WO2007002384A2 - Procedes de depistage par genotype d'une souche deposee par un porteur adsorbant - Google Patents

Procedes de depistage par genotype d'une souche deposee par un porteur adsorbant Download PDF

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Publication number
WO2007002384A2
WO2007002384A2 PCT/US2006/024461 US2006024461W WO2007002384A2 WO 2007002384 A2 WO2007002384 A2 WO 2007002384A2 US 2006024461 W US2006024461 W US 2006024461W WO 2007002384 A2 WO2007002384 A2 WO 2007002384A2
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WIPO (PCT)
Prior art keywords
well
samples
screening
remote user
container
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PCT/US2006/024461
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English (en)
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WO2007002384A3 (fr
Inventor
Timothy A. Hodge
Phillip E. Mark
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Transnetyx, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Priority claimed from US11/166,990 external-priority patent/US20050239125A1/en
Priority claimed from US11/170,693 external-priority patent/US20060014186A1/en
Application filed by Transnetyx, Inc. filed Critical Transnetyx, Inc.
Publication of WO2007002384A2 publication Critical patent/WO2007002384A2/fr
Publication of WO2007002384A3 publication Critical patent/WO2007002384A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B50/00—ICT programming tools or database systems specially adapted for bioinformatics
    • G16B50/30—Data warehousing; Computing architectures
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B50/00—ICT programming tools or database systems specially adapted for bioinformatics

Definitions

  • This invention relates to methods for genotype screening. More specifically, this invention relates to various methods to detect or screen for at least one designated genetic sequences in a plurality of biological samples, disposed on an adsorbent carrier.
  • Genomic modification resulting from mutations in the DNA of an organism can be transferred to the progeny if such mutations are present in the gametes of the organism, referred to as germ-line mutations.
  • These mutations may arise from genetic manipulation of the DNA using recombinant DNA technology or may be introduced by challenging the DNA by chemical or physical means.
  • DNA introduced via recombinant DNA technology can be derived from many sources, including but not limited to DNA from viruses, mycoplasm, bacteria, fungi, yeast, and chordates including mammals such as humans.
  • Random introduction of DNA into a cell can be achieved by technologies such as transfection (including electroporation, lipofection), injection (pronuclear injection, nuclear transplantation) or transduction (viral infection).
  • Random mutations point mutations, deletions, amplifications
  • LET linear energy transfer irradiation
  • Targeted addition, deletion or replacement of DNA in an organism is achieved via homologous recombination.
  • Inducible systems employ sequence-specific recombinases such as Cre-LoxP (US patent numbers 5,654,182 and 5,677,177) and FLP/FRT (US patent number 5,527,695).
  • Transgenic organisms are organisms that carry DNA sequences (be it genes or gene segments) derived from another or the same species, stably integrated randomly into their genome.
  • Transgenic mammals are generally created by microinjection of DNA into the pronucleus of fertilized eggs, a technique in which the number of DNA copies or the integration site of the DNA into the host genome is uncontrollable.
  • a transgenic line or strain refers to an organism that transmits the foreign DNA sequences to its offspring.
  • Genotype screening is used to determine if a genome possesses specific genetic sequences that exist endogenously or have been modified, mutated or genetically engineered. Genomic nucleic acid is screened for these modifications, mutations or endogenous conditions.
  • Genomic nucleic acid is challenging to work with because of its size.
  • the genomic nucleic acid includes both coding and noncoding regions. Therefore, the genomic nucleic acid contains exons and introns, promoter and gene regulation regions, telomeres, origins or replication and nonfunctional intergenic nucleic acid.
  • the genomic nucleic acid is a double stranded molecule which is methylated.
  • cDNA and PCR-amplicons differs in that the molecules are much smaller. Additionally, biochemical modification events, such as methylation, do not occur with the smaller molecules. Shena, M (2000) DNA Microarrays: A Practical Approach. Oxford University Press, New York, NY.
  • Genotype screening is currently done manually.
  • the present manual system is time-consuming and can provide variable results depending on the laboratory and even depending on skill of laboratory workers.
  • a researcher using Southern blot technology may require greater than a week to screen a tissue sample for a transgene or a targeted mutation.
  • PCR polymerase chain reaction
  • transgenic species are of vital importance.
  • An emerging technique in mouse mutant breeding is producing 'homozygous' transgenic conditions.
  • the transgene sequence integrates randomly into the host genome.
  • the number of transgene insertions also varies.
  • the preferred mechanism for getting both chromosomes to have the transgene(s) is by breeding two transgenic animals from the same strain together.
  • the present invention provides a unique solution to the above-described problems by providing a method for rapid genotype screening.
  • this invention provides a method to rapidly report screening results to a remote user from a screening laboratory for a plurality of biological samples disposed on an adsorbent carrier. Efficient screening of a plurality of biological samples can be achieved by placing the sample to be screened in a well of a microwell container. The biological samples in the microwell containers are lysed to release at least a portion of intact genomic nucleic acid and cellular debris.
  • a standard concentration of purified genomic nucleic acid is obtained by saturating the binding ability of the magnetic particles and by regulating the amount of genomic nucleic acid released.
  • the purified genomic nucleic acid are screened to obtain screening results.
  • the screening results are reported to a remote user. These screening results can include information on whether a designated genetic sequence is present in an organism and the zygosity of designated genetic sequences. Additionally, the zygosity of a transgene can be quantitatively determined and reported to a remote user.
  • FIG. 1 is an illustrative overview of the remote automated testing procedures of the present invention.
  • FIG. 2 is a block diagram of one embodiment of the system.
  • FIG. 3 is a block diagram of the ordering procedure.
  • FIG. 4 is a block diagram of account registration.
  • FIGS. 5-6 illustrate the survey of work and sample identification sections.
  • FIG. 7A is a block diagram of the laboratory process system.
  • FIG. 7B is a block diagram of the laboratory process system.
  • FIG. 7C is a block diagram of the laboratory process system.
  • FIG. 7D is a block diagram of the laboratory process system.
  • FIG. 8 is a block diagram of standard laboratory stations.
  • FIG.9 is a screen display illustrating a document on the transgenic screening laboratory 20 's web site relating to an outcome file.
  • FIG. 10 is a graphical representation of the results.
  • FIG. 11 is a graphical representation of signal magnitude.
  • FIG. 12 is a graphical representation of signal magnitude.
  • FIG. 13 is a graphical representation of signal magnitude.
  • FIGS. 14 and 15 illustrate a preferred device for performing the functions of a
  • FIG. 16 illustrates a preferred device for performing the functions of an
  • FIG. 17 illustrates a preferred device for drying samples.
  • FIG. 18 illustrates a preferred device for performing the functions of a
  • FIG. 19 illustrates a preferred device for performing the functions of a
  • FIG. 2OA shows a schematic diagram of two swab holders.
  • FIG. 2OB shows a cross-sectional view of a swab holder.
  • FIG. 21 shows a schematic diagram of a kit.
  • FIGS. 22-25 show a representative screening result for human data.
  • the present invention provides a method for high volume genotype screening.
  • This invention provides a method for rapid identification of an organism, whose genome possesses specific genetic sequences that exist endogenously or has been modified, mutated or genetically engineered. All patents, patent applications and articles discussed or referred to in this specification are hereby incorporated by reference. 1. Definitions:
  • Probe 2 TCTCTCTCCTAACTTC (SEQ ID NO. 13)
  • copy number the number of transgenes that have randomly integrated into the genome.
  • designated genetic sequence - includes a transgenic insert, a selectable marker, microsatellite loci, recombinant site or any gene or gene segment.
  • DNA deoxyribonucleic acid
  • nucleic acid consisting of a long, unbranched macromolecule formed from one, or more commonly, two, strands of linked deoxyribonucleotides, the 3"-phosphate group of each constituent deoxyribonucleotide being joined in 3', 5'-phosphodiester linkage to the 5'-hydroxyl group of the deoxyribose moiety of the next one.
  • embryonic stem cells a cell of the early embryo that can replicate indefinitely and which can differentiate into other cells; stem cells serve as a continuous source of new cells.
  • genomic nucleic acid includes both coding and noncoding regions. Therefore, the genomic nucleic acid contains exons and introns, promoter and gene regulation regions, telomeres, origins or replication and nonfunctional intergenic nucleic acid.
  • the genomic nucleic acid is a double stranded molecule which is methylated. cDNA and PCR-amplicons differs in that the molecules are much smaller. Additionally, biochemical modification events, such as methylation, do not occur with the smaller molecules. Shena, M (2000) DNA Microarrays: A Practical Approach. Oxford University Press, New York, NY.
  • genotype - genetic constitution of an individual cell or organism that can include at least one designated gene sequence.
  • heterozygosity the state of having two different genes (alleles) at one or more corresponding loci on homologous chromosomes.
  • homozygosity The state of having the same genes (alleles) at one or more corresponding homologous chromosomes.
  • internet a collection of interconnected (public and/or private) networks that are linked together by a set of standard protocols to form a global, distributed network.
  • the World Wide Web refers to both a distributed collection of interlinked, user viewable hypertext documents (commonly referred to as web pages) that are accessible via the Internet and the user and server software components which provide user access to such documents using standard Internet protocols.
  • line - A line is a group of organisms bred for a genotype (i.e. at least one designated genetic sequence).
  • mutation - a heritable change in DNA sequence resulting from mutagens.
  • plate controls - are wells that include the house-keeping probe without nucleic acid sample.
  • recombination The process by which offspring derive a combination of genes different from that of either parent. In higher organisms, this can occur by crossing over.
  • recombinant DNA A combination of DNA molecules of different origin that are joined using recombinant DNA technologies.
  • RNA - on of the two main types of nucleic acid consisting of a long, unbranched macromolecule formed from ribonucleotides, the 3 '-phosphate group of each constituent ribonucleotide (except the last) being joined in 3', 5' - phosphodiester linkage to the 5'-hydroxyl group on each ribose moiety renders these phosphodiester bonds susceptible to hydrolytic attack by alkali, in contrast to those of DNA.
  • the RNA chain has polarity, with one 5' end and on 3' end.
  • Two purines, adenine and guanine, and two pyrimidines, cytosine and uracil, are the major bases usually present.
  • RNA is fundamental to protein biosynthesis in all living cells. Oxford Dictionary of Biochemistry and Molecular Biology; p. 577.
  • screening reference - are probes that are run on every sample submitted to screen laboratory.
  • the probe is one that is found in every mouse, mutant or not.
  • strain - a group of organisms bred for a genotype (at least one designated genetic sequence).
  • strain controls - are biomatter samples submitted by a remote user 1. Strain controls are controls positive and negative sent to the screen laboratory as the remote user that discloses the genotype.
  • transgenic this term describes an organism that has had genes from an organism or additional elements of it our sequence put into its genome through recombinant DNA techniques. These organisms are usually made by microinjection of DNA in the pronucleus of fertilized eggs, with the DNA integrating at random.
  • transgenic line - a transgenic mouse or organism strain in which the transgene is stably integrated into the germline and therefore inherited in Mendelian fashion by succeeding generation.
  • web site - a computer system that serves informational content over a network using the standard protocol of the World Wide Web.
  • a web site corresponds to a particular Internet domain name such as TransnetYX.com.
  • wild type the phenotype that is characteristic of most of the members of a species occurring naturally and contrasting with the phenotype of a mutant.
  • zygosity This term reflect the genetic makeup of an individual. When identical alleles exist at a loci it is said to be homozygous; when alleles are different the alleles are said to be heterozygous.
  • the present invention provides methods for genotype screening. More specifically, the present application relates to a method to rapidly screen biological samples for at least one designated genetic sequence. Various aspects of genotype screening involve: sample collection, lysing of the biological sample, isolation of purified genomic nucleic acid and nucleic acid screening. Additionally, the method operating according to the features described herein can provide screening results to a remote user 1 from the screening laboratory 20 within 24 hours of receiving the biological samples.
  • the designated genetic sequence In order to screen for a designated genetic sequence, that sequence must first be determined or identified. Only when the designated sequence is known can a test be devised to search for its existence in the biological samples provided by the remote user 1 to the screening laboratory 20. [0084] There are a variety of ways the designated genetic sequence can be acquired by the remote user 1 or by the screening laboratory 20. For example, if the sequence of bases that makeup the designated genetic sequence is known by the remote user 1, the sequence can be directly communicated to the screening laboratory 20 via an electronic link, such as any of the electronic communication links identified herein, and particularly the communication links extending between the remote user's computer and the screening laboratory 20.
  • the remote user 1 can indirectly communicate the designated genetic sequence to the screening laboratory 20 by communicating a publication, journal article, a gene name, a sequence name, a line or strain name (if the designated genetic sequence is found in animals of that line or strain), or the name of a mutation having the designated genetic sequence to the screening laboratory 20.
  • the remote user 1 can communicate to the screening laboratory 20 the sequence of a primer set or probe that corresponds to a target genetic sequence of the designated genetic sequence. These primer sets or probes will have previously been created or defined to indicate the presence of the designated genetic sequence.
  • the indirect references may provide the entire sequence.
  • the screening laboratory 20 may take the information from the references or from the remote user 1 and use it to search public genetic databases such as The National Center for Biotechnology Information (NCBI), Ensembl, or The Wellcome Trust Sanger Institute database.
  • NCBI National Center for Biotechnology Information
  • Ensembl Ensembl
  • Wellcome Trust Sanger Institute database The screening laboratory 20 can also search proprietary databases, such as the database provided by Celera Bioscience (Rockville, MD).
  • Another indirect method that may be used to acquire or identify the designated genetic sequence is to use a third party who has specific knowledge of the sequence.
  • the screening laboratory 20 can receive the name of a transgenic animal line or strain from the remote user 1, then contact the company that engineers that line or strain. The company can then transmit the sequence of bases that constitute the particular genetic sequence corresponding to that line or strain back to the screening laboratory 20.
  • These companies include such firms as Lexicon Genetics (Woodland, TX) or Charles River Laboratories (Wilmington, Mass.). Even further, individual researchers who have developed the line or strain, or who work with the same line or strain at another laboratory may provide the designated genetic sequence, the primer sets or the probes necessary to identify the designated genetic sequence.
  • the screening laboratory 20 may use scientific methods. If the remote user 1 has a working genotyping assay, and they are performing PCR and separating fragments in a gel, the appropriate bands can be cut from the gel, purified and sequenced to determine the sequence of bases in that band. The company sequencing the bands can directly communicate the base sequence to the screening laboratory 20 or to the remote user 1, who in turn can communicate the base sequence to the screening laboratory 20.
  • the screening laboratory 20 must then select a target genetic sequence of the designated genetic sequence for which a primer set and/or probe can be constructed.
  • the sequence of the primer set and probe is determined using software such as Primer Express® (Applied Bio Systems).
  • the target genetic sequence may be directly selected from the designated genetic sequence by the screening laboratory 20.
  • the base sequence corresponding to the target genetic sequence is communicated to an oligonucleotide vendor, who manufactures the probe and primer sets and transmits them to the screening laboratory 20.
  • the screening laboratory 20 preferably keeps a supply of probes and primer sets on hand so each future request by the remote user need not require special production of probes and primer sets.
  • a special probe or primer set may be required.
  • the screening laboratory 20 may not select the target genetic sequence itself, but may communicate to a third party specific areas in the designated genetic sequence that are important for mutation detection.
  • the third party is typically an oligonucleotide vendor, who in turn will select the target genetic sequence, manufacture the probes and primer sets, and send the probes and primer sets to the screening laboratory 20.
  • the screening laboratory 20 requests that the remote user 1 provide both the base sequence of the designated genetic sequence of the mutation as well as the DNA sequence of the endogenous location. The endogenous DNA sequence is disrupted if a mutation has occurred.
  • two primer-probe sets are designed. The first primer-probe set determines if the sequence of the mutation is present, irrespective of the number of times it is present. The second primer-probe set determines if the endogenous DNA sequence is present. It is these two primer-probe sets that the oligonucleotide vendor designs and transmits to the screening laboratory 20.
  • a remote user 1 can contact the screening laboratory 20 and provide information for a human mutation or suspected endogenous condition of interest. This information may include the remote user's interest in wanting to know if the sample is from a human or a mouse and if it is from a human what gender is the sample.
  • the screening laboratory 20 can acquire primers and probe that can distinguish between humans and mice. This is accomplished by identifying areas of genetic sequence in the mouse genome that are not homologous with the genetic sequence in the Homo sapiens genome. With no input from the remote user 1, the screening laboratory 20 can query a database such as Ensembl that would discriminate between the sex chromosomes in humans (X and Y).
  • This query would yield sequence data for the Y chromosome, which is the designated genetic sequence.
  • the screening laboratory 20 can take the designated genetic sequence, or portion thereof, and send it to a vendor indicating where to build the primer set and probe as to be informative for screening. Moreover, where there are a large number of nucleotides that are unique on the human Y chromosome, the screening laboratory 20 may send the sequence of bases to the vendor and have them build primer sets and probe anywhere inside the sequence.
  • the remote user l's Internet web-based account will have a field populated that represents these reagents with an identifier such as the genetic line identification 84. The remote user 1 will use the identifier (strain name or profile name) to indicate that these specific reagents are to be used on subsequent samples.
  • a remote user 1 can contact the screening laboratory 20 and provide a literature reference of the mutation which discloses the mutation name.
  • a mutation name query of the Mouse Genome Informatics website yields links to different databases such as Ensenbl and National Center for Biotechnology Information that provides sequence data.
  • This sequence data is the designated genetic sequence. Knowing the endogenous nucleotide and the mutant nucleotide, the screening laboratory 20 can take the designated genetic sequence, or portion thereof, and send it to a vendor indicating specifically where to build the primers and probes as to be informative for screening.
  • the screening laboratory 20 may indicate to the reagent vendor to build a SNP assay targeting the 239 th nucleotide.
  • the reagent vendor will then supply to the screening laboratory 20, the primers and probes to specifically discriminate between a nucleotide change at the 239 th position of the designated genetic sequence.
  • the remote user 1 's Internet web-based account will have a field populated that represents these reagents with an identifier such as a name or number, or what is commonly referred to as the genetic line identification 84.
  • the remote user 1 will use the genetic line identification 84 to indicate that these specific reagents are to be used on subsequent samples.
  • the probes and primer sets if they are new and have not before been tested against a sample containing the designated genetic sequence, must then be tested, preferably by the screening laboratory 20.
  • the screening laboratory 20 preferably receives both a positive and a negative strain control samples from the remote user 1 and tests them against the probes and primer sets to confirm that they can be used successfully to determine whether the designated genetic sequence can be detected.
  • These controls include one positive and one negative control for each mutation found in the strain of interest.
  • the screening laboratory 20 updates the website and the order management software to provide the remote user 1 with a web-based selection for sample testing using those tested probes and primer sets. These selections among which the remote user 1 can select are one of the screening parameter selections identified below.
  • the screening laboratory 20 can immediately add a selection to the website and does not need to test controls with the probes and primer sets.
  • the strain controls are used to tell LIMS 24 a signal magnitude that is then associated with a positive or negative sample.
  • the remote user 1 may send these controls together with the samples to be tested to the screening laboratory 20 in a single shipment. Alternatively, the controls may be sent separately from the samples to be tested.
  • the screening laboratory 20 tests the strain controls using the process described herein for testing samples. At the end of this testing process, the signal values for the strain controls are recorded into LIMS 24.
  • the magnitude of the signal provided by the positive control indicates the expected signal level for subsequently tested samples having the designated genetic sequence.
  • the magnitude of the signal provided by the negative control indicating the expected signal level for subsequently tested samples that do not have the designate genetic sequence.
  • the computer at the screening laboratory 20 is configured to compare the test results (i.e. signal levels) for every sample that it subsequently tests for that designated genetic sequence with these multiple control signal levels and, based on that determination, to decide whether that sample has or does not have the designated genetic sequence. Positive and negative strain controls for a line therefore do not need to be resubmitted for each subsequent order but can be referenced by the screening laboratory 20 computer when later samples are tested for the same designated genetic sequence.
  • transgenic zygosity genotyping additional controls (not just a positive and a negative) are required to indicate each possible variation such as: a homozygous control, a heterozygous control and a wild type control.
  • the sample Upon receipt of the primers and probe from a vendor, the sample, if available, will be screened using these reagents. Once a determination is made that there is discrimination between different genetic conditions, then the reagents will be placed in the inventory. Additionally, the screening laboratory 20 will populate a data field on the order management system, allowing the remote user 1 to select this primer sets and probe combination(s) for subsequent samples. This data filed will be populated with an indicator such as a mutation name, strain name or genetic line identification that will represent these reagents or combination of reagents that will be used in subsequent samples of this strain. This allows the remote user 1 to select the indicator of the reagents and prevents the need to transfer genetic information with each order.
  • an indicator such as a mutation name, strain name or genetic line identification that will represent these reagents or combination of reagents that will be used in subsequent samples of this strain. This allows the remote user 1 to select the indicator of the reagents and prevents the need to transfer genetic information with each order.
  • FIGS. 1-3 present an overview of certain features of the present invention.
  • the present invention allows a remote user 1 with access to a computer 5 to order genotype screening of samples they submit to screening laboratory 20.
  • the remote user 1 uses the Internet or other communication link 7, the remote user 1 sends an access request from the remote user's computer 5 to a screening laboratory 20 computer 9 via an electronic communication link 7, such as the Internet.
  • the screening laboratory 20 website 19 will transmit an access enabling response to the remote user 1 via electronic communication link 7.
  • This response includes three distinct sections. The three sections are Account Registration 21, Survey ofWork 23 and Sample Identification and Designation 25 (FIG. 3).
  • a remote user 1 can access screening laboratory 20 website 19 via communication link 7.
  • the website 19 can be housed by an order manager 22.
  • An order manager is a software-based order management system.
  • the order manager 22 is an order management system developed by "Big Fish", a software development company in Memphis, TN.
  • the order manager 22 functions to manage the placement of the order.
  • the order received from the remote user 1 is transmitted to website 19, which reports the order to order manager 22.
  • Manager 22 is in electronic communication via link 7 with screening laboratory 20 computer 9.
  • Screening laboratory 20 computer 9 includes LIMS 24, which is communicatively coupled to a process controller 26.
  • LIMS 24 is the generic name for laboratory information management system software.
  • the function of LIMS 24 is to be a repository for data, to control automation of a laboratory, to track samples, to chart work flow, and to provide electronic data capture.
  • LIMS 24 can also, in another embodiment, be in direct communication with the remote user 1 via an electronic communications link 7.
  • Any standard laboratory information management system software can configured to be used to provide these functions.
  • a standard relational database management system such as Oracle (Oracle Corp., Redwood Shores, CA) or SQL Server (Microsoft Corp., Redmond, WA) either alone or in combination with a standard LIMS system can be used.
  • the Nautilus® program (Thermo LabSystems, a business of Thermo Electron Corporation, Beverly, MA) is used.
  • the process controller 26 is communicatively coupled to the workstation 14.
  • the process controller provides commands to any portions of the workstation 14 that are amenable to automation.
  • process controller 26 directs the delivery of the probes and primers to the Screening Station 95.
  • the workstation 14 is communicatively linked 28 to LIMS 24.
  • the workstation 14 can provide data to LIMS 24 for the formulation of the outcome report 249, and then, via link 7 to the order manager 22 or remote user 1.
  • remote user 1 at remote user computer 5 can be linked 7 to the screening laboratory 20 by a direct phone line, cable or satellite connection.
  • the user's Account Registration section 21 begins with logging into the system 30.
  • a remote user 1 accesses an existing account by entering an account identification 31, which is, for example, an e-mail address.
  • the user will then enter a password 37. If a valid password is entered, the user can place a new order 39. Alternatively, the user can check an order status 41 by providing an order number 43 and can proceed to order tracking 45. Alternatively, a new account 47 can be opened by providing an institution name, principal investigator, address, phone number, fax number, electronic mail address, billing information, and other authorized user names 49. The user can enter a password 51, confirm the password 53 and enter this billing information 55.
  • the remote user 1 will be presented with the Sample Identification and Designation section 25.
  • the user (among other things) identifies where he will place each sample to be tested in an actual (physical) container 2 (FIG. 1) by associating each sample with a corresponding well of a virtual 96 well container displayed on the computer screen of computer 5 as described below.
  • the Sample Identification and Designation section 25 includes 96 well container locations.
  • the remote user 1 designates which sample was or will be placed into each well. If the remote user 1 has more than 96 samples, subsequent 96 source well containers and designations are available. With respect to FIG. 6, a 96 well source well container 2 having a barcode accession number 3 (FIG. 1) will be shown (FIG.
  • FIGS. 5 and 6 together illustrate the Survey of Work section 23 and the
  • the remote user 1 is asked to provide: source well container 2 accession number 82, which the remote user 1 gets from the accession number 3 on the physical source well container 2 at his facility (FIG. 1) that he intends to fill (or has filled) with the samples, number of lines 83, genetic line identification 84, number of samples 85, and well location 88.
  • the remote user 1 is also asked for any internal sample identification number 91.
  • the positive strain control and the negative strain control samples are designated and deposited in wells of a microwell container.
  • the remote user 1 indicates that a sample is a control sample at 89. This assumes, of course, that the strain controls were not earner provided to tne screening laboratory 20 as described above. If a control is deposited in source well container 2, remote user 1 can also designate the zygosity, mosaic nature and copy number of the sample.
  • the remote user has completed the Survey of Work section 23 and the Sample Designation section 25 of FIGS. 5-6 and is ready to transmit the screening parameter selections gathered in those sections to website 19 and thence to screening laboratory 20 computer 9.
  • the remote user 1 transmits his or her order including the completed screening parameter selections to the screening laboratory 20 via link 7 such as the Internet or a direct line.
  • the remote user 1 can transmit the selected screening parameter selections to LIMS 24 in screening laboratory 20 via electronic communications link 7.
  • This link 7 can be direct or indirect.
  • the screening parameters are first transmitted to web site 19, wherein order manager 22 receives the order and then provides LIMS 24 with the screening parameter selections.
  • remote user 1 at computer 5 transmits a request for a home web page served by screening laboratory 20 web site 19 via the electronic communication link 7.
  • Web site 19 serves a home web page to computer 5 that includes information identifying the source of the web page and including a login button.
  • Remote user 1 at computer 5 clicks on the login button displayed on his computer screen, transmitting a signal to web site 19 requesting access to the web site.
  • This request is transmitted over communications link 7 to web site 19, which responds with a second web page having fields for the entry of an account identifier (in the preferred embodiment an e-mail address), and a password.
  • an account identifier in the preferred embodiment an e-mail address
  • Remote user 1 enters the remote user 1 e-mail address and password, and transmits this information to web site 19 to gain access to the web site.
  • Web site 19 receives this access request and compares the account identifier and password against its database of pre-existing accounts in the order manager 22 to determine whether the user is permitted to access the web site 19. If so, computer order manager 22 serves up a further web page, called an order manager web page, which includes several user selectable choices including an "order status" button for tracking previous orders and results (if any have been received), a "supply request” button for requesting supplies, and an "order” button for ordering additional tests. [00115] To order genetic testing, user 1 clicks on the "order” button displayed on the screen of computer 5. Computer 5 transmits the user 1 request to web site 19.
  • Web site 19 receives this request, and transmits a first ordering web page to computer 5.
  • the first of these widgets is list box including two selectable entries for requesting the speed of service. In the preferred embodiment there are two speeds of service: 24-hour service and 72 hour service.
  • the second of these widgets is a list box providing several entries, each entry in the box corresponding to a strain for which the sample is to be tested.
  • the third widget is a text box for entering the number of samples of the selected strain to be tested.
  • the fourth widget is a text box for entering the accession number (typically a bar code number) of the source well container 2 in which the samples are to be placed for shipping to the screening laboratory 20.
  • the remote user 1 types in the number of samples to be tested.
  • the samples are taken from transgenic animals, each sample typically corresponding to one animal to be tested. Typically several animals are tested to determine if they received the transgenic gene from their parents.
  • Each strain of animal is defined by one or more designated genetic sequence.
  • the remote user 1 selects the one or more designated genetic sequences associated with that sequence.
  • the remote user 1 can also select or deselect each individual probe and primer set that is used to screen for the designated sequences in the strain or line of the biological sample.
  • the remote user 1 Once the remote user 1 has entered the number of samples to be tested, he or she then enters the name of the strain that the samples are to be tested for. Again, by selecting a strain the remote user 1 indicates the designated genetic sequence for which the samples are to be tested, since each strain is bred to have that sequence.
  • Samples of the same color from the same group are grouped together thus producing several different contiguous groups of wells, each group of wells have the same color different from the color of the adjacent groups.
  • each well contains a sample, such as a tissue sample, taken from an individual animal.
  • the purpose of the testing performed on the samples in the wells is to determine the genetic characteristics of the animal from which each sample was taken.
  • the user In order to relate the test results performed on each sample back to the animal from which the sample was taken, the user must make a record of the animal source of each sample (i.e. the animal from which each sample was taken).
  • remote user 1 selects a button on the third ordering web page.
  • This button signals computer 9 to generate an additional web page.
  • This web page lists each well in the well plate that was previously identified as containing a sample. Thus, if the first group of samples were 13 in number, there would be 13 entries listed in the additional web page.
  • the web page itself is arranged as a single column of entries. Each entry in the column of entries includes a well identifier (called well location 88, above), which is a string of alphanumeric characters that uniquely identifies one well of source well container 2.
  • a preferred well identifier for the 96 well plate is an alphabetic character followed by a numeric character.
  • a text box is adjacent to each well identifier on the additional web page.
  • the user enters alphanumeric characters in the text box that are uniquely associated with each sample.
  • This identifier is typically a short string of consecutive alphabet or numeric characters, a practice commonly used by research facilities to identify individual animals used for testing.
  • Animals in a particular group of animals having (presumed) common genetic characteristics will typically be identified by tattoos, tags, or other permanent means by consecutive or sequential numbers, characters, or combinations of numbers and characters (for example “Al”, “A2", “A3”, or “101", “102", 103", or “AA”, AB”, “AC”, etc.).
  • user 1 enters each animal number into the text box as a sample ID 91.
  • Animals may also be identified by a unique combination of disfigurements such as cutting or cropping toes, tails or ears that can also be approximated to a progressive alphanumeric sequence.
  • a button is provided to automatically fill several consecutive text boxes based upon the alphanumeric characters typed into a few text boxes from the group. For example, if the user types in "B7" in the first text box of a group, then types in "B8" in the second text box of a group, computer 5 is configured to automatically generate consecutive alphanumeric strings to fill the remaining text boxes of the group based upon these two manually typed-in entries. In this case, computer 5 would automatically generate the alphanumeric strings "B9", “BlO”, "BIl”, etc. and insert these characters sequentially into the remaining text boxes of the group in the additional web page.
  • the computer can be configured to automatically generate alphanumeric characters for all the groups at once and to fill the text boxes of all the groups all at once.
  • the user Once the user has finished identifying all of the groups of samples and filling out all of the sample ID's 91 in the text boxes on the screen of computer 5, he clicks on a button labeled "next".
  • Computer 5 transmits this request to website 19, which responsively generates another web page in which the user 1 enters shipping and tracking information.
  • This page, called the order confirmation page includes a text box for entering a character string. This character string provides access to a web-based shipment tracking system of a commercial shipping company.
  • the character string is a tracking number used by the shipping company to track the samples from the remote user 1 to the screening laboratory 20.
  • the tracking number is provided to the user together with the source well container 2 and the packaging materials in which the user places the source well container 2 for shipment to the screening lab 20.
  • the order confirmation page also includes an invoice that lists the different tests requested by the operator in the foregoing steps on the screen of computer 5. Each test or group of tests is displayed on the screen adjacent to the price or prices for those tests. A total price of all the tests is displayed as well.
  • the order confirmation page has a second text box in which the remote user 1 can type the expected shipping date.
  • the expected shipping date is the date on which remote user 1 intends to give the samples in their packaging materials to the delivery service associated with the tracking number.
  • the order once the order has been transmitted to the order manager 22, the order generates two electronic messages, which will be sent to different locations.
  • the first message is cross-referenced in LIMS 24 with a list of stocked probes. If the probe designated by the user is not stocked, an order message is sent to a supplier 11, such as a contracted probe provider.
  • This request can be transmitted from remote user 1 to screening laboratory 20 via any form of electronic communication, and then via a form of electronic communication 10 to suppliers' computer 8, or in the alternative, the order message can go from user 1 via any form of electronic communication link 12 to suppliers' computer 8.
  • the supplier 11 creates the primer sets and probe based on the designated genetic sequence designated by the remote user 1 or the screening laboratory 20.
  • the made to order probe can be referred to as the target-binding probe.
  • This supplier 11 will then barcode and overnight ship 13 the primer sets and target-binding probes 17 to the screening laboratory 20.
  • the barcodes on the primer sets and target-binding probes are scanned into LIMS 24.
  • the LIMS 24 records the date and time the primers and target-binding probes were received along with the quality control data provided from the probe provider.
  • the primer sets and target-binding probes are placed in workstation 14 and LIMS 24 will record the barcode of the probe and record its specific location on the deck of the workstation 14, as will be discussed in more detail with respect to the Screening Station 95. Additionally, the screening laboratory 20 and the LIMS 24 system correlates which target-binding probes will be used on which samples, as will be discussed in more detail with regard to the Screening Station 95.
  • the second message in the preferred embodiment, that is generated from the order placement of the remote user 1 insures that the remote user 1 has the proper supplies to package and ship their samples.
  • This message sent via link 12, will define the barcode number of well container(s), shipping labels tracking number and amount of reagents needed for the user.
  • supplier 11 will package 18 supplies for remote user 1 and ship 14A the supplies back to remote user 1.
  • the remote user 1 procures or receives these supplies, the remote user 1 places the appropriate samples into the source well containers 2 previously identified in the order sent to website 19, order manager 22 and LIMS 24. In other words, the remote user 1 fills each well of source well container 2 such that each well contains the same sample with the same sample ID 91 that the user previously identified in the order previously sent to website 19. Alternatively, if the user already had sufficient supplies when the user placed the order the user need not wait for a source well container 2 to be sent by a supplier, but can fill the source well container 2 when the user creates the order, or even before the order is created. What is important is that the contents of the actual 96 source well container 2 that the user fills exactly matches the description of the samples and has the same accession number as the order the user previously sent to website 19.
  • the samples can be obtained from prokaryotic or eukaryotic organisms.
  • the samples may be a tissue, cells or biological fluid such as blood, lymph or semen sample from a mouse 8 A, but can also come from other animals (including humans), plants and viruses.
  • mouse oral cavity swabs or anal cavity swabs provide a sample.
  • Source well container 2 is a 96 well plate or the like that receives the sample in each well of the well plate.
  • a sufficient amount of lysis reagent can be added to cover the sample.
  • the lysis reagent is added prior to transit to the screening laboratory 20.
  • the lysis reagent is added at the screening laboratory 20 at Lysing Station 92.
  • a biological sample can be collected in a variety of ways to facilitate rapid screening.
  • the collection method involves swabbing the oral, nasal or anal cavity of an animal to be tested, such as a mouse, to collect cells for screening.
  • swab tips are removed by the remote user 1 and placed in individual wells of a multi-well container for transport to the screening laboratory 20.
  • Many different swab materials may be used including polyester, cotton, acrylamide, nylon and calcium alginate.
  • Microbrush® (Graftin, WI) swabs are used.
  • a multi-well container as shown in FIG.
  • the remote user 1 operates computer 5 to enter a variety of data regarding the samples placed in the source well container. Once all of the samples in all of the wells have been identified in this manner, the remote user sends the source well container 2 containing a plurality of biological samples to a screening laboratory 20 for screening.
  • FIG. 2OA and 2OB an apparatus to swab the subject and to facilitate placement of the swab into a source well container 2 is disclosed.
  • a swab holder 300 with disposable swab 301 is shown.
  • the swab 301 has a proximal and a distal end with respect to a swab holder 300.
  • the distal end of the swab 301 is made of a sufficient amount of flocking to collect a biological sample.
  • the proximal end of the swab 301 has at least one annulus 305.
  • the function of the at least one annulus 305 is to secure the swab 301 to the swab holder 300 during swabbing of a subject.
  • the swab holder 300 preferably includes an elastomeric, rigid plastic grip area, metal or the like on outer surface with metal, metallized plastic or the like main body.
  • the body of the swab holder 300 is linear with respect to the swab 301 to facilitate collection of biomatter.
  • a spring loaded plunger 306 has a release button 307 on opposite end from swab 301. The action is like that of a retractable ball point pen but without the latch function.
  • the swab holder 300 has an internal section configured to retain at least one annulus of a swab 301.
  • the internal section 304 is deformable.
  • This section can be elastomeric, serving as a swab grip, which receives and holds disposable swab 301 until released by the spring plunger 306.
  • the mounting end of the swab tip has at least one annulus 305 which, upon insertion into the swab grip, deforms or squeezes into the elastomer sufficiently to retain the swab 301 during its function.
  • three annuli are shown in the FIG 2OA, it would be possible for one elongated annulus to serve the purpose.
  • the swabs 301 are composed of a plastic material that measures approximately 1 inch long with a diameter of approximately 0.050 inches.
  • the distal portion of the swab 301 is flocked with nylon fibers.
  • the proximal end of the swab 301 shaft is designed to fit into the swab holder 300.
  • the swab 301 is seated in the swab holder 300 the remaining portion of the swab 301 shaft and flocking are inserted into an orifice of a subject to collect biomatter.
  • the swab 301 and/or swab holder 300 may be rotated to facilitate the collection of biomatter.
  • a mechanism 307 is depressed on the swab holder 300, such as a button that ejects the swab 301 from the distal end of the swab holder 300.
  • the ejector mechanism is then loaded with a new swab 301 and the process is repeated as many times as necessary.
  • the biological sample is a blood sample collected by nicking the animal to be tested and blotting the blood on a filter paper.
  • the blotted filter paper is placed in individual wells of source well container 2 by the remote user 1 and transported to the screening laboratory 20.
  • the biological sample is disposed on an absorbent carrier.
  • the swab holder apparatus 300, swab 301 and a source well container 2 can be packaged in a kit 310 and sent to a remote user 1.
  • the kit 310 does not need to be sterilized.
  • source well container 2 has an accession number 3 affixed to the side of the container.
  • the accession number is used by LIMS 24 to track the source of source well container 2.
  • the remote user 1 places the appropriate samples into the well locations in source well container 2 that they had previously designated while placing their order in FIG. 6.
  • the remote user 1 will add lysis reagent 4 to each well of the source well container 2.
  • the lysis reagent 4 should completely cover the samples.
  • the remote user 1 places a seal on the top of the source well container 2 preventing samples from leaking.
  • the remote user 1 then places a plastic lid on the seal for transportation.
  • the remote user 1 places the source well container 2 into an overnight delivery service package and shipped frozen 15.
  • the remote user 1 will then seal the package and ship 16 to screening laboratory 20, and apply a barcode shipping label.
  • FIG. 7A-D the preferred embodiment of the present invention is shown.
  • the source well containers 2 arrive 101 at the screening laboratory 20.
  • the tracking number of the shipping label is read with a barcode reader 103. If the shipping label is unreadable 105, the tracking numbers are manually entered 107.
  • the scanning of the tracking number is received 104 in LIMS 24 and a received message is posted to the user's account as shown in tracking field.
  • the source well container 2 are removed from the package and taken to a clean room 109.
  • the source well containers 2 contain the raw biological matter and in one embodiment lysis reagent.
  • the source well containers 2 individual barcodes are scanned by the barcode reader 111 and recorded 106 in LIMS 24 as accession numbers.
  • LIMS 24 can send 106 a probe order to supplier 11 through the order manager 22. If the source well containers 2 individual barcodes are unable to be scanned 113, the accession numbers are entered manually 115. If the tracking number, accession number, user order and worklist properly correlate, LIMS 24 will activate (not shown) an active record number for the containers.
  • the source well containers 2 are loaded 116 into a transportation apparatus in a clean room.
  • a transportation apparatus is any device that holds well containers and that can dock with the workstation.
  • the transportation apparatus in the preferred embodiment, includes several rigid trays stacked vertically in a housing unit that is mobile. This transportation apparatus can be moved between different automated stations, docked and the rigid trays can be removed in an automated fashion and processed on the deck of a workstation.
  • Each rigid tray consists of nine locations for source well containers 2. Each of these nine locations per tray has a unique barcode designating its specific location inside the trays of the transportation module.
  • Source well container 2 accession number 3 is scanned with a barcode reader and the bar-coded source well container 2 location in the transportation apparatus trays is scanned.
  • the barcodes of source well containers 2 are married 117 in LIMS 24 with the unique barcode locations in the transportation apparatus trays for tracking purposes. LIMS 24 records and associates each well container to this location.
  • LIMS 24 will generate a worksheet for laboratory personnel (not shown).
  • the worksheet outlines the probes and primer sets that the operator will need to prepare or gather in order to test the latest samples.
  • the LIMS 24 worklist will generate a single file.
  • the file format may include, but is not limited to, ASCII, XML or HTML.
  • the file will be written into a specified directory on the network drive.
  • the name of the file will be unique and will correlate to a run number.
  • the extension will be unique for worklist files.
  • a transportation apparatus includes a housing unit provided to support several trays, each tray having nine different locations for nine source well containers 2.
  • the housing unit can be eliminated.
  • the source well containers 2 can be manually transported throughout the workstation in trays from functional station to functional station. In this system, operator at the laboratory loads source well containers into the trays after the source well containers 2 are received at the screening laboratory 20 and are scanned into LIMS 24 as described above for transportation to workstation 14.
  • source well containers 2 can be transported individually to workstation 14 and be placed in a tray or trays that are already located at workstation 14.
  • FIG. 8 depicts one embodiment of the workstation 14.
  • Standard laboratory stations are logical groupings of laboratory operations. These groupings, however, do not necessarily refer to different physical stations. These logical groupings include: Lysing Station 92, Automated Accessioning Station 93, Isolation/Purification Station 94, Screening Station 95 and Detection Station 96, all of whom make up the workstation 14.
  • the Screening Station 95 can include other screening processes such as PCR.
  • Lysing Station 92 is an alternative step provided to lyse the samples in containers 2 in the event user 1 does not choose to lyse the samples by adding a lysis reagent before sending them to laboratory 20. The functions of the various logical stations are described below in connection with the steps shown in FIGS. 7A-D.
  • remote user 1 need not add a lysis reagent to the samples before shipping them to screening laboratory 20. Instead, the samples may be shipped un- lysed (frozen) and may be lysed at laboratory 20 by piercing the cover 121 of the container 2 and treating each of the samples with a lysis reagent after docking the tray in the workstation 119 in the lysing station 92. The samples are incubated 123 to produce a lysate containing cellular debris including at least a portion of intact genomic nucleic acid.
  • the preferred embodiment is to have the swabs shipped without lysis solution.
  • a sufficient amount of a lysis reagent such as SV Lysis reagent or Nucleic Lysing Solution (Promega Corporation, Madison, Wisconsin) is added to each well of source well containers 2 to cover the swab tips at the screening laboratory. Swabs do not need to be incubated for three hours, however they are voretexed for ten minutes in the lysis solution.
  • a sufficient amount of a lysis reagent such as Nuclei Lysing Solution (Promega Corporation, Madison, Wisconsin) is added to each well of source well containers 2 to cover the filter paper after shipment.
  • Nuclei Lysing Solution Promega Corporation, Madison, WI
  • the source well container 2 is treated under conditions to facilitate rapid lysis of the biological sample. In the preferred embodiment, these conditions are heating at 55°C for three hours.
  • Lysing Station 92 includes loading source well containers 2 into the tray 9206 and taking the rigid tray to Lysing Station 92 to be lysed.
  • Lysing Station 92 includes a liquid handler 9220, such as Genesis Tecan (Raleigh Durham, North Carolina) or Multimeck Beckman (Indianapolis, Indiana).
  • An example of a preferred Lysing Station 92 is shown in FIG. 14. It includes a frame 9202, on which a deck 9204 is mounted to provide a horizontal working surface, which supports tray 9206, which supports and positions up to nine source well containers 2.
  • a material handler 9214 is fixed to frame 9202 and extends upward and across the top surface of deck 9204.
  • a computer 9208 is coupled to material handler 9206 to direct the movement and operation of pipettes 9210.
  • a trough or reservoir 9212 is provided on deck 9204, from which computer 9208 commands the material handler 9214 to aspirate lysis reagent into pipettes 9210 and to deposit the reagent into wells of container 2.
  • computer 9208 commands the material handler 9214 to aspirate lysis reagent into pipettes 9210 and to deposit the reagent into wells of container 2.
  • [u ⁇ iszj me operator tirst carries a plurality of source well containers 2 and places them on deck 9204 in one of the nine positions on the rigid tray 9206 that support and orient source well containers 2 thereby docking them 119 into the workstation 14. The operator then enters the number of wells that are filled with samples in each of the source well containers 2 into computer 9208 in combination with the location of that container with respect to tray 9206.
  • computer 9208 then directs material handler 9214 to move the pipettes 9210 to each source well container 2 in turn, piercing 121 the barrier sealing mechanism and filling each of the wells of source well containers 2 containing a sample with lysis reagent.
  • material handler 9214 By providing the location and the number of samples, computer 9208 is configured to fill only the wells containing samples with lysis reagent and to leave the empty wells empty of lysis reagent.
  • the operator moves the entire tray or trays 9206 containing the samples to an oven 9216 (FIG. 15), where the samples are incubated 123 by heating for a period of about three hours at a temperature of 55° C (described above). Once the incubation process is complete, the operator moves source well containers 2 supported on the tray or trays 9206 to Automated Accessioning Station 93.
  • An Automated Accessioning Station 93 provides a device to remove liquid from the source well container 2 to the primary master well container 6.
  • the primary master well container 6 is the container in which the nucleic acid is isolated. It is preferably a 384 well plate (Fisher Scientific #NC9134044). Any commercially available automated accessioning device can perform this function such as Genesis® Tecan (Raleigh-Durham, NC) or Multimeck® Beckman (Indianapolis, IN). These devices are referred to as liquid handlers.
  • the source well containers 2 barcode accession numbers 3 are re-scanned 127. This measurement will be recorded and posted 108 into the LIMS 24 database and reflected in the outcome report 249.
  • LIMS 24 ensures 108 that source well containers 2 are consistent from transportation apparatus to the Automated Accessioning Station 93. Error codes will be generated if a sufficient amount of raw testing material is not available.
  • the liquid handler utilizes stainless steel, or the like, pipette tips that are washed between each sample transfer. Alternatively, disposable pipette tips may be used.
  • the nucleic acid lysate is transferred 129 to clean well containers, called primary master well containers 6.
  • Each of the containers 6 has a scannable accession number, preferably a barcode accession number, called "barcodes" or "accession numbers” below.
  • the barcodes of the primary master well containers 6 are scanned 131 and LIMS 24 marries 102 the barcodes for the primary master well containers 6 to the scanned barcode accession numbers 3 of the source well plates 2.
  • the automated process accessioning continues until all of the day's pending samples are accessioned into the primary master well containers 6.
  • the preferred method of performing the above steps at Accessioning Station 93 includes taking the rigid tray 9206 and the source well containers 2 from the incubating oven 9216 back to the same liquid handler 9220 that performs the functions of Lysing Station 92. This liquid handler 9220 is also preferably configured to function as Accessioning Station 93.
  • tray 9206 places tray 9206 back on deck 9204 generally in the same location it was in when the lysis reagent was inserted into each well containing a sample.
  • the operator commands computer 9208 to fetch the work list from LIMS 24 and electronically stores it in the computer memory of process controller 26.
  • the work list includes the accession numbers of each source well container 2 that is in tray 9206, together with the probe type that should be used for each well.
  • the work list uniquely associates the location of the well, the accession number of source well container 2 from which the well is from, the probe type that is to be used with the sample in that source well container 2, and the quantity of probe to be added to that sample.
  • computer 9208 fetches the work list, computer 9208 directs the operator to electronically scan 127 the accession numbers of all the source well containers 2 that are in rigid tray 9206 on deck 9204 of liquid handler 9220 using scanning device 9218 coupled to computer 9208.
  • Scanning device 9218 is preferably a glyph scanner, character scanner, bar code scanner, dot matrix scanner, or RFID tag scanner, depending upon the form of the accession identifier (typically a barcode accession number 3) on source well container 2.
  • accession identifier typically a barcode accession number 3
  • Process controller 26 preferably includes an instrument database to which each of the computers of Lysing Station 92, Automated Accessioning Station 93, Isolation/Purification Station 94, Screening Station 95 and Detection Station 96 transmit their data in order to maintain an ongoing record of the testing process and the location of materials and samples throughout that process.
  • the database is preferably implemented using Microsoft's SQL Server, although any relational database (e.g. Oracle), may be used.
  • Computer 9208 then commands material handler 9206 to transfer 129 the contents of each well (i.e. lysate) in source well containers 2 to a corresponding well in the primary master well container 6 using pipettes 9210.
  • Computer 9208 directs the operator to scan 131 the accession numbers on the primary master well container 6.
  • the accession number on the primary master well container 6 may be any electronically scannable indicia or device.
  • Computer 9208 transmits the accession numbers to process controller 26, which sends them to LIMS 24. In this manner, LIMS 24 maintains a record of each sample and its location in each source well container 2 and in each primary master well container 6.
  • LIMS 24 and process controller 26 correlate the accession number of each primary master well container 6 with the identity of each sample it contains, the strain for which each sample is to be tested, the designated genetic sequence or sequences that identify or indicate that strain, the probes and primer sets necessary to test for those designated genetic sequences and the results of the testing.
  • the tray of primary master well containers is moved by the transportation apparatus to the Isolation/Purification Station 94.
  • the genomic nucleic acid will be isolated and purified using a separation method such as magnetic or paramagnetic particles.
  • Purified genomic nucleic acid, substantially free of protein or chemical contamination is obtained by adding a sufficient amount of magnetic particles to each of the well containers that bind to a predefined quantity of nucleic acid.
  • the term "magnetic" in the present specification means both magnetic and paramagnetic.
  • the magnetic particles can range from 0.1 micron in mean diameter to 100 microns in mean diameter.
  • the magnetic particles can be functionalized as shown by Hawkins, U.S. Patent No. 5,705,628 at col. 3 (hereinafter '628 patent hereby incorporated by reference).
  • the magnetic particles are purchased from
  • a measured amount of magnetically responsive particles are added 133 to the lysate mixture with or without the presence of a chaotropic salt 135.
  • 13 ⁇ l amounts of 1 micron silica magnetic particles with chaotrope 113 ⁇ l are added to each well of the microwell container.
  • the fixed volume of particles becomes saturated with nucleic acid if there is enough nucleic acid in the lysate. It has been observed that the resulting nucleic acid concentration between samples is very consistent if there is an excess nucleic acid is present in the lysate.
  • a standard A 260 is 0.2 OD units.
  • a standard concentration range of 0.1 to 0.3 O.D. units is disassociated from the magnetic particles to yield purified genomic nucleic acid.
  • Table 1 shows that with increasing amounts of magnetic particles, the nucleic acid concentration also increases.
  • nucleic acid concentration is consistent between samples treated with the same protocol, several factors may increase or decrease the resulting standard concentration of genomic nucleic acid. These factors include: the starting amount of nucleic acid in each lysate preparation, the binding reagent, the number of purification washes, and the solution that is used to elute the nucleic acid.
  • the preferred binding solution for the magnetic particles obtained from Promega (Madison, WI) is a chaotropic salt, such as guadinium isothiocyanate.
  • binding reagents such as 20% polyethylene glycol (PEG) 8000, 0.02% sodium azide and 2.5M sodium chloride may be used to nonspecifically bind the genomic nucleic acid to the surface chemistry of the functionalized magnetic particles.
  • the preferred binding solution is PEG.
  • the PEG or chaotropic guadinium isothiocyanate allows for the disruption of hydrogen binding of water, which causes binding of the nucleic acid to the particles.
  • the preferred washing procedure to remove contaminants includes two chaotrope washes, after the initial chaotrope binding step, followed by four 95% ethanol washes.
  • Aqueous solutions, or the like, are the best elution solutions. These solutions include water, saline sodium citrate (SSC) and Tris Borate EDTA (ie. IxTBE).
  • the amount of DNA isolated from the swabs and blood is less than the DNA yield recovered from tissue.
  • the tissue lysate has enough DNA content to saturate the binding ability of the fixed volume of beads.
  • the swab and blood lysate does not have enough DNA to saturate the binding ability of the fixed amount of beads.
  • the housekeeping (cjun) CT values for tissue isolations are approximately 26 whereas the approximate CT for housekeeping (cjun) for the blood isolations are approximately 35.
  • This nine cycle difference represents approximately a 512 (2 A 9) fold difference in the amount DNA present.
  • This non- saturated DNA yield does not present a problem for results because the housekeeping probe normalizes the results.
  • CT Cjun For each sample, the CT values for the wells containing the housekeeping probe, cjun, are averaged (CT Cjun ).
  • RCN RCN 2
  • the RCN values are calculated by comparing the test probe (i.e. Cre or MNlTEL) signal to the housekeeping gene signal average for each of the two test probe wells (CT 1 and CT 2 ), the following equation is applied:
  • Isolation/Purification Station 94 is a liquid handler 9402 identical in general construction to the liquid handler 9220 identified above for use as the Lysing Station 92 and the Accessioning Station 93 that has been configured to automatically transfer the various reagents and other liquids as well as the magnetic particles in the manner described below.
  • FIG. 16 illustrates a preferred embodiment of the liquid handler 9402.
  • Handler 9402 comprises a frame 9404 on which is mounted a deck 9406, which is surmounted by material handler 9408, which supports and positions pipettes 9410 and is coupled to and controlled by computer 9412, which is in turn coupled to process controller 26 to communicate information to and from LIMS 24.
  • Liquid handler 9402 includes a syringe pump 9414 that is coupled to and driven by computer 9412 to dispense magnetic particles via a 16x24 array of 384 pipettes 9410 simultaneously into all 384 wells of the primary master well container 6 under the command of computer 9412.
  • Liquid handler 9402 also includes a second syringe pump 9416 that is configured to dispense a binding buffer into wells of the primary master well container 6 under computer control.
  • the liquid handler also includes a magnet 9418 mounted in deck 9406 as well as a conveyor 9420 that is coupled to and controlled by computer 9412 to move the primary master well container 6 in tray 9206 back and forth between a first position 9422 in which the container is within the magnetic field and a second position 9424 in which the container is outside the magnetic field.
  • the operator Before the functions of the Isolation and Purification Station 94 can be performed, the operator must first move the primary master well container 6 from Accessioning Station 93 to deck 9406 of liquid handler 9402 and place it in a predetermined location on the deck. Once the operator has placed the primary master well container 6, the operator starts an isolation/purification program running on computer 9412. This program drives the operations of liquid handler 9402 causing it to dispense magnetic particles 133 into all the wells of the primary master well container 6 containing lysed samples. Computer 9412 signals syringe pump 9414 to dispense the particles using pipettes 9410 into the primary master well container 6 when container 6 is in position 9424, away from the magnetic field created by magnet 9418.
  • a chaotropic salt such as guadinium isothiocyanate
  • computer 9412 then mixes the contents of the wells by signaling the pipettes 9410 to alternately aspirate and redispense the material in each of the wells. This aspiration/redispensing process is preferably repeated three or four times to mix the contents in each well.
  • computer 9412 pauses for two minutes to permit the particles, binding reagent, and raw biological material in the wells to incubate at room temperature in position 9424.
  • computer 9412 commands the conveyor 9420 to move tray 9206 from position 9424 to position 9422, directly above magnet 9418 at 137. In this position the magnet draws the magnetic particles in each of the wells downward to the bottom of the wells of the primary master well container 6.
  • Computer 9412 keeps tray 9206 and the primary master well container 6 over the magnet and within the magnetic field for 2-6 minutes, or until substantially all the magnetic particles are drawn to the bottom of each well and form a small pellet.
  • computer 9412 signals the conveyor to move the primary master well container 6 on tray 9206 to the nonmagnetic position 9424.
  • the foregoing process of adding chaotropic salt, mixing the combination, pausing, drawing the magnetic particles down and aspirating the supernatant is repeated two more times.

Abstract

L'invention concerne un procédé de dépistage rapide de génotype dans une pluralité d'échantillons biologiques déposés sur un porteur adsorbant dans un récipient à microcupules à des fins d'utilisation à distance par un laboratoire de dépistage. L'invention concerne également un kit de génotypage comprenant au moins un support d'écouvillon, au moins un écouvillon et un récipient à microcupules.
PCT/US2006/024461 2005-06-24 2006-06-23 Procedes de depistage par genotype d'une souche deposee par un porteur adsorbant WO2007002384A2 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US11/166,990 US20050239125A1 (en) 2000-09-06 2005-06-24 Methods for genotype screening
US11/166,990 2005-06-24
US11/170,693 2005-06-29
US11/170,693 US20060014186A1 (en) 2001-09-04 2005-06-29 Methods for genotype screening of a strain disposed on an adsorbent carrier

Publications (2)

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WO2007002384A2 true WO2007002384A2 (fr) 2007-01-04
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Publication number Priority date Publication date Assignee Title
CN111631754A (zh) * 2020-05-26 2020-09-08 清华大学 一种咽拭子自动采样系统
CN112300911A (zh) * 2020-10-27 2021-02-02 广州和实生物技术有限公司 一种核酸检测仪及核酸检测方法

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US20060014186A1 (en) * 2001-09-04 2006-01-19 Hodge Timothy A Methods for genotype screening of a strain disposed on an adsorbent carrier

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US4283809A (en) * 1979-11-05 1981-08-18 Prost Claude D Swab holding tool
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111631754A (zh) * 2020-05-26 2020-09-08 清华大学 一种咽拭子自动采样系统
CN111631754B (zh) * 2020-05-26 2021-07-09 清华大学 一种咽拭子自动采样系统
CN112300911A (zh) * 2020-10-27 2021-02-02 广州和实生物技术有限公司 一种核酸检测仪及核酸检测方法

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