EP1468111A1 - Procede de mise en oeuvre d'un ordinateur et/ou d'un reseau informatique en vue de distribuer des produits biotechnologiques - Google Patents

Procede de mise en oeuvre d'un ordinateur et/ou d'un reseau informatique en vue de distribuer des produits biotechnologiques

Info

Publication number
EP1468111A1
EP1468111A1 EP02784321A EP02784321A EP1468111A1 EP 1468111 A1 EP1468111 A1 EP 1468111A1 EP 02784321 A EP02784321 A EP 02784321A EP 02784321 A EP02784321 A EP 02784321A EP 1468111 A1 EP1468111 A1 EP 1468111A1
Authority
EP
European Patent Office
Prior art keywords
sequence
accordance
probe
assay
design
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP02784321A
Other languages
German (de)
English (en)
Other versions
EP1468111A4 (fr
Inventor
Ryan T. Koehler
Kenneth J. Livak
Junko Stevens
Francisco M. De La Vega
Michael Rhodes
Laurent R. Bellon
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Applied Biosystems Inc
Original Assignee
Applera Corp
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.)
Filing date
Publication date
Application filed by Applera Corp filed Critical Applera Corp
Publication of EP1468111A1 publication Critical patent/EP1468111A1/fr
Publication of EP1468111A4 publication Critical patent/EP1468111A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the present invention relates computer systems and networks, and more particularly to methods for utilizing such computer systems and networks for accepting and filling orders for products and services, including, for example, biotechnology laboratory assays and services.
  • SNPs single nucleotide polymorphisms
  • Some configurations of the present invention therefore provide a method for providing a product to a consumer.
  • the method includes utilizing a computer network to interact with the consumer to obtain information associated with at least one target nucleic acid sequence; providing a forward primer sequence, a reverse primer sequence and a probe sequence, wherein the forward primer sequence and the reverse primer sequence together define an amplicon sequence, the amplicon lies within the target nucleic acid sequence, and the probe sequence is complementary to a portion of the amplicon sequence; manufacturing at least one assay that includes a forward primer in accordance with the forward primer sequence, a reverse primer in accordance with the reverse primer sequence, and a probe in accordance with the probe sequence; validating one or more of the forward primer, the reverse primer, and the probe; and delivering the manufactured assay to the consumer.
  • Some configurations of the present invention provide a method for providing a product to a consumer, wherein the method includes: utilizing a computer network to interact with the customer to obtain information associated with at least one target nucleic acid sequence; providing a forward primer sequence, a reverse primer sequence and a probe sequence having specified characteristics, wherein the forward primer sequence and the reverse primer sequence together define an amplicon sequence, the amplicon lies within the target nucleic acid sequence, and the probe sequence is complementary to a portion of the amplicon sequence; manufacturing at least one assay comprising a forward primer in accordance with the forward primer sequence, a reverse primer in accordance with the reverse primer sequence, and a probe in accordance with the probe sequence; validating one or more of the forward primer, the reverse primer, and the probe; and delivering the manufactured assay to the customer.
  • Various configurations of the present invention provide a method for operating a computing system.
  • the method includes: receiving information that includes at least one target nucleic acid sequence via a computer communication network; attempting to design an assay that includes a forward primer sequence, a reverse primer sequence, and a probe sequence, wherein the forward primer sequence and the reverse primer sequence together define an amplicon sequence, the amplicon sequence lies within the target nucleic acid sequence, and the probe sequence is complementary to a portion of the amplicon sequence; validating at least one of the forward primer sequence, the reverse primer sequence, and the probe sequence against a genome database; recording each design attempt in a log file, including whether each of the design attempts succeeded or failed to meet constraints set by the design metrics and scoring metrics; and utilizing the log file to generate output sequence data for at least one forward primer sequence, reverse primer sequence, and probe sequence.
  • Some configurations of the present invention provide a method for operating a computing system.
  • the method includes: receiving information comprising at least one target nucleic acid sequence via a computer communication network; providing assay design metrics and scoring metrics; attempting to design an assay comprising a forward primer sequence, a reverse primer sequence, and a probe sequence, wherein the forward primer sequence and the reverse primer sequence together define an amplicon sequence, the amplicon sequence lies within the target nucleic acid sequence, and the probe sequence is complementary to a portion of the amplicon sequence; validating at least one of the forward primer sequence, the reverse primer sequence, and the probe sequence against a genome database; recording each design attempt in a log file, including whether each said design attempt succeeded or failed to meet constraints set by the design metrics and scoring metrics; and utilizing the log file to generate a batch of output sequence data for at least one forward primer sequence, reverse primer sequence, and probe sequence.
  • Figure 1 is a block diagram representation of various configurations of a computing system of the present invention that is useful for distributing biotechnology products to a consumer.
  • Figure 2 is a flow chart representative of various method configurations of the present invention that can be performed by computing system configurations such as those represented in Figure 1 , or by other computing system configurations.
  • Figure 3 is a flow chart representative of various configurations of the present invention that provide a method for operating a computer system to distribute a product to a consumer.
  • Figure 4 is a block diagram representative of components and data flow in various configurations of an assay design system.
  • Figure 5 is a diagram representative of various configurations of assay design program logic suitable for use in assay design system configurations represented by Figure 4.
  • Figure 6 is a diagram representative of various configurations of reagent design procedures suitable for use in assay design program logic configurations represented by Figure 5.
  • Figure 7 is a diagram representative of various configurations of probe placing procedures suitable for use in reagent design procedure configurations represented by Figure 6. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • the terms “distribute” and “provide” may be used synonymously, and are intended encompass selling, marketing, or otherwise providing a product or service.
  • the terms “distributor” and “provider” thus encompass sellers, marketers, and other providers of such products and services, and the term “consumer” encompasses customers and other users of the products and services. Unless explicitly stated otherwise, it is permitted but not required that configurations of the present invention precondition distribution on receipt of a payment or a promise to pay for the distributed products or services.
  • a probe is intended to include two bi-allelic probes in the case of SNP assays.
  • a computing system 10 comprising a plurality of computers 12, 16 is utilized to distribute a product to a consumer 18.
  • a first computer 12 i.e., a distributor computer
  • a computer network 14 e.g., a public network, such as the Internet
  • a second computer 16 i.e., a consumer computer
  • information that is associated with a human or nonhuman target DNA (or RNA) sequence including SNP and/or exon locations, i.e., the sequence itself, the SNP and/or exon locations themselves, or other information from which these items may be determined such as, for example, a gene name, accession number, etc.
  • this interaction is initiated by consumer 18 typing a uniform resource locator (URL) into a web browser running on consumer computer 16 and downloading a hypertext mark-up language (HTML) or other type of web page from a server 20 running on distributor computer 12.
  • the web page displayed on consumer computer 16 may include various types of introductory and sales information, provide a login for authorized user/purchasers, and solicit the DNA (or RNA) sequence and other information, as is necessary or desirable.
  • the initial web page is one of several web pages provided by server 34 that interact with consumer 18 to obtain information.
  • the initial web page accessed by consumer 18 is a corporate web site that provides information for consumer 18 as well as a form in which consumer 18 types identifying information using consumer computer 16.
  • Distributor computer 12 receives the information entered by consumer 18 and sent by consumer computer 16 via computer network 14.
  • distributor computer 12 verifies the identity of consumer 18 and his or her qualifications to access a sales page and to purchase assays from the distributer. For example, this verification may be performed by a web application server 22 (for example, the IBM ® WEBSPHERE ® Application Server available from International Business Machines Corporation, Armonk NY) running on distributor computer 12 with reference to a consumer database 24 of qualified consumers and consumer identifications. If consumer 18 cannot be verified or is not qualified to make a purchase, this information may be returned by web application server 22 and web page server 20 via computer network 14 to consumer 18, and consumer 18 will not be allowed to complete a purchase and/or to access additional information.
  • a web application server 22 for example, the IBM ® WEBSPHERE ® Application Server available from International Business Machines Corporation, Armonk NY
  • consumer 18 specifies information including at least one target nucleic acid sequence.
  • the target nucleic acid sequence is a target DNA sequence
  • the information provided by consumer 18 and obtained by distributor computer 12 may also include an exon or a portion thereof, and/or a single nucleotide polymorphism (SNP).
  • Other information that may be provided by consumer 18 and obtained by distributor computer 12 may include a SNP location and/or an exon location.
  • Information from consumer 18 may, for example, be provided on a web page form by consumer 18 using consumer computer 16 sent to and received by distribution computer 12 via computer network 14.
  • a variant configurator 26 (such as SELECTICA ® ConfiguratorTM, available from Selectica, Inc., San Jose, CA) interacts with consumer 18 via network 14 to produce a list of specified characteristics, as discussed below.
  • Configurator 26 is essentially an automated decision tree that produces the input for assay design program 28 and that ensures that input parameters to assay design program 28 are within bounds that can be handled by program 28. If there are no errors, assay design program 28 then uses a lookup process, a design process, or another suitable method to provide a forward primer sequence, a reverse primer sequence, and a probe sequence that have the specified characteristics.
  • the forward primer sequence and the reverse primer sequence define an amplicon sequence, which itself lies within the target nucleic acid sequence, and the probe sequence is complementary to a portion of the amplicon sequence.
  • Some configurations of the present invention utilize distributor computer 12 to validate or "BLAST" the results from assay design program 28 against a genome database 30, for example, a human genome database.
  • BLAST as used herein is intended to refer to a Basic Local Alignment Search Tool such as was developed by Altschul et al. (J Mol Biol 215:403-10, 1990) which comprises a fast search algorithm to search DNA databases based upon sequence similarities.
  • Such a BLAST search tool is useful for various validation methods that can be used for the probe and primer sequences.
  • the validation process is intended to verify that the probe and primer sequences are selective for the target region, i.e. they will hybridize to the target region but not to other regions of the genome.
  • oligo factory 32 accepts the order from consumer 18, manufactures at least one assay having components including a forward primer, a reverse primer and a probe and ships the manufactured assay to the consumer.
  • the manufactured forward primer, reverse primer, and probe are manufactured in accordance with the validated sequences.
  • the assay is shipped as a homogenous assay in a single tube format with a two-dimensional bar code.
  • the manufactured assay is tested prior to delivering to the consumer to verify that the assay meets the specified characteristics.
  • the testing may include performing mass spectroscopy on the assay to determine that an oligonucleotide sequence is correct, and/or the testing may include performing a functional test to determine that an amplification has occurred and at least one allelic discrimination is confirmed.
  • the probe in the assay shipped to the consumer includes a non-fluorescent dye that is configured to reduce background fluorescence and increase quenching efficiency.
  • the assay is particularly suitable and provides a substantial benefit to consumers using PCR sequence detection systems such as the Applied Biosystems PRISM® 7900HT Sequence Detection System, enabling high-throughput SNP genotyping in which approximately 250,000 genotypes per day are analyzed, each needing only a small amount of sample DNA.
  • various configurations of the present invention perform a method 34 for distributing a biotechnology product to a consumer. More particularly, the method includes utilizing a computer network 14 to interact at 36 with a consumer 18 to obtain information associated with (i.e., indicative of) at least one nucleic acid sequence.
  • the target nucleic acid sequence obtained from the consumer is, for example, a target RNA or DNA sequence, which itself may include an exon or a portion thereof, and/or a single nucleotide polymorphism (SNP).
  • the information may further include information associated with a SNP location and/or an exon location.
  • the provided nucleic acid sequence is analyzed at 38 for format errors.
  • further interaction at 36 may be performed to correct the format errors.
  • consumer 18 prior to interacting at 36 with consumer 18 to obtain information comprising a nucleic acid sequence, consumer 18 is required to verify his or her identity via computer network 14, and/or confirm his qualifications to place an order.
  • various methods of the present invention provide, at 40, a forward primer sequence, a reverse primer sequence, and a probe sequence having specified characteristics.
  • the specified characteristics may include size limits, target T m (melting temperature), minimum T m , total matching bases in a hairpin stem, contiguous matching groups in a hairpin stem, combined G and C content, runs of G bases, runs of non-G bases, and/or G content not greater than C content (G ⁇ C).
  • the forward primer sequence and the reverse primer sequence together define an amplicon sequence.
  • the amplicon lies within the target nucleic acid sequence.
  • the probe sequence is complementary to a portion of the amplicon sequence.
  • one or more of the forward primer sequence, the reverse primer sequence, and the probe sequence are validated at 42, using, for example, a genome database such as database 30. Validation may include BLASTing of one or more of the sequences, as described above.
  • At least one assay is manufactured at 44.
  • the manufactured assay comprises a forward primer in accordance with the forward primer sequence, a reverse primer in accordance with the reverse primer sequence, and a probe in accordance with the probe sequence.
  • the forward primer sequence, the reverse primer sequence, and/or the probe sequence is a validated sequence from 42.
  • the assay is shipped at 48 to consumer 18.
  • Some configurations of the present invention ship the assay in a single tube format with a two-dimensional bar code.
  • the probe in the manufactured assay comprises a non-fluorescent dye configured to reduce background fluorescence and increase quenching efficiency.
  • the assay itself is suitable for use in a sequence detection system.
  • Tests at 46 may include, for example, performing mass spectroscopy on the manufactured assay to determine that an oligonucleotide sequence is correct, and/or performing a functional test to determine that an amplification has occurred and at least one allelic discrimination is confirmed.
  • a method 50 for operating a computer system includes receiving, at 52, information including at least one target nucleic acid sequence via a computer communication network such as network 14 of Figure 1.
  • Assay design metrics i.e. specified characteristics of probe and primers as well as scoring values for the specified characteristics are provided at 54, for example, from local storage.
  • the scoring approach involves assessing how closely the probe and primers conform to each of the specified characteristics and, then, arriving at an overall value for conformance which can be used to determine whether the probes and primers are acceptable.
  • the computer system is used to attempt to design, at 58, an assay that comprises a forward primer sequence, a reverse primer sequence, and a probe sequence.
  • the forward primer sequence and the reverse primer sequence together define an amplicon sequence.
  • the amplicon sequence lies within the target nucleic acid sequence, and the probe sequence is complementary to a portion of the amplicon sequence.
  • Some configurations also attempt to apply, at 60, design metrics or constraints to the resulting assay design, or portions thereof. For example, constraints may include size limits, target T m , minimum T m , total matching bases in a hairpin stem, contiguous matching groups in a hairpin stem, combined G and C content, runs of G bases, and runs of non-G bases.
  • Attempting to design the assay at 58 may also comprise, in some configurations, attempting to design at least one of the primer sequences in accordance with at least one constraint including a limit on G+C at a 3' end (5 bases) of the primer sequence.
  • each design attempt is recorded at 62 in a log file, including whether each design attempt succeeded or failed to meet constraints set by the design metrics and scoring metrics. If the design at 64 fails to meet these constraints, another attempt to design the assay at 58 may be made. Otherwise, if the design is adequate, the log file is used to generate output sequence data at 66 for at least one forward primer sequence, reverse primer sequence, and probe sequence. Some configurations also assign, at 68, a serial number to each batch of output sequence data. [0034] In some configurations and referring to Figure 4, an assay design system 70 is provided as computer software that allows automated, high-throughput design of TAQMAN ® primers and probes for allelic discrimination and gene expression assays in a batch format.
  • Assay design program 28 is a non-interactive pipeline of algorithms for the design of TAQMAN ® probe and primer reagents. In some configurations, heuristic rules are utilized in assay design program 28. Pre- and post-processing utility programs and wrapper scripts are utilized as components of the complete assay design system 70.
  • Components and dataflow of assay design system 70 include two data files 72 and 73.
  • a sequence input file 72 contains formatted and annotated sequence data.
  • a parameter file 73 contains keyword- associated settings that govern rules and scoring applied during designs. Prior to attempting any designs, the format of supplied sequence data is checked at 74 for errors. If errors are found at 76 in the sequence data from input file 72, they are reported to an error log 88 and the process terminates.
  • Assay design program 28 starts by parsing a parameter file 73 to set up rules and scoring schemes. If there are any errors encountered during this initialization phase, they are reported to log file 88 and assay design program 28 stops. Initialization errors may be caused by conflicting options or incorrect file names or formats. Following successful initialization, assay design program 28 sequentially attempts to design assay sets for each target site in each sequence listed in the input sequence data from parameter file 73. As designs are processed, they are recorded in a design log file 80. Design attempts that fail are also recorded in log file 80. Design failure occurs when no acceptable set of reagents satisfying all rules and scores is found for a sequence target.
  • design log file 80 may be used to generate output sequence data in a number of different formats.
  • Log pick program 84 performs this post-processing of design log 80 data to produce formatted outputs 86.
  • a script can be implemented in the UNIX operating system to integrate the whole system by tying together all of the processes shown in Figure 4. The script also logs each runs of process 70 and assigns each output batch a serial number for tracking purposes.
  • Input to assay design program 28 includes a parameter file 73 that specifies design rules and one or more sequence data files 72.
  • Output includes a log file 80 that reports system settings and attributes describing each successful reagent design (including probe, primer, and amplicon sequences). Additional output indicating a system status is reported to a display screen as the program is running, in some configurations.
  • Probe constraints include limits on size (i.e., probe length), T m (target, minimum, and maximum temperatures), internal loops (total and contiguous matching bases in a "hairpin stem"), G+C content (i.e., combined G and C percentage), and runs of a given base, such as G.
  • Analogous constraints are also separately applied to primers, which have an additional limit on G+C at the 3' end (5 bases) of the primers.
  • Constraints applied to amplicons include length (including primers), G+C content, and the number of ambiguous bases (note that ambiguous bases are never allowed within probes or primers).
  • the primers defining applicons are constrained to limit the maximal size of internal priming sites (i.e., the number of contiguous matching bases starting at the 3' end of one primer that complements any part of the other primer).
  • system 70 may apply either a filter or a score.
  • a constraint When applied as a filter, a constraint will be either satisfied or not with the corresponding design being either accepted or rejected.
  • attributes When applied as a score, attributes may be given a graded value that reflects how "optimal" a given design is. For example, a design with all constrained attributes near optimum values will be favored over one with attributes deviating from the optimum values. Scoring provides finer tuning of the constraints that system 70 will use to evaluate and select designs.
  • an initialization phase 98 reads parameter data from parameter file 73 and sequence data from sequence data file 72. (As shown in Figure 4, sequence data 72 may be checked for errors at 74 and 76 before being read by assay design program 28.)
  • Initialization 98 includes parsing parameter file 73 and setting up for subsequent probe design. If any problems are encountered at 100 as a result of initialization 98, assay design program 28 reports a diagnostic message and stops at 104. Otherwise, processing continues.
  • most parameter file 73 options have default values and may be superceded by command line options. Options actually used during design are reported in log file header 102, which is or becomes part of design log 80 of Figure 4.
  • Assay design program 28 attempts to acceptably design assay sets for each target site at 110. These designs are logged at 112. An attempt is made to identify acceptable designs at 114 for each input sequence record from sequence data file 72. When records are exhausted at 106, assay design program 28 is done at 104. Otherwise, for each record, each target is tried at 110 in the order listed. If no target information is supplied, the sequence midpoint (if the sequence contains no SNP annotations) or the first SNP (if annotated) is used as a target. When no targets are left for a given record at 108, assay design program 28 progresses at 106 to the next record.
  • assay design program 28 For target sites, assay design program 28 identifies, at 114, successful and unsuccessful designs, according to the design metrics and scoring metrics. If it fails to design for a target, this fact along with the corresponding unsuccessful design is reported to log file 80 and the program progresses at 108 to the next target associated with the record. If it succeeds to design for a target, the details of the chosen record are reported to log file 80. Normally, a single successful design causes assay design program 28 to move to the next record at 106. However, in some configurations, if an option to evaluate all targets listed for each record is enabled, assay design program 28 progresses, at 116, to the next target at 108 rather than to the next record at 106 following a successful design.
  • design for record/target program 110 extracts design "windows" at 124, e.g., one or two subsequences around the target are extracted.
  • design "windows" e.g., one or two subsequences around the target are extracted.
  • two separate windows are extracted at 124 around the SNP target site, one for each allele.
  • any other SNP that is known to be within the sequence of the window is masked by converting it to an N, which represents any nucleotide.
  • a single subsequence window is extracted at 124. Windows are limited in size by the supplied input sequence length or by the maximum allowable amplicon size.
  • probe placement at 130 yields either one or two acceptable probes (non-SNP and SNP cases, respectively), or not. If acceptable probes are not identified at 132, target design process 110 fails at 146. Otherwise, bounds are set for primers at 134.
  • a central mask region corresponding to coordinates of the probes is defined. Bounds for the mask region may be explicitly designed relative to target site coordinates. For example, in some configurations, a command line option (such as "-pm") is used to specify that the mask region is to be explicitly designed relative to target site coordinates. In this case, the actual mask region is the larger of the specified bounds or the mask formed by the probes. Fixing the central mask region determines the two sub regions where primers may be designed.
  • the "upstrand” sub-region begins at the start of the window and extends to the start of the mask region.
  • the “downstrand” sub-region follows the mask and extends to the end of the window.
  • the three sub-regions of the window i.e., upstrand, mask, and downstrand do not overlap.
  • design procedure 110 attempts to collect a number of primers in each sub- region at 136. Forward primers are taken from the upstrand region and reverse primers are taken from the downstrand region. Potential primers are evaluated at each nucleotide position starting from the coordinates closest to the mask (i.e., the end and start coordinates of the upstrand and downstrand regions, respectively). Such evaluation may, for example, determine whether a potential primer is acceptable according to standards known and recognized in the art. In some configurations, design procedure 110 collects up to ten forward and ten reverse primers, but by setting a command line option (such as "-np"), the limit of ten can be changed to another number.
  • a command line option such as "-np”
  • design process 110 fails at 146. With two lists of primers, design process 110 next attempts to identify an acceptable forward/reverse pair at 140. If no acceptable primer pair is identified at 142, design process 110 fails at 146. Otherwise, a complete design has been found at 144.
  • Minimal target overlap is an input parameter. For non-SNP targets this value may be negative, allowing a larger sequence region to be sampled. For SNP targets, the minimum value of target overlap is two bases, but the overlap may be increased. Probes targeting both forward and reverse strands are evaluated. Probes may not start with G and normally the requirement that G content does not exceed C content is applied, but an option is provided to eliminate the G ⁇ C rule. In some configurations, and in some cases, both forward and reverse strands are considered explicitly for T m delineation. If probe scoring is being applied, the best scoring probe is selected. Otherwise, the constraint satisfying probe most overlapping the target site is selected. From this determination of whether a probe is acceptable or not at 174 or 176, probes are selected that pass, at 178, or alternatively are selected against, at 180, for failing the criteria.
  • sequences corresponding to both alleles are explicitly constructed and the best probes for both strands of both allele sequences are identified as described above.
  • An acceptable pair of SNP probes must target the same sequence strand. If acceptable probe pairs are found for both strands, the strand yielding the pair with the largest total score is selected.
  • the non-targeted SNP sites are masked (i.e., set to base N) when the sequences for each explicitly targeted allele are constructed.
  • a list of upstream (forward) and downstream (reverse) primers are delineated starting immediately before and after the probe position. These are delineated via T m (in some configurations using a different algorithm than used for probe design), and filtered or scored. If SNP probe pairs are being designed, primers are delineated starting immediately before and after the footprint corresponding to both SNP-targeting probe positions. At least one forward and one reverse primer must be identified. By default, up to ten forward and ten reverse primers are collected, but the number of upstream and downstream primers may be changed, such as by using a command line switch. Failure to identify any forward or any reverse primers results in probe placement process 130 to report the problem and continue with the next target coordinate or next sequence as described above.
  • Forward and reverse primers are checked for pair-wise compatibility and the corresponding amplicons are filtered or scored.
  • the compatibility check includes screening the 3' ends of the primers across the amplicon associated with a given primer pairing. If too great a 3' match is identified, the primers may not be paired. The pair of primers with the best score, by default, the shortest amplicon, is chosen. Failure to select an acceptable primer pair results in probe placement process 130 reporting the problem and continuing as described above.
  • Acceptable designs comprising one or two probe sequences (such as, for example, probe sequences that can be used to make TAQMAN ® probes) together with corresponding forward and reverse primer sequences are recorded in the log file.
  • auxiliary data e.g., tracking information
  • various method configurations of the present invention provide a method for operating a computer system and for using a computer system to distribute products to consumers.
  • These products can include customized and quality-validated assays for single nucleotide polymorphism (SNP) genotyping and gene expression studies.
  • SNP single nucleotide polymorphism
  • This service can save consumers substantial time, money, and manual labor that home-brew assay design requires, thereby enabling a new level of high- throughput, low-cost SNP genotyping or gene-expression research studies.
  • human or nonhuman target DNA sequences can be provided by the consumer as starting information.
  • Various configurations provide ready-to-use, functionally tested assays that confirm performance, and some configurations provide probes in a single tube format with a two- dimensional bar code for easy sample tracking.
  • the use of a non-fluorescent dye in some configurations eliminates background fluorescence, provides superior quenching efficiency, and increases signal-to-noise ratio, which provides a substantial benefit to consumers using sequence detection systems such as the Applied Biosystems, Inc. PRISM ® 7900HT Sequence Detection System, thereby enabling high-throughput SNP genotyping where approximately 250,000 genotypes per day can be analyzed, each requiring only a small quantity of sample DNA.

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Abstract

L'invention concerne un procédé destiné à distribuer un produit à un consommateur et consistant à utiliser un réseau informatique pour interagir avec le consommateur en vue d'obtenir des informations comprenant au moins une séquence d'acides nucléiques cible, et à fournir une séquence amorce avant, une séquence amorce inverse et une séquence sonde présentant des caractéristiques spécifiées, la séquence amorce avant et la séquence amorce inverse définissant ensemble une séquence amplicon, l'amplicon se trouvant dans la séquence d'acides nucléiques, la séquence sonde étant complémentaire d'une partie de la séquence amplicon. Ledit procédé consiste ensuite à fabriquer un produit d'analyse comprenant une amorce avant selon la séquence amorce avant, une amorce inverse selon la séquence amorce inverse et une sonde selon la séquence sonde, à valider l'une au moins de l'amorce avant, de l'amorce inverse et de la sonde, puis à distribuer le produit d'analyse à un consommateur.
EP02784321A 2002-01-25 2002-10-30 Procede de mise en oeuvre d'un ordinateur et/ou d'un reseau informatique en vue de distribuer des produits biotechnologiques Withdrawn EP1468111A4 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US35203902P 2002-01-25 2002-01-25
US352039P 2002-01-25
US35235602P 2002-01-28 2002-01-28
US352356P 2002-01-28
PCT/US2002/034599 WO2003064694A1 (fr) 2002-01-25 2002-10-30 Procede de mise en oeuvre d'un ordinateur et/ou d'un reseau informatique en vue de distribuer des produits biotechnologiques

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EP1468111A1 true EP1468111A1 (fr) 2004-10-20
EP1468111A4 EP1468111A4 (fr) 2005-05-25

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EP1706826A4 (fr) 2002-01-25 2008-01-30 Applera Corp Procedes permettant de placer, d'accepter et de classer des commandes relatives a des produits et des services
JP2006134188A (ja) * 2004-11-08 2006-05-25 Japan Science & Technology Agency オリゴヌクレオチドデータ管理装置、オリゴヌクレオチドデータ管理システム、オリゴヌクレオチドデータ管理プログラムおよび記録媒体
KR101613612B1 (ko) 2010-07-29 2016-04-20 다카라 바이오 가부시키가이샤 표적 염기를 검출하기 위한 rna를 함유한 프로브를 제조하기 위한 방법
US10467594B2 (en) * 2012-08-03 2019-11-05 Label Independent, Inc. Systems and methods for designing, developing, and sharing assays

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US6300058B1 (en) * 1992-01-29 2001-10-09 Hitachi Chemical Research Center, Inc. Method for measuring messenger RNA
AU2001240991A1 (en) * 2000-03-15 2001-09-24 Genset Methods, software, and apparati for designing, ordering, pricing, tracking and directing production of custom biologicals

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JP2005516296A (ja) 2005-06-02
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CA2474481A1 (fr) 2003-08-07

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