expanded state for a predetermined number of cycles. Generally, the one or more reaction solutions are at from about 1 to about 150 ° C / sec, or from about 1 to about 100 ° C / sec; or from about 1 to about 80 ° C / sec; or from about 1 to about 60 ° C / sec Or; about 1 to about 40 ° C / sec; or about 1 to about 30 ° C / sec; or about 1 to about 20 ° C / sec; about 4 to about 150 ° C / sec, or about 4 to about 100 ° C / sec; or about 4 to about 80 ° C / sec; or about 4 to about 60 ° C / sec; or about 4 to about 40 ° C / sec; or about 4 to about 30 ° C / sec; Or from about 4 to about 20 ° C / sec; or from about 10 to about 150 ° C / sec; or from about 10 to about 100 ° C / sec; or from about 10 to about 80 ° C / sec; or from about 10 to about 60 The first cooling rate of ° C / sec; or about 10 to about 40 ° C / sec; or about 10 to about 30 ° C / sec; or about 10 to about 20 ° C / sec is cooled from the denatured state to the bonded state. The one or more reaction solutions may be from about 1 to about 150 ° C / sec, or from about 1 to about 100 ° C / sec; or from about 1 to about 80 ° C / sec; or from about 1 to about 60 ° C / sec. Or about 1 to about 40 ° C / sec; about 1 to about 30 ° C / sec; about 1 to about 20 ° C / sec; 4 to about 150 ° C / sec, or about 4 to about 100 ° C / sec Or; about 4 to about 80 ° C / sec; or about 4 to about 60 ° C / sec; or about 4 to about 40 ° C / sec; about 4 to about 30 ° C / sec; about 4 to about 20 ° C / sec; or about 10 to about 150 ° C / sec; or about 10 to about 100 ° C / sec; or about 10 to about 80 ° C / sec; or about 10 to about 60 ° C / sec; From about 10 to about 40 ° C / sec; or from about 10 to about 30 ° C / sec; or from about 10 to about 20 ° C / sec, the first heating rate is heated from the bonded state to the expanded state; and/or one may be Or a plurality of reaction solutions at from about 1 to about 150 ° C / sec or from about 1 to about 100 ° C / sec; or from about 1 to about 80 ° C / sec; or from about 1 to about 60 ° C / sec; or about 1 Up to about 40 ° C / sec; from about 1 to about 30 ° C / sec; from about 1 to about 20 ° C / sec; from about 4 to about 150 ° C / sec or from about 4 to about 100 ° C / sec; 4 to about 80 ° C / sec; or about 4 to about 60 ° C / sec Or from about 4 to about 40 ° C / sec; from about 4 to about 30 ° C / sec; from about 4 to about 20 ° C / sec; or from about 10 to about 150 ° C / sec; or from about 10 to about 100 ° C /second; or about 10 to about 80 ° C / sec; or about 10 to about 60 ° C / sec; or about 10 to about 40 The second heating rate of ° C / sec; or about 10 to about 30 ° C / sec; or about 10 to about 20 ° C / sec is heated from the expanded state to the denatured state. Finally, the reaction solution is maintained in a final state to provide one or more amplified nucleic acid products.

The denaturation state can generally range from about 90 to 99 ° C for a period of time ranging from about 1 to 30 seconds. The actual time and temperature depend on the enzyme, primer and target. For the Applied Biosystems (AB) multiple 7 STR kit used to amplify human genomic DNA, a temperature of about 95 ° C for about 5 seconds is preferred.

Adhesion temperature and time affect the specificity and efficiency of primers that bind to a particular locus within a target nucleic acid and are particularly important for multiplex PCR reactions. Accurate binding of the entire set of primer pairs during the binding step allows for the multiplex amplification of multiple loci, such as one or more complete STR maps with acceptable PHR and signal intensity balance within the locus. For a given pair of primers, the bond state can range from about 50 ° C to 70 ° C and for about 1 to 30 seconds. The actual time and temperature depend on the enzyme, primer and target. For an AB multiple STR set to amplify human genomic DNA, a temperature of about 59 ° C for 15 seconds is preferred.

The extended temperature and time primarily affect the allele product yield and are inherent properties of the enzyme under investigation. It should be noted that the rate of expansion reported by the manufacturer is usually set for a single reaction; the rate of expansion of multiple reactions may be much slower. For a given enzyme, the expanded state can be in the range of about 60 to 75 °C and the time is about 1 to 30 seconds. The actual time and temperature depend on the enzyme, primer and target. For AB multiple STR sets used to amplify human genomic DNA, a temperature of about 72 ° C for about 5 seconds is preferred. Preferably, the reaction solution is, for a predetermined number of cycles, from about 1 to about 150 ° C / sec or from about 1 to about 100 ° C / sec; or from about 1 to about 80 ° C / sec; or from about 1 to about 60 ° C / sec; or about 1 to about 40 ° C / sec; or about 1 to about 30 ° C / sec; or about 1 to about 20 ° C / sec; 4 to about 150 ° C / sec or about 4 Up to about 100 ° C / sec; or about 4 to about 80 ° C / sec; or about 4 to about 60 ° C / sec; or about 4 to about 40 ° C / sec; or about 4 to about 30 ° C / sec Clock; or about 4 to about 20 ° C / sec; or about 10 to about 150 ° C / sec; or about 10 to about 100 ° C / sec; or about 10 to about 80 ° C / sec; or about 10 to about 60 ° C / sec; or about 10 to about 40 ° C / sec; Or from about 10 to about 30 ° C / sec; or from about 10 to about 20 ° C / sec at a third rate from the expanded state to the denatured state. Typically, the predetermined number of cycles is selected to be from about 10 to about 50 cycles, although fewer or more cycles may be used as desired.

Until the incomplete NTA begins to increase, the final expansion time can be significantly reduced. For a given enzyme, the final extension temperature can range from about 60 to 75 °C and the time is from about 0 to 300 seconds. The actual time and temperature depend on the enzyme, primer and target. For an AB multiple STR kit to amplify human genomic DNA, a temperature of about 72 ° C for about 90 seconds is preferred.

In addition to the 3-step thermal cycling method set forth above, this method is also suitable for a 2-step thermal cycling process. In this method, the reaction solution is sequentially cycled between the denatured state and the bonded/extended state for a predetermined number of cycles. The method uses primers designed to bond at extended temperatures such that the bonding and spreading steps share the same temperature. A reduced number of temperature transitions further reduces cycle time.

In certain embodiments, a plurality of amplified nucleic acid products can be obtained in about 5 to about 20 minutes. In certain other embodiments, the plurality of amplified nucleic acid products can be obtained in about 5 to 10 minutes, about 1 to 5 minutes, or less than 5 minutes. Each amplified nucleic acid product can be produced starting from less than about 10 ng of the target nucleic acid. Preferably, it can be less than about 5 ng or less than about 2 ng of nucleic acid, or less than about 1 ng of nucleic acid, or less than about 0.5 ng of nucleic acid, or less than about 0.2 ng of nucleic acid, or less than about 0.1 ng of nucleic acid, or less than about 0.05 ng of nucleic acid, or less. Approximately 0.006 ng of nucleic acid is initiated to produce an amplified nucleic acid product.

In other embodiments, such as identifying a biological weapon agent in a clinical or environmental sample or diagnosing a bacterial, viral or fungal infection in a human, plant or animal, the amplified nucleic acid product can be produced from at least one copy of the target nucleic acid. . For example, prior to the multiplex amplification reaction, the sample to be analyzed may comprise less than 1000 copies of the target nucleic acid (eg, 1-1000) Replica), less than 400 copies, less than 200 copies, less than 100 copies, less than 50 copies, less than 30 copies, less than 10 copies, or 1 copy.

In addition, if a target nucleic acid locus is present in more than one copy of the genome, amplification of the DNA may be performed using less than a single genome equivalent.

As will be readily appreciated by those skilled in the art, in any of the foregoing methods, the thermal cycle can be performed for a predetermined number of cycles to achieve sufficient amplification of the locus in the target nucleic acid. For example, the predetermined number of cycles may be between about 10 and about 50 cycles and preferably between about 20 and 50 cycles. Additionally, in any of the foregoing methods, at least two loci of one or more nucleic acids can be simultaneously amplified. Depending on the desired application, more than 4, 5 to 10, 10 to 20, 20 to 30 or about 10 to 250 loci are amplified simultaneously. For example, to amplify an STR locus, 10-20 loci may be preferred.

Preferably, the temperature of the reaction solution is circulated by the thermal cycler (described above) of the present invention. Although it is possible to use a commercial module thermal cycler by compensating for the hysteresis reaction of the PCR solution by setting the module temperature above the desired solution temperature of the heating step and setting the module temperature below the desired solution temperature for the cooling step. Rapid thermal cycling, but this mode of operation is cumbersome to implement when the temperature set point required to compensate for the slow ramping reaction must be determined empirically. In addition, since feedback and control are still performed by the module and the solution temperature is not monitored, the repeatability and reproducibility of the distribution can be affected by factors including the outer boundary of the room temperature change. Therefore, the temperature distribution of the solution is not reproducible.

Many commercially available polymerases are suitable for use in rapid PCR applications using the methods described herein. Typically, the nucleic acid polymerase has an expansion rate of at least 100 bases per second. A large number of polymerases that can be used for PCR amplification include Thermus aquaticus (Taq), Pyrococcus furiosus (Pfu), Pyrococcus woesei (Pwo), and Yellow Thermus ( Thermas) Flavus , Tfl), Themus thermophilus (Tth), ( Thermus litoris , Tli ) and Thermotoga maritime (Tma). Such enzymes, modified forms of such enzymes, and combinations of enzymes are commercially available from suppliers including Roche, Invitrogen, Qiagen, Strategene, and Applied Biosystems. Representative enzymes include PHUSION (New England Biolabs, Ipswich, MA), Hot MasterTaq TM (Eppendorf), PHUSION Mpx (Finnzymes), PyroStart (Fermentas), KOD (EMD Biosciences), Z-Taq (TAKARA) and CS3AC / LA ( KlenTaq, University City, MO). STR genotyping PCR for amplification of the Taq polymerase enzyme is widely used, and the system becomes TaqGold to Identifiler ^TM, Profiler ^TM and COfiler ^TM kit supplied.

In certain embodiments, the PCR conditions provided herein can produce a full STR map from a human target nucleic acid with high efficiency, although a complete map is not required to be produced. The complete map of the autosomal chromosome STR may comprise, for example, amelogenin, D8S1179, D21S11, D7S820, CFS1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, FGA or a variety thereof Locus. Other STR loci include small STR and Y-STR analyses. The criteria for the optimization scheme include generating a complete map, signal strength, dynamic range, signal intensity balance within the locus, PHR, incomplete NTA, shadow band, and total cycle time.

According to one embodiment, the total cycle time of the biowafer and tube reaction can be reduced to 17.3 and 19.1 minutes, respectively, using the SpeedSTAR enzyme and the thermal cycler of the present invention to produce a complete STR pattern. In this embodiment, the denatured state is about 98 ° C for about 4 seconds, the bonded state is about 59 ° C for about 15 seconds, the expanded state is about 72 ° C for about 7 seconds, and the final state is about 70 ° C for about 90 seconds. Seconds.

In certain embodiments, the total cycle time of at least 10, 20 or 30 multiplex PCR cycles can range from about 1 minute to about 90 minutes. Preferably, the total cycle time of at least 10, 20 or 30 multiplex PCR cycles may range from about 1 minute to about 90 minutes; or from about 1 minute to about 85 minutes; or from about 1 minute to about 80 minutes; or about 1 Minutes to about 75 minutes; or from about 1 minute to about 70 minutes; or from about 1 minute to about 65 minutes; or from about 1 minute to about 60 minutes; or from about 1 minute to about 55 minutes; or from about 1 minute to about 50 minutes Or from about 1 minute to about 45 minutes; or From about 1 minute to about 40 minutes; or from about 1 minute to about 35 minutes; or from about 1 minute to about 30 minutes; or from about 1 minute to about 25 minutes; or from about 1 minute to about 20 minutes; or from about 1 minute to about 15 minutes; or about 1 minute to about 10 minutes or about 1 minute to about 5 minutes. In other embodiments, the total cycle time of at least 10, 20 or 30 multiplex PCR cycles is less than about 90 minutes. Preferably, the total cycle time of at least 10, 20 or 30 multiplex PCR cycles is less than about 89, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20 , 15, 10, 5, 4, 3, 2 or 1 minute.

The present invention encompasses an integrated biochip comprising one or more microfluidic systems for performing multiplex PCR amplification of nucleic acid samples and multiple loci in at least one other sample preparation, and/or within the same biochip platform Analytical method. For example, in each microfluidic system on a biochip (each having a flow direction from the inlet to the outlet), the system can include a plurality of reaction chambers, wherein the first reaction chambers of the plurality of reaction chambers are in fluid communication with the inlet, and The final reaction chamber of the plurality of reaction chambers is in fluid communication with the outlet, and the at least one microchannel is fluidly coupled to the adjacent reaction chambers in the flow direction. At least one of the reaction chambers within each microfluidic system can be less than 200 [mu]m from the contact surface of the biowafer substrate to facilitate thermal communication with the thermal cycler of the present invention for performing multiplex PCR within the reaction chamber.

Each of the remaining reaction chambers within each microfluidic system of the biochip can be suitable for nucleic acid extraction, nucleic acid purification, nucleic acid hybridization, nucleic acid ligation, pre-PCR nucleic acid scavenging, post-PCR scavenging, pre-sequencing scavenging, sequencing, post-sequencing removal, Isolation and detection, reverse transcription, pre-reverse clearance and/or post-reverse clearance, electrophoretic separation, nucleic acid detection. The term "clearing" as used herein means the removal of a reaction component (including anions, cations, oligonucleotides, nucleotides, preservatives, enzymes or inhibition) that can interfere with any of the reaction chamber processes listed above. Agent).

The following examples illustrate specific embodiments of the invention and its various uses. It is intended to be illustrative only and should not be taken as limiting the invention.

Instance Example 1

Conventional thermal cycler and microfluidic biochip

The reaction temperature of the PCR solution by rapid, controllable and reproducible heating and cooling, using the heat cycle of the present invention shown in FIG. 1A for rapid cycling. The instrument accepts a 16-chamber microfluidic biochip and consists of a high output thermoelectric cooler/heater mounted on a high efficiency heat sink. Each of the 16 PCR reaction solutions was placed in an individual chamber of the microfluidic biochip, and coupled to the heat pump by applying a compression pressure of 0.2 MPa with a clamping mechanism. Figure 1B shows a photograph of 16 sample disposable plastic microfluidic biochips. Each PCR chamber was 500 μm deep and approximately 1 mm wide and contained 7 μL of PCR reaction solution.

Instrument and temperature distribution

In the following examples, to Eppendorf Mastercyler ^TM ep gradient S (Eppendorf North America, Westbury, NY) amplification reactions within all of the tubes. The module temperature distribution of the above instrument was obtained using a 127 μm diameter K-type thermocouple sensor directly connected to the module. For the reaction solution distribution, a 127 μm diameter K-type thermocouple sensor was placed in a 20 μL reaction solution in a thin-walled PCR tube. Data acquisition was performed with the Omega HH506RA Multilogger thermometer set to obtain data at a rate of 100 Hz.

The amplification reaction in the biochip was carried out using the 16-sample plastic biochip as a reaction vessel using the thermal cycler of Example 1. The temperature of the solution within the microfluidic biochip is monitored by inserting a thermocouple into the sensing chamber within the biochip.

PCR reaction mixed components and cycling conditions

Multiplex PCR reactions were performed using the AmpF l STR® Profiler Plus® ID PCR Amplification Kit (Profiler Plus ID Set) (Applied Biosystems, Foster City, CA) using 9947A genomic DNA (Promega, Madison, WI) as a template. The polymerase used for amplification kit Profiler Plus ID supply of AmpliTaq Gold® DNA polymerase (TaqGold ^TM) or other polymerases: SpeedSTAR HS DNA polymerase (SpeedSTAR) (Takara BIO USA Inc. , Madison, WI), KOD Hot Start DNA polymerase (KOD) (EMD Biosciences Inc., Gibbstown, NJ) or ^{PyroStart TM Fast PCR Master Mix (PyroStart} ) (Fermentas Inc., Glen Burnie, MD). Multiplex PCR of other polymerases was performed using a labeled multiplexer set from the Profiler Plus ID kit and polymerase-specific buffer and dNTPs. In the thin-walled 0.2mL PCR tubes (Eppendorf North America, Westbury, NY ) using Eppendorf Mastercyler ^TM ep gradient S of all PCR tubes. All biochip reactions were amplified using 16 sample biochips in the thermal cycler of Figure 1A .

The following PCR reaction mixtures were prepared and used for thermal cycling:

Standard TaqGold ^TM reaction:

TaqGold ^TM standard multiplex reaction mixture in a reaction volume of 25μL 9.55μL Profiler Plus ID, 1ng 9947A genomic DNA, 5μL Profiler Plus ID group and 2.25U TaqGold ^TM primer composition. Cycle conditions (module temperature and time) were selected according to the manufacturer's recommendations and set to initial 95 °C for 11 min (hot start), followed by 1 min (denaturation) at 94 °C, 1 min (bonding) at 59 °C, at 72 °C The next 1 min (expansion) and the final expansion of 28 cycles at 45 ° C for 45 min.

Optimize TaqGold ^TM reaction:

Optimized for rapid cycling TaqGoldTM reactions in a 10 [mu]L reaction volume containing 3.82 [mu]L of Profiler Plus ID reaction mix, 1 ng of 9947A genomic DNA, 2 [mu]L of Profiler Plus ID primer set and 0.9 U TaqGold ^{( TM)} . The reaction was cycled at 95 ° C for 11 min, 28 cycles: 10 s at 98 ° C; 45 s at 59 ° C; 30 s at 72 ° C and 15 min at 72 ° C.

SpeedSTAR tube reaction:

The SpeedSTAR PCR mix for tube PCR is: 2 μL Profiler Plus ID primer set in 10 μL reaction volume, 9947A genomic DNA, 1× fast buffer I (Takara BIO USA Inc., Madison, WI), 200 μM dNTP and 0.315 U SpeedSTAR. The rapid cycling conditions were set to 1 min at 95 ° C (enzyme activation), followed by 4 s at 98 ° C, 15 s at 59 ° C, 5 s at 72 ° C and 1 ° at 72 ° C. The final cycle of min is extended by 28 cycles.

SpeedSTAR Biochip Reaction:

For biochip PCR, 7 μL of the reaction mixture contained 1.4 μL of Profiler Plus ID primer set, 9947A genomic DNA, 1× fast buffer I buffer, 200 μM dNTP, and 0.42 U SpeedSTAR. The cycle parameters were set to 70 s at 95 ° C for 28 s: 4 s at 98 ° C, 15 s at 59 ° C; 7 s at 72 ° C and a final extension at 70 ° C 1:30 min.

KOD reaction:

KOD amplification with 2 μL Profiler Plus ID primer set, 1×KOD buffer (EMD Biosciences Inc., Gibbstown, NJ), 200 μM dNTP, 1 ng 9947A genomic DNA, 1.5 mM MgSO ₄ , 0.2 U KOD in 10 μL reaction volume . The cycling conditions were: 2 minutes at 95 ° C, followed by 28 cycles of final expansion at 98 ° C for 4 s, at 59 ° C for 30 s, at 72 ° C for 10 s and at 72 ° C for 1 min.

PyroStart Reaction:

The reaction mixture with 1× final concentration of PyroStart also contained 2 μL of Profiler Plus ID primer set and 1 ng of 9947A genomic DNA in 10 μL of reaction, and was cycled as follows: 1 min and 28 cycles at 95 ° C: 4 s at 98 ° C; 20 s at 59 ° C; 30 s at 72 ° C, followed by a final extension of 1 min at 72 ° C.

Multiplex PCR for other STR typing sets:

SpeedSTAR above with respect to the conditions of the reaction, to Profiler Plus ID kit in test tube and Biological wafer SpeedSTAR other STR genotyping kit (AmpF l STR® Identifiler® (Identifiler) , AmpF l STR® COfiler® PCR The suitability of the complete STR map is generated by the COfiler (Applied Biosystems). In these reactions, the Profiler Plus ID primer set was replaced by a primer set from each set.

Reproducibility

In TaqGold ^TM and ApeedSTAR, genomic DNA using 1ng 9947A tube and Biological Research reproducibility of wafer performed as a template. For tube reproducibility, five individual reactions were prepared. Biochip reproducibility was determined in three biochip PCR runs with 8 reactions each.

Sensitivity

Sensitivity studies of SpeedSTAR amplification in tubes and biochips using the following amounts of 9947A template DNA: tubes: 4 ng, 2 ng, 1.5 ng, 1 ng, 0.5 ng, 0.25 ng, 0.125 ng, 0.1 ng, 0.05 ng, 0.03 Ng, 0.02 ng, 0.01 ng, 0.006 ng; in biochip: 4 ng, 2 ng, 1.5 ng, 1 ng, 0.5 ng, 0.25 ng, 0.1 ng, 0.05 ng, 0.025 ng, 0.02 ng, 0.015 ng, 0.01 ng, 0.006 Ng. The reaction of each template level was performed in duplicate.

STR separation and detection instrument

Use Network Biosystem Genebench-FX ^TM Series 100 (Pyzowski and Tan, Advances in Biochip-Based Analysis: A Rapid Field-Based Approach 59th Annual Meeting of the American Academy of Forensic Sciences) TX, February 19-24, 2007) Isolation and detection of amplified products. This instrument was developed and optimized to be specifically used for STR analysis. 2.7μL to each amplification product was added 10.2μL Hi-Di ^TM A Amides and 0.1μL Genescan 500 LIZ internal standard ribbon (both Applied Biosystems, Foster City, CA) . After denaturation at 95 ° C for 3 min and rapid cooling on ice, the sample was loaded into a separate wafer and electrophoretically moved into the separation channel by applying an electric field of 350 V/cm for 90 seconds. Thereafter, an electric field of 150 V/cm was applied along the separation channel to separate the DNA fragment. All separations were carried out at 50 °C.

date analyzing

Data were analyzed using GeneMarker ^® HID STR Human Identification Software Version 1.51 (SoftGenetics LLC, State College, PA). The signal intensity is normalized to the internal ribbon standard and the percentage of shadow bands, incomplete NTA, and PHR is determined. PHR is calculated by dividing the lower signal intensity allele within the locus by the higher signal strength allele. The level of incomplete NTA was calculated by dividing the signal intensity of the template fragment (-A) by the signal intensity of the adenylated fragment (+A).

Example 2

Temperature distribution of thermal cycling instruments and reaction solutions in conventional PCR tubes and microfluidic biochips

The amplification reaction was carried out using a commercial thermal cycler in a thin-walled PCR tube and using the thermal cycler of Example 1 in a microfluidic biowafer. For the reaction tube, using Eppendorf Mastercyler ^TM. 2A shows the module and temperature of the reaction solution in one of the 28 thermal cycles in the tube using a conventional STR cycle scheme. Mastercycler ^TM-based heating and cooling system with an integrated heat pump module for insertion of tube-based. Time and temperature set points were used for denaturation at 98 ° C for 1 minute, for 1 minute at 59 ° C for bonding and 1 minute at 72 ° C for expansion. A comparison of the temperature profile of the heating module with the reaction solution demonstrates the reaction lag of the solution temperature relative to the module temperature. The measured heating and cooling rates of the modules were 5.6 ° C / sec and 4.9 ° C / sec, and the measured heating and cooling rates of the solutions were 4.8 ° C / sec and 3.3 ° C / sec. The module allowed the temperature to transition from self-expansion (72 ° C) to denaturation (98 ° C) in 14 seconds, but the solution did not reach the set point temperature for 39 seconds. For the module and solution, the transition between the denaturation and bonding steps (59 ° C) took 10 and 27 seconds, respectively, and the transition between the bonding and expansion steps took 7 and 24 seconds, respectively.

The ^TM rapid temperature cycling conditions were 28 thermal cycles of Eppendorf Mastercyler one module and the distributed reaction solution is shown in Figure 2B. The time and temperature set points were for denaturation at 98 ° C for 5 seconds, for bonding at 59 ° C for 15 seconds and for expansion at 72 ° C for 5 seconds. However, the delay and decay reactions of the solution prevent it from reaching the desired set point temperature.

The heat pump and the temperature distribution of the reaction solution of one of the 28 thermal cycles of the thermal cycler of the present invention using rapid cycle conditions were also measured ( Fig. 3 ). To determine the temperature of the reaction solution, a sensing chamber within the biochip is used. The time and temperature set points were for denaturation at 95 ° C for 4 seconds, for 15 seconds at 59 ° C for bonding and 7 seconds at 72 ° C for expansion. The measured heat and cooling rates of the heat pump were 21.5 ° C / sec and 21.7 ° C / sec and the measured heating and cooling rates of the reaction solution were 14.8 ° C / sec and 15.4 ° C / sec.

Therefore, the thermal cycler of the present invention is capable of heating and cooling the reaction solution at a rate three to five times faster than that of the commercial module base circulator. The transition time between the expansion, denaturation and bonding steps of the heat pump was 1.7, 2.1 and 0.7 seconds and the transition time between the expansion, denaturation and bonding steps of the solution was 2.7, 4.5 and 2.2 seconds. The thermal cycler of the present invention allows the reaction solution to reach a desired temperature about 7 times faster than the modular base circulator, resulting in a defined and controlled incubation temperature and time under rapid cycling conditions.

Example 3

Evaluation of PCR enzymes in tubes

The potential use of a large number of polymerases for rapid multiple STR analysis was evaluated and candidates were selected based in part on the hot initiation activation time and expansion rate. The recommendation conditions TaqGold ^TM compared with the reported properties selected for evaluation of the four kinds of experiments polymerase is provided in Table 1 (A).

The enzyme is evaluated to have a reported rate of expansion in the range of about 15-200 nucleotides per second; in general, the reported rate of expansion is based on single amplification and can be somewhat lower for multiple applications.

The in-tube PCR conditions of these four enzymes were initially investigated with the goal of achieving a complete STR map in a minimum time. For all reactions, using primers from the Profiler Plus ID kit and buffers and enzymes of the concentration recommended by the supplier 1ng to amplify the human genome the DNA, and using Genebench-FX ^TM Series 100 separating the resulting spectra, the detection , size and quantity. Determination of denaturation, adhesion and various time and temperature of the expansion step is adapted to obtain a signal strength for SpeedSTAR STR explanation purposes to 19.13 minutes for TaqGold ^TM terms of ranges of 71.7 minutes total time PCR amplification [TABLE 1 (B)]. Process conditions of the present invention allows to 10 times faster than for the amplification of the recommended conditions TaqGold ^TM.

Further assessment of the forensic-related efficacy of enzymes included signal intensity, shadow band levels, and incomplete NTA and PHR [Table 1 (B)]. All enzymes are capable of high multiplex amplification using the Profiler Plus ID primer. SpeedSTAR, Pyrostart, KOD and optimization TaqGold ^TM signal strength of the reaction are substantially the same or greater than the signal strength of their TaqGold using standard PCR conditions arising ^TM.

About incomplete NTA, SpeedSTAR and PyroStart and optimization of reaction TaqGold ^TM display up to three times higher than the standard level of TaqGold ^TM reaction. For most alleles, the level falls below 15% to explain the threshold. As discussed below, their alleles with higher incomplete NTA levels can be reduced to below the 15% interpretation threshold. KOD polymerase has 3'-5' exonuclease activity and does not produce fragments with A overhang; therefore, all alleles are 1 nucleotide shorter than their allele ladder counterparts.

For optimizing the shadow TaqGold ^TM, SpeedSTAR and PyroStart reaction of stutter observed relative levels of TaqGold ^TM reacted with the similarly generated with standard range; stutter arising in KoD range slightly higher than the stutter TaqGold ^TM standard.

Example 4a

Rapid PCR protocol using SpeedSTAR polymerase in tubes and biochips

Based on the results provided above, SpeedSTAR polymerase was selected for further evaluation in biochips with the goal of minimizing total cycle time and achieving signal strength, PHR, incomplete NTA, and shadow band explain the complete STR map required.

The time and temperature set point for amplification on the thermal cycler of the present invention using a SpeedSTAR in a microfluidic 16 sample biochip was 70 seconds at 95 °C for thermal initiation activation, followed by 28 cycles: at 95 4 seconds at ° C for denaturation, 15 seconds at 59 ° C for bonding and 7 seconds at 72 ° C for expansion. The final expansion was carried out at 72 ° C for 90 seconds with a total solution time of 17.3 minutes. The tube reaction in the Eppendorf Mastercycler was carried out in 19.13 minutes, which was set to an initial activation time of 1 minute at 95 ° C, 28 cycles: 4 seconds at 98 ° C, 15 seconds at 59 ° C and at 72 5 seconds at °C, followed by the final expanded module time and temperature at 72 °C for 1 minute.

Figures 4A and 4B show STR spectra generated using 0.5 ng of DNA in a 7 μL biofilm ( Figure 4A ) and 10 μL tube reaction ( Figure 4B ) using the aforementioned SpeedSTAR cycling conditions, and Table 2 provides standard protocols from SpeedSTAR biochips and tubes. and the reaction ^(TM) TaqGold signal strengths of all of the inner tube the Profiler Plus ID of alleles. 0.5ng SpeedSTAR signal intensity ratio of the reaction biochips TaqGold ^TM standard reaction 1ng an average of about 2, although the signal strength of the signal strength 0.5ng SpeedSTAR the reactor to react with substantially the same average TaqGold ^TM.

Example 4b

Allelic Characterization of Rapid PCR Using SpeedSTAR Polymerase in Tubes and Biochips

To characterize the product from the rapid PCR reaction of Example 4a, quantification of PHR, incomplete NTA, and shadow bands was performed. SpeedSTAR polymerase using bio-reactor chip and display the locus as compared to the peak height more unbalanced TaqGold ^TM reaction. The PHR line of the alleles produced in the biochip reaction is between 0.70 and 0.95 and is approximately the same within the tube; all conform to acceptable interpretation criteria. The reaction using SpeedSTAR than their measured for the reaction is a standard TaqGold ^TM PHR lower by about 10%. Similarly, the level of incomplete NTA SpeedSTAR use of biochips and most of the reaction tube is substantially the same alleles (2.0 and 10.6%); both of control than TaqGold ^TM about 2 times higher than the reaction. With the exception that incomplete D3S1358 alleles of NTA, 4.8 to 7 times higher than when it has TaqGold ^TM when having SpeedSTAR; Even in this case, for SpeedSTAR enzyme, incomplete NTA level of less than 12 %. Finally, the shadow of biochips using SpeedSTAR reaction tube with a horizontal base line is between about 6.0 and 14.1%, TaqGold ^TM tube reactor than the standard average of about 1.6 times higher.

Microfluidic biochips 0.5ng of template DNA than their reaction 1ng template using standard reaction of TaqGold ^TM signal strength of about 2-fold higher signal strength. This result indicates that the conventional enzyme SpeedSTAR wafer of known biological effect similar to the reaction efficiency of the enzyme TaqGold ^TM; of the DNA concentration in the biological wafer is approximately 1.8 times the concentration of the tube, this corresponds to 2-fold increase in signal strength. In contrast, the reaction tube proximal ^TM rapid reaction inefficient than control TaqGold; product yield may be reduced about 40% in commercial thermal cycler for the result of poor circulation pattern arising during rapid thermal cycling. Even in this case, the signal strength is completely higher than the level required for interpretation and can be significantly increased by increasing the extension time of each cycle by a few seconds (data not shown). The repeatability and reproducibility of the signal intensity of rapid PCR reactions in biochips and tubes are similar to those in the conventional reactions.

Locus within the reactor and the flash of the biochip allele signal intensity display as compared to ^TM TaqGold reaction with a higher degree of imbalance. The signal intensity balance within the locus is affected by a variety of factors including primer concentration, binding temperature and time, and locus molecular weight. Such experiments for the STR amplification kit having a set of primers concentration recommended for TaqGold ^TM Enzymes optimized in terms of the program cycle. May be adjusted by the concentration of amplification primers used in the reaction to the change of the signal strength locus (Henegariu et al., Biotechniques 1997,23,504-11).

The relationship between expected signal intensity and template levels for rapid biowafer and tube reactions is that signal intensity typically increases with the template. Good peak morphology was observed for all alleles at a high template level of 4 ng, which produced an allele with a signal intensity greater than 12,000 RFU. Some allelic loss occurred at template levels of 0.03 ng and below 0.03 ng. This effect was observed when the amplification reaction was carried out with a limited number of template DNA strands in a solution that produced random amplification (Walsh et al ., PCR Methods Appl. 1992, 1, 241-250). In the template 0.006ng level, the high signal strength and low signal strength allele allele can easily detect the presence of the side signal separation and detection of proof quickly associated with a biological reactor and the wafer Genebench-FX ^TM Series 100 The high sensitivity proves the utility of this system for low copy number analysis. In summary, this data also indicates that the rapid PCR method and thermal cycler and Genebench instrument of the present invention have a high template dynamic range.

Example 4c

DNA template level and allelic characteristics in rapid PCR reactions

The effect of template DNA on the signal intensity of a rapid PCR reaction using SpeedSTAR polymerase in the ( Fig. 5A ) biochip and ( Fig. 5B ) tube reaction is provided in Figures 5A and 5B . The allele selected for analysis was the enamel protein, with the highest signal level allele in the STR map and FGA 23 and 24 and D7S820 10 and 11, with the lowest signal level allele in the map. When the DNA template level in the SpeedSTAR biochip-tube reaction increased from 0.006 ng to 4 ng, the signal intensity of all alleles increased. At a template level of 0.006 ng, the enamel protein peak signal intensity of 111 RFU for biochips and the enamel protein peak signal intensity of 58 RFU for tube reaction were observed. At a template level of 4 ng, the signal intensity of 12680 RFU is visible for the biowafer and the signal intensity of 5570 RFU is visible for the tube reaction. All alleles observed in both reaction types exhibited good peak morphology.

For fast biowafer reactions ( Fig. 6A ), the PHR range is between 0.6 and 1.0 for template levels in the range of 0.05 to 4.0 ng. For template levels below 0.05 ng, PHR decreased to 0.025 ng, and when allele loss occurred, a PHR of zero was observed. Similar results were observed for the flash tube reaction, although it typically showed a slightly lower PHR than the biochip reaction ( Figure 6B ). For biochip reactions, the level of incomplete NTA was 15% or less at template levels of 2.0 ng and below. For tube reactions, incomplete NTA levels exceeded 15% at a template level of 1 ng and a significant increase of 4 ng.

The two main differences between biowafer and tube reactions are the temperature distribution of the reaction solution and the relative concentration of template and polymerase. When more polymerase is available, the level of incomplete NTA is reduced. For biochip reactions, the experimental data is shown to be in the range of 0.5-4.0 ng of DNA template. When the amount of SpeedSTAR polymerase is increased from 0.3 U to 1.2 U, the level of incomplete NTA is reduced by about 50% ( Figures 7A and 7B). ). The level of shadow bands for fast biochip and tube reactions is relatively constant and is typically less than 15% for all alleles in the template level range of 0.25-4.0 ng ( Figures 8A and 8B ).

The speed of the STR amplification reaction is only relevant if the reaction itself produces arbitrable data that meets the forensic interpretation criteria. The FBI has general guidelines for STR interpretation, and individual laboratories set thresholds that must be met before they can be considered acceptable based on their validation studies (Holt et al ., J. Forensic Sci. 2002, 47(1), p. 66-96; LaFountain et al ., J. Forensic Sci. , 2001 , 46 (5), 1191-8).

The conditions provided herein can produce a fast STR map that satisfies these criteria. The PHR of 0.5 ng of template in the biowafer and tube reaction meets the interpretation criteria of 0.6 or above and is consistent with previously reported results (Leclair et al , J. Forensic Sci. 2004, 49, 968-80). The amount of DNA template in terms of high, the PHR remains relatively constant, but less than their PHR 1ng TaqGold ^TM of the reaction. For low copy numbers, PHR is controlled by amplification due to random fluctuations.

The level of incomplete NTA is based on the ability of the polymerase to completely adeninate all STR amplicons. For conventional amplification, this is accompanied by a "pigtail" that is attached to the primer and increases the final extension time. The incomplete NTA levels of the 0.5 ng biochip and tube reactions described herein are within the interpretation criteria.

The level of incomplete NTA increases with increasing DNA template (result of increased ratio of DNA to polymerase) and can be reduced by increasing the amount of polymerase, the extension time per cycle, and the final expansion time. The latter two methods are not fully suitable for rapid multiplex amplification due to increased reaction time. Increasing the polymerase concentration is effective and compatible with rapid PCR. The shadow band is the result of DNA strand slip during expansion (Walsh et al , Nucleic Acids Res. 1996, 24(14), 2807-12). The level of shadow band described herein for the 0.5 ng biochip and tube reaction is within the interpretation criteria and also conforms to previously cited reports. As expected, the shadow band level appears to be independent of the DNA template level.

Example 4d

Reproducibility and reproducibility study

The repeatability of the reaction of the fast biochip (Table 3A) and tube (Table 3B) using SpeedSTAR polymerase was evaluated by performing 24 identical PCR reactions in 3 PCR biochips and performing 5 identical tube reactions. Reproducibility. For biochip reactions, the signal strength confidence value (CV) is in the range of 17% to 24% and in the range of 15% to 34% for the tube reaction. CV standard TaqGold ^TM reaction of between 6% and 21%.

With regard to the scope of the observed standard reaction TaqGold ^TM compared to 5-10%, PHR CV for the purposes of biochips for up to 14% and up to 21% in terms of the reaction tube. The CV line of incomplete NTA in the biowafer reaction varied between 6% and 28% and varied between 2% and 13% for tube reactions. Further, such variation range similar to the standard regarding the observed reaction TaqGold ^TM of 4-28%. CV biological wafer with the shadow of 4-9%, in the pipe is 2-6% Qieyi similar range about 4% to 13% of the observed response TaqGold ^TM standard.

Example 4e

Compatibility with other commercially available STR kits

Using the same conditions and pipe fast biochip, using primers from COfiler ^TM Identifiler ^TM kit and evaluate a series of samples of the group. Figures 9A and 9B show the use of such primer sets to achieve a complete map using the thermal cycler of the present invention, the SpeedSTAR enzyme, and the protocol described herein. Each is also suitable for such commercial packages. Despite the complete map, an imbalance in the signal intensity of the locus was observed.

Example 5

Quick sequence with thermal cycler

Thermal cycling instruments and methods can also be applied to rapid DNA sequencing reactions. In this embodiment of the rapid thermal cycler, the instrument and biochip are identical to the instruments and biochips used in PCR. Different reaction solutions, polymerases, and cycle temperatures are suitable for the sequencing reaction. The currently commercially available sequencing reaction took 49 minutes (for ^{GE Amersham DYEnamic TM ET Terminato Cycle Sequencing} Kit terms) and 2.25 hours (for the purposes of AB Big Dye V3.1). Using a NetBio thermal cycler, the sequencing reaction time can be reduced to 21 minutes with conventional reagents.

Fast sequencing has been achieved using the thermal cycler disclosed herein and a biochip containing 16 ribbons. The final reaction volume of the wafer was 7 μL. From GE Healthcare to the DYEnamic ^TM ET Terminator Cycle Sequencing Kit set half-strength sequencing reaction according to manufacturer & scheme. Therefore, all volumes were scaled down to accommodate 7 μL of final volume. The reaction is a template having a 343bp fragment size 0.1pmol of Bacillus subtilis (B.subtilus).

Prove three cycle schemes, the first scheme consists of 30 cycles (20s at 95°C, 15s at 50°C and 60s at 60°C) (total cycle time is 51.7min), and the second option consists of 30 Cycle (5 s at 95 ° C, 15 s at 50 ° C and 30 s at 60 ° C) (total cycle time 29 min) and the third option consists of 30 cycles (5 s at 95 ° C, 10 s at 50 ° C and Composition at 60 ° C for 20 s (total cycle time 21.6 min). Each sequencing reaction was cleared by ethanol precipitation and separated on a Genebench FX Series 100. The average PHRED scores for the sequencing 343 bp PCR products of the three cycling protocols were 282, 287, and 279, respectively; demonstrating that the sequencing of the 343 bp product can be achieved in the wafer in less than 22 min. Figure 10 shows the DNA sequence of a rapid DNA sequencing protocol.

In general, biofilm-based multiplex amplification of one or more nucleic acids using the systems and methods described herein has a significantly shorter response to reactions in thin-walled tubes and using current commercial thermal cycling units. The advantage of providing an amplified nucleic acid product over the total reaction time.

Claims (31)

A method for simultaneously amplifying a plurality of loci in a nucleic acid solution, comprising: a single solution contained in one or more reaction chambers provided in a biochip, comprising at least 10 and at most 250 to be amplified a sample of the target nucleic acid template with at least 10 and up to 250 different primer pairs, each primer pair hybridizing to one of the at least 10 and up to 250 loci to be amplified, the solution further comprising: (i One or more buffers; (ii) one or more salts; (iii) a nucleic acid polymerase; and (iv) nucleotides; and a temperature control element (TCE) comprising a heating and cooling member, at least one heat An inductor, a controller for receiving signals from the at least one thermal sensor, and a power source, the at least one thermal sensor being positioned and mounted to measure an effective temperature of each individual solution in the reaction chamber in the biochip, And providing feedback to the TCE to set or maintain the solution at a desired temperature; simultaneously amplifying the plurality of loci using the at least one thermal sensor, the controller, and the TCE, heating at 4 ° C - 150 ° C / sec And cooling rate, in turn The temperature of the reaction chamber in the nucleic acid solution of the denatured state, adhesive (annealing) heat cycle and the state of a predetermined number of cycles between the expanded state for 97 minutes or less to obtain a plurality of amplification of the loci in the reaction chamber. The method of claim 1, further comprising: maintaining the one or more reaction solutions in a final state to provide one or more amplified nucleic acid products. A method for simultaneously amplifying 5 or more loci in a nucleic acid solution, comprising: Providing a single solution contained in one or more reaction chambers in a biochip, having a sample of at least 5 target nucleic acid templates to be amplified and at least five different primer pairs, each pair of primers hybridizing to the one or more In one of at least five loci in the nucleic acid template, the solution further comprises: (i) one or more buffers; (ii) one or more salts; (iii) a nucleic acid polymerase; and (iv) a nucleoside And providing a TCE comprising a heating and cooling member, at least one thermal sensor, a controller for receiving signals from the at least one thermal sensor, and a power source, the at least one thermal sensor being positioned and mounted to measure the The effective temperature of each individual solution in the reaction chamber in the biochip, and provides feedback to the TCE to set or maintain the solution at the desired temperature; simultaneous amplification using the at least one thermal sensor, controller, and the TCE The plurality of loci sequentially heat the temperature of the nucleic acid solution in each reaction chamber to a predetermined number of cycles between a denatured state, a bonded state, and an expanded state at a heating and cooling rate of 4 ° C to 150 ° C /sec. And most Extending between the transition time, and the bonding state and the denatured state in 97 minutes or less to give more than 5 or amplification of the loci in the reaction chamber. The method of claim 3, wherein the nucleic acid solution is present in the biochip. The method of claim 3, wherein the nucleic acid solution is present in a thin-walled tube. The method of claim 3, wherein the 5 or more amplified loci are produced in less than about 97 minutes. The method of claim 3, wherein the amplified loci are produced in less than about 45 minutes. The method of claim 3, further comprising before the sequential thermal cycle: Heating the one or more reaction solutions to a first temperature suitable for thermal initiation activation of the nucleic acid polymerase; and maintaining the one or more reaction solutions at the first temperature for a period of time suitable for the nucleic acid polymerase The first period of time during which the thermal initiation is activated. The method of claim 8, wherein the first time period is less than 90 seconds. The method of claim 8, wherein the first temperature is from about 90 ° C to about 99 ° C. The method of claim 8, wherein the thermal cycle is provided by a thermal cycler comprising a thermal sensor and a temperature control element (TCE), whereby the thermal sensor provides feedback to the TCE to apply the solution Set or maintain at the desired temperature. The method of claim 7, wherein the nucleic acid polymerase has an expansion rate of at least 100 bp/second. The method of claim 7, wherein each reaction chamber has a volume of less than 100 μL. The method of claim 7, wherein each of the reaction chambers is separated from the thermal cycler by less than 200 μm. The method of claim 7, wherein each nucleic acid solution comprises from about 1 to about 1000 copies of a target nucleic acid. The method of the requested item 7, wherein the nucleic acid polymerase is ^{SpeedSTAR, PHUSION, Hot MasterTaq TM,} PHUSION Mpx, PyroStart, KOD, Z-Taq or CS3AC / LA. The method of claim 7, wherein the analysis of each amplified nucleic acid product satisfies a forensic interpretation criterion. The method of claim 7, wherein the denatured state is about 4 seconds at about 95 °C. The method of claim 7, wherein the bonding state is about 15 seconds at about 59 °C. The method of claim 7, wherein the expanded state is about 7 seconds at about 72 °C. The method of claim 7, wherein the final state is about 90 seconds at about 70 °C. The method of claim 7, wherein the one or more reaction solutions are at about 10 to about 50 ° C / sec. The first cooling rate of the bell is cooled from the denatured state to the bonded state. The method of claim 7, wherein the one or more reaction solutions are heated from the bonded state to the expanded state at a first heating rate of from about 10 to about 50 ° C/second. The method of claim 7, wherein the one or more reaction solutions are heated from the expanded state to the denatured state at a second heating rate of from about 10 to about 50 ° C/second. The method of claim 7, wherein the one or more amplified nucleic acid products are obtained in about 10 to about 90 minutes. The method of claim 7, wherein each of the reaction solutions comprises from about 0.005 to about 10 ng of the target nucleic acid. The method of claim 7, wherein the target nucleic acid comprises a human nucleic acid, a microbial nucleic acid or a viral nucleic acid. The method of claim 7, wherein 10 to 250 loci are simultaneously amplified. The method of claim 28, wherein the loci comprise amelogenin, D8S1179, D21S11, D7S820, CFS1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, FGA or A variety of them. The method of claim 7, wherein the predetermined number of cycles is between about 10 and about 50 cycles. The method of claim 7, wherein the one or more thin wall reaction tubes comprise the one or more reaction chambers.