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 provides methods for combining target amplification reactions with signal amplification reactions to achieve rapid and sensitive detection of small quantities of nucleic acids, particularly in unpurified bodily fluids (e.g. blood).
• the present invention also provides methods to optimize multiplex amplification reactions.
• the present invention also provides methods to perform highly multiplexed PCR in combination with the INVADER assay.
• the present invention further provides methods to perform PCR in combination with the
• PCR Polymerase Chain Reaction
• the present invention provides methods and routines for developing and optimizing nucleic acid detection assays for use in basic research, clinical research, and for the development of clinical detection assays.
• the present invention provides methods comprising; a) providing target sequence information for at least Y target sequences, wherein each of the target sequences comprises; i) a footprint region, ii) a 5' region immediately upstream of the footprint region, and iii) a 3' region immediately downstream of the footprint region, and b) processing the target sequence information such that a primer set is generated, wherein the primer set comprises a forward and a reverse primer sequence for each of the at least Y target sequences, wherein each of the forward and reverse primer sequences comprises a nucleic acid sequence represented by 5'-N[x]-N[x-l]- ....-N[4]-N[3]-N[2]-N[l]-3', wherein N represents a nucleotide base, x is at
• the present invention provides methods comprising; a) providing target sequence information for at least Y target sequences, wherein each of the target sequences comprises; i) a footprint region, ii) a 5' region immediately upstream of the footprint region, and iii) a 3' region immediately downstream of the footprint region, and b) processing the target sequence information such that a primer set is generated, wherein the primer set comprises a forward and a reverse primer sequence for each of the at least Y target sequences, wherein each of the forward and reverse primer sequences comprises a nucleic acid sequence represented by 5'-N[x]-N[x-l ]- ....-N[4]-N[3]-N[2]-N[l]-3', wherein N represents a nucleotide base, x is at least 6, N[l] is nucleotide G or T, and N[2]-N[l]-3' of each of the forward and reverse primers is not complementary to N[2]-N[l
• a method comprising; a) providing target sequence information for at least Y target sequences, wherein each of the target sequences comprises; i) a footprint region, ii) a 5' region immediately upstream of the footprint region, and iii) a 3' region immediately downstream of the footprint region, and b) processing the target sequence information such that a primer set is generated, wherein the primer set comprises; i) a forward primer sequence identical to at least a portion of the 5' region for each of the Y target sequences, and ii) a reverse primer sequence identical to at least a portion of a complementary sequence of the 3' region for each of the at least Y target sequences, wherein each of the forward and reverse primer sequences comprises a nucleic acid sequence represented by 5'-N[x]-N[x-l]- ....-N[4]- N[3]-N[2]-N[l]-3', wherein N represents a nucleotide base, x is at least 6, N
• the present invention provides methods comprising a) providing target sequence information for at least Y target sequences, wherein each of the target sequences comprises; i) a footprint region, ii) a 5' region immediately upstream of the footprint region, and iii) a 3' region immediately downstream of the footprint region, and b) processing the target sequence information such that a primer set is generated, wherein the primer set comprises; i) a forward primer sequence identical to at least a portion of the 5' region for each of the Y target sequences, and ii) a reverse primer sequence identical to at least a portion of a complementary sequence of the 3' region for each of the at least Y target sequences, wherein each of the forward and reverse primer sequences comprises a nucleic acid sequence represented by 5'-N[x]-N[x-l]- ....-N[4]-N[3] ⁇ N[2]-N[ll-3', wherein N represents a nucleotide base, x is at least 6,
• the present invention provides methods comprising a) providing target sequence information for at least Y target sequences, wherein each of the target sequences comprises a single nucleotide polymorphism, b) determining where on each of the target sequences one or more assay probes would hybridize in order to detect the single nucleotide polymorphism, such that a footprint region is located on each of the target sequences, and c) processing the target sequence information such that a primer set is generated, wherein the primer set comprises; i) a forward primer sequence identical to at least a portion of the target sequence immediately 5' of the footprint region for each of the Y target sequences, and ii) a reverse primer sequence identical to at least a portion of a complementary sequence of the target sequence immediately 3' of the footprint region for each of the at least Y target sequences, wherein each of the forward and reverse primer sequences comprises a nucleic acid sequence represented by 5'-N[x]-N " [x-l]- ....-N[4]
• the present invention provides methods comprising a) providing target sequence information for at least Y target sequences, wherein each of the target sequences comprises a single nucleotide polymorphism, b) determining where on each of the target sequences one or more assay probes would hybridize in order to detect the single nucleotide polymorphism such that a footprint region is located on each of the target sequences, and c) processing the target sequence information such that a primer set is generated, wherem the primer set comprises; i) a forward primer sequence identical to at least a portion of the target sequence immediately 5' of the footprint region for each of the Y target sequences, and ii) a reverse primer sequence identical to at least a portion of a complementary sequence of the target sequence immediately 3' of the footprint region for each of the at least Y target sequences, wherein each of the forward and reverse primer sequences comprises a nucleic acid sequence represented by 5'-N[x]-N[x-l]- ....-N[4]-
• the primer set is configured for performing a multiplex PCR reaction that amplifies at least Y amplicons, wherein each of the amplicons is defined by the position of the forward and reverse primers.
• the primer set is generated as digital or printed sequence information.
• the primer set is generated as physical primer oligonucleotides.
• N[3]-N[2]-N[l]-3' of each of the forward and reverse primers is not complementary to N[3]-N[2]-N[l]-3' of any of the forward and reverse primers in the primer set.
• the processing comprises initially selecting N[l] for each of the forward primers as the most 3' A or C in the 5' region.
• the processing comprises initially selecting N[l] for each of the forward primers as the most 3' G or T in the 5' region. In some embodiments, the processing comprises initially selecting N[l] for each of the forward primers as the most 3' A or C in the 5' region, and wherein the processing further comprises changing the N[l] to the next most 3' A or C in the 5' region for the forward primer sequences that fail the requirement that each of the forward primer's N[2]-N[l]-3' is not complementary to N[2]-N[l]-3' of any of the forward and reverse primers in the primer set.
• the processing comprises initially selecting N[l] for each of the reverse primers as the most 3' A or C in the complement of the 3' region. In some embodiments, the processing comprises initially selecting N[l] for each of the reverse primers as the most 3' G or T in the complement of the 3' region.
• the processing comprises initially selecting N[l] for each of the reverse primers as the most 3' A or C in the 3' region, and wherein the processing further comprises changing the N[l] to the next most 3' A or C in the 3' region for the reverse primer sequences that fail the requirement that each of the reverse primer's N[2]-N[l]-3' is not complementary to N[2]-N[l]-3' of any of the forward and reverse primers in the primer set.
• the footprint region comprises a single nucleotide polymorphism. In some embodiments, the footprint comprises a mutation.
• the footprint region for each of the target sequences comprises a portion of the target sequence that hybridizes to one or more assay probes configured to detect the single nucleotide polymorphism. In certain embodiments, the footprint is this region where the probes hybridize. In other embodiments, the footprint further includes additional nucleotides on either end. In some embodiments, the processing further comprises selecting N[5]-N[4]-N[3]-N[2]-
• the processing comprises selecting x for each of the forward and reverse primers such that each of the forward and reverse primers has a melting temperature with respect to the target sequence of approximately 50 degrees Celsius (e.g. 50 degrees, Celsius, or at least 50 degrees Celsius, and no more than 55 degrees Celsius).
• the melting temperature of a primer is at least 50 degrees Celsius, but at least 10 degrees different than a selected detection assay's optimal reaction temperature.
• the forward and reverse primer pair optimized concentrations are determined for the primer set.
• the processing is automated.
• the processing is automated with a processor.
• the present invention provides a kit comprising the primer set generated by the methods of the present invention, and at least one other component, (e.g. cleavage agent, polymerase, INVADER oligonucleotide).
• the present invention provides compositions comprising the primers and primer sets generated by the metlrods of the present invention.
• the present invention provides methods comprising; a) providing; i) a user interface configured to receive sequence data, ii) a computer system having stored therein a multiplex PCR primer software application, and b) transmitting the sequence data from the user interface to the computer system, wherein the sequence data comprises target sequence information for at least Y target sequences, wherein each of the target sequences comprises; i) a footprint region, ii) a 5' region immediately upstream of the footprint region, and iii) a 3' region immediately downstream of the footprint region, and c) processing the target sequence information with the multiplex PCR primer pair software application to generate a primer set, wherein the primer set comprises; i) a forward primer sequence identical to at least a portion of the target sequence immediately 5' of the footprint region for each of the Y target sequences, and ii) a reverse primer sequence identical to at least a portion of a complementary sequence of the target sequence immediately 3' of the footprint region for each of the at least Y target sequences
• the present invention provides methods comprising; a) providing; i) a user interface configured to receive sequence data, ii) a computer system having stored therein a multiplex PCR primer software application, and b) transmitting the sequence data from the user interface to the computer system, wherein the sequence data comprises target sequence information for at least Y target sequences, wherein each of the target sequences comprises; i) a footprint region, ii) a 5' region immediately upstream of the footprint region, and iii) a 3' region immediately downstream of the footprint region, and c) processing the target sequence information with the multiplex PCR primer pair software application to generate a primer set, wherein the primer set comprises; i) a forward primer sequence identical to at least a portion of the target sequence immediately 5' of the footprint region for each of the Y target sequences, and ii) a reverse primer sequence identical to at least a portion of a complementary sequence of the target sequence immediately 3' of the footprint region for each of the at least Y target sequences
• the present invention provides systems comprising; a) a computer system configured to receive data from a user interface, wherein the user interface is configured to receive sequence data, wherein the sequence data comprises target sequence information for at least Y target sequences, wherein each of the target sequences comprises; i) a footprint region, ii) a 5' region immediately upstream of the footprint region, and iii) a 3' region immediately downstream of the footprint region, b) a multiplex PCR primer pair software application operably linked to the user interface, wherein the multiplex PCR primer software application is configured to process the target sequence information to generate a primer set, wherein the primer set comprises; i) a forward primer sequence identical to at least a portion of the target sequence immediately 5' of the footprint region for each of the Y target sequences, and ii) a reverse primer sequence identical to at least a portion of a complementary sequence of the target sequence immediately 3' of the footprint region for each of the at least Y target sequences, wherein each of the forward and reverse
• the present invention provides systems comprising; a) a computer system configured to receive data from a user interface, wherein the user interface is configured to receive sequence data, wherein the sequence data comprises target sequence information for at least Y target sequences, wherein each of the target sequences comprises; i) a footprint region, ii) a 5' region immediately upstream of the footprint region, and iii) a 3' region immediately downstream of the footprint region, b) a multiplex PCR primer pair software application operably linked to tlie user interface, wherein the multiplex PCR primer software application is configured to process the target sequence information to generate a primer set, wherein the primer set comprises; i) a forward primer sequence identical to at least a portion of the target sequence immediately 5' of the footprint region for each of the Y target sequences, and ii) a reverse primer sequence identical to at least a portion of a complementary sequence of the target sequence immediately 3' of tlie footprint region for each of the at least Y target sequences, wherein each of
• the computer system is configured to return the primer set to the user interface.
• the present invention provides methods for conducting target and signal amplification reactions in a single reaction vessel.
• the target amplification reactions are PCR reactions.
• the signal amplification reactions are invasive cleavage (INVADER) assays.
• reagents for the combined target and signal amplification reactions are added prior to initiation of either reaction.
• the target amplification reactions are terminated after 20 cycles.
• the target amplification reactions are terminated after 15 cycles.
• the target amplification reactions are terminated after 11 cycles.
• the predispensed reagents are dried in the reaction vessel.
• the predispensed reagents comprise one or more INVADER assay reagents
• the reaction vessel comprises a microfluidic card.
• the reaction vessel comprises a microfluidic card configured for centrifugal or centripetal distribution or manipulation of fluid reactions and reaction components.
• the present invention provides methods and compositions for conducting multi-dye multiplex FRET INVADER assays, e.g., in a single reaction or reaction vessel.
• the multiplex FRET assays are carried out on synthetic targets. In other preferred embodiments, the multiplex FRET assays are carried out on nucleic acid fragment targets, e.g., PCR amplicons. In some particularly preferred embodiments, multiplex FRET assays are carried out on genomic DNA targets. In still other prefened embodiments, multiplex FRET assays are carried out on RNA targets. In some particularly preferred embodiments, the multiplex FRET assays are tetraplex reactions. In some embodiments one or more the INVADER assay reagents may be provided in a predispensed format (i.e., premeasured for use in a step of the procedure without re-measurement or re-dispensing).
• selected INVADER assay reagent components are mixed and predispensed together.
• predispensed assay reagent components are predispensed and are provided in a reaction vessel (including but not limited to a reaction tube or a well, as in, e.g., a microtiter plate).
• predispensed INVADER assay reagent components are dried down (e.g., desiccated or lyophilized) in a reaction vessel.
• the INVADER assay reagents are provided as a kit.
• kit refers to any delivery system for delivering materials.
• kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and/or supporting materials.
• fragment kit refers to delivery systems comprising two or more separate containers that each contains a subportion of the total kit components. The containers may be delivered to the intended recipient together or separately.
• a first container may contain an enzyme for use in an assay, while a second container contains oligonucleotides.
• fragment kit is intended to encompass kits containing Analyte specific reagents (ASR's) regulated under section 520(e) of the Federal Food, Drug, and Cosmetic Act, but are not limited thereto. Indeed, any delivery system comprising two or more separate containers that each contains a subportion of the total kit components are included in the term “fragmented kit.”
• a “combined kit” refers to a delivery system containing all of the components of a reaction assay in a single container (e.g., in a single box housing each of the desired components).
• kit includes both fragmented and combined kits.
• the present invention provides INVADER assay reagent kits comprising one or more of the components necessary for practicing the present invention.
• the present invention provides kits for storing or delivering the enzymes and/or the reaction components necessary to practice an INVADER assay.
• the kit may include any and all components necessary or desired for assays including, but not limited to, the reagents themselves, buffers, control reagents (e.g., tissue samples, positive and negative control target oligonucleotides, etc.), solid supports, labels, written and/or pictorial instructions and product information, inhibitors, labeling and/or detection reagents, package environmental controls (e.g., ice, desiccants, etc.), and the like.
• kits provide a sub-set of the required components, wherein it is expected that the user will supply the remaining components.
• the kits comprise two or more separate containers wherein each container houses a subset of the components to be delivered.
• a first container e.g., box
• an enzyme e.g., structure specific cleavage enzyme in a suitable storage buffer and container
• a second box may contain oligonucleotides (e.g., INVADER oligonucleotides, probe oligonucleotides, control target oligonucleotides, etc.).
• Figure 1 shows a schematic diagram of an embodiment of the INVADER assay.
• the target molecule hatchched rectangle
• the primary probe which includes the target-specific region (open rectangle) and the 5' flap (filled rectangle).
• the overlap-flap is cleaved by the structure-specific 5' nuclease.
• the cleavage site of the overlap-flap structure shown by the arrow is located after the 5' terminal nucleotide of the target-specific region of the primary probe.
• the overlap between the probes is positioned opposite the polymorphic site (X). If the X nucleotide is not complementary to the primary probe, no specific cleavage occurs.
• the cleaved 5' flap forms the overlap-flap structure with FRET cassette (gray line) labeled with a dye (D) and quencher (Q). Cleavage of the FRET cassette by the 5' nuclease releases the unquenched dye.
• the semicircular anows indicate the oligonucleotide turnover process essential for signal amplification.
• FIG. 2 is a graph showing the dependence of the logarithm of the amplification factor IgF on the number of PCR cycles n for the PCR 5.
• Figure 3A is a graph showing the effect of primer concentration c on IgF for the PCR 1
• Figure 3B is a graph showing the relationship between ln(2-F° 0S ) and c using the data shown in 3 A.
• Figure 4 shows scatter plots of the net FAM and RED INVADER assay signals for eight genomic DNA samples in reactions as described in Example 7.
• Figure 5 shows the net RED fluorescence signal normalized per allele for the 161 successful INVADER assays as a function of PCR target length, in reactions as described in Example 7.
• Figure 6 shows scatter plots of the net FAM and RED signals for the eight DNA samples in reactions as described in Example 7.
• Figure 7 shows a graph displaying the results of a combined target and signal amplification reaction according to the methods of Example 8.
• Figure 8 shows a flow chart outlining the steps that may be performed in order to generated a primer set useful in multiplex PCR.
• Figure 9 shows a graph displaying the results of a combined multiplex target and signal amplification reaction according to the methods of Example 8.
• Figure 10 shows a graph displaying the results of a tetraplex INVADER assay as described in Example 9.
• Figures 11A-11G show graphs displaying the results of INVADER assay detection of multiplex PCR amplified target DNA in a microfluidic card.
• Figures 12A-12G show graphs displaying the results of combined multiplex PCR and INVADER assay signal amplification reactions in a microfluidic card.
• SNP single nucleotide polymorphisms
• SNPs single nucleotide polymorphisms
• SNPs can be located in a portion of a genome that does not code for a gene.
• a “SNP” may be located in the coding region of a gene.
• the “SNP” may alter the structure and function of the RNA or the protein with which it is associated.
• allele refers to a variant form of a given sequence (e.g., including but not limited to, genes containing one or more SNPs).
• a large number of genes are present in multiple allelic forms in a population.
• a diploid organism canying two different alleles of a gene is said to be heterozygous for that gene, whereas a homozygote carries two copies of the same allele.
• linkage refers to the proximity of two or more markers (e.g., genes) on a chromosome.
• allele frequency refers to the frequency of occurrence of a given allele (e.g., a sequence containing a SNP) in given population (e.g., a specific gender, race, or ethnic group). Certain populations may contain a given allele within a higher percent of its members than other populations. For example, a particular mutation in the breast cancer gene called BRCA1 was found to be present in one percent of the general Jewish population. In comparison, the percentage of people in the general U.S. population that have any mutation in BRCA1 has been estimated to be between 0.1 to 0.6 percent.
• in silico analysis refers to analysis performed using computer processors and computer memory.
• insilico SNP analysis refers to the analysis of SNP data using computer processors and memory.
• gene type refers to the actual genetic make-up of an organism (e.g., in terms of the particular alleles carried at a genetic locus).
• the term "locus” refers to the position of a gene or any other characterized sequence on a chromosome.
• disease or “disease state” refers to a deviation from the condition regarded as normal or average for members of a species, and which is detrimental to an affected individual under conditions that are not inimical to the majority of individuals of that species (e.g., dianhea, nausea, fever, pain, and inflammation etc).
• treatment in reference to a medical course of action refers to steps or actions taken with respect to an affected individual as a consequence of a suspected, anticipated, or existing disease state, or wherein there is a risk or suspected risk of a disease state. Treatment may be provided in anticipation of or in response to a disease state or suspicion of a disease state, and may include, but is not limited to preventative, ameliorative, palliative or curative steps.
• therapy refers to a particular course of treatment.
• gene refers to a nucleic acid (e.g., DNA) sequence that comprises coding sequences necessary for the production of a polypeptide, U A (e.g., rRNA, tRNA, etc.), or precursor.
• the polypeptide, RNA, or precursor can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or functional properties (e.g., ligand binding, signal transduction, etc.) of the full-length or fragment are retained.
• the term also encompasses the coding region of a structural gene and the including sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb on either end such that the gene conesponds to the length of the full-length mRNA.
• the sequences that are located 5' of the coding region and which are present on the mRNA are refened to as 5' untranslated sequences.
• genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed "introns” or “intervening regions” or “intervening sequences.”
• Introns are segments included when a gene is transcribed into heterogeneous nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript; introns therefore are generally absent in the messenger RNA (mRNA) transcript.
• the mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide. Variations (e.g., mutations, SNPS, insertions, deletions) in transcribed portions of genes are reflected in, and can generally be detected in conesponding portions of the produced RNAs (e.g., hnRNAs, mRNAs, rRNAs, tRNAs).
• RNAs e.g., hnRNAs, mRNAs, rRNAs, tRNAs.
• genomic forms of a gene may also include sequences located on both the 5' and 3' end of the sequences that are present on the RNA transcript. These sequences are refened to as "flanking" sequences or regions (these flanking sequences are located 5' or 3' to the non-translated sequences present on the mRNA transcript).
• the 5' flanking region may contain regulatory sequences such as promoters and enhancers that control or influence the transcription of the gene.
• the 3' flanking region may contain sequences that direct the termination of transcription, post-transcriptional cleavage and polyadenylation.
• wild-type refers to a gene or gene product that has the characteristics of that gene or gene product when isolated from a naturally occurring source.
• a wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designed the "normal” or “wild-type” form of the gene.
• the terms "modified,” “mutant,” and “variant” refer to a gene or gene product that displays modifications in sequence and or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally-occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.
• nucleic acid molecule encoding As used herein, the terms “nucleic acid molecule encoding,” “DNA sequence encoding,” and “DNA encoding” refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. In this case, the DNA sequence thus codes for the amino acid sequence.
• DNA and RNA molecules are said to have "5' ends” and "3' ends” because mononucleotides are reacted to make oligonucleotides or polynucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage.
• an end of an oligonucleotides or polynucleotide refened to as the "5' end” if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring and as the "3' end” if its 3' oxygen is not linked to a 5' phosphate of a subsequent mononucleotide pentose ring.
• a nucleic acid sequence even if internal to a larger oligonucleotide or polynucleotide, also maybe said to have 5' and 3' ends.
• an oligonucleotide having a n ⁇ cleotide sequence encoding a gene and "polynucleotide having a nucleotide sequence encoding a gene,” means a nucleic acid sequence comprising the coding region of a gene or, in other words, the nucleic acid sequence that encodes a gene product.
• the coding region may be present in either a cDNA, genomic DNA, or RNA form.
• the oligonucleotide or polynucleotide may be single-stranded (i.e., the sense strand) or double-stranded.
• Suitable control elements such as enhancers/promoters, splice junctions, polyadenylation signals, etc. may be placed in close proximity to the coding region of the gene if needed to permit proper initiation of transcription and/or conect processing of the primary RNA transcript.
• the coding region utilized in the expression vectors of the present invention may contain endogenous enhancers/promoters, splice junctions, intervening sequences, polyadenylation signals, etc. or a combination of both endogenous and exogenous control elements.
• the terms "complementary” or “complementarity” are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules.
• Complementarity may be “partial,” in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids.
• the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods that depend upon binding between nucleic acids.
• the term “homology” refers to a degree of complementarity. There may be partial homology or complete homology (i.e., identity).
• a partially complementary sequence is one that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid and is refened to using the functional term "substantially homologous.”
• the inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency.
• a substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous to a target under conditions of low stringency.
• low stringency conditions are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction.
• the absence of non-specific binding may be tested by the use of a second target that lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non-complementary target.
• low stringency conditions factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of low stringency hybridization different from, but equivalent to, the above listed conditions.
• conditions that promote hybridization under conditions of high stringency e.g., increasing the temperature of the hybridization and/or wash steps, the use of formamide in the hybridization solution, etc.).
• substantially homologous refers to any probe that can hybridize to either or both strands of the double-stranded nucleic acid sequence under conditions of low stringency as described above.
• a gene may produce multiple RNA species that are generated by differential splicing of the primary RNA transcript.
• cDNAs that are splice variants of the same gene will contain regions of sequence identity or complete homology (representing the presence of the same exon or portion of the same exon on both cDNAs) and regions of complete non-identity (for example, representing the presence of exon "A” on cDNA 1 wherein cDNA 2 contains exon "B” instead).
• the two cDNAs contain regions of sequence identity they will both hybridize to a probe derived from the entire gene or portions of the gene containing sequences found on both cDNAs; tlie two splice variants are therefore substantially homologous to such a probe and to each other.
• substantially homologous refers to any probe that can hybridize (i.e., it is the complement of) the single-stranded nucleic acid sequence under conditions of low stringency as described above.
• hybridization is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, the T m of the formed hybrid, and the G:C ratio within the nucleic acids.
• T m is used in reference to the "melting temperature.”
• the melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands.
• stringency is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. Those skilled in the art will recognize that “stringency” conditions may be altered by varying the parameters just described either individually or in concert. With “high stringency” conditions, nucleic acid base pairing will occur only between nucleic acid fragments that have a high frequency of complementary base sequences (e.g., hybridization under "high stringency” conditions may occur between homologs with about 85-100% identity, preferably about 70-100% identity).
• nucleic acid base pairing will occur between nucleic acids with an intermediate frequency of complementary base sequences (e.g., hybridization under "medium stringency” conditions may occur between homologs with about 50-70% identity).
• conditions of "weak” or “low” stringency are often required with nucleic acids that are derived from organisms that are genetically diverse, as the frequency of complementary sequences is usually less.
• “High stringency conditions” when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42 C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH 2 P0 4 H 2 0 and 1.85 g/1 EDTA, pH adjusted to 7.4 with
• “Medium stringency conditions” when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42 C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH 2 P0 4 H 2 0 and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5X Denhardt's reagent and 100 ⁇ g ml denatured salmon sperm DNA followed by washing in a solution comprising 1.OX SSPE, 1.0% SDS at 42 C when a probe of about 500 nucleotides in length is employed.
• Low stringency conditions comprise conditions equivalent to binding or hybridization at 42 C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH 2 PC>4 H 2 0 and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5X Denhardt's reagent [50X Denhardt's contains per 500 ml: 5 g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)] and 100 g ml denatured salmon sperm DNA followed by washing in a solution comprising 5X SSPE, 0.1% SDS at 42 C when a probe of about 500 nucleotides in length is employed.
• 5X SSPE 43.8 g/1 NaCl, 6.9 g/1 NaH 2 PC>4 H 2 0 and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH
• 5X Denhardt's reagent 50X Denhardt's contains per 500
• reference sequence is a defined sequence used as a basis for a sequence comparison; a reference sequence maybe a subset of a larger sequence, for example, as a segment of a full-length cDNA sequence given in a sequence listing or may comprise a complete gene sequence. Generally, a reference sequence is at least 20 nucleotides in length, frequently at least 25 nucleotides in length, and often at least 50 nucleotides in length.
• two polynucleotides may each (1) comprise a sequence (i.e., a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) may further comprise a sequence that is divergent between the two polynucleotides
• sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.
• a “comparison window,” as used herein, refers to a conceptual segment of at least 20 contiguous nucleotide positions wherein a polynucleotide sequence may be compared to a reference sequence of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
• Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman [Smith and Waterman, Adv. Appl. Math.
• sequence identity means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison.
• percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, deterrmriing the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
• the term "substantial identity” denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 25-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison.
• the reference sequence may be a subset of a larger sequence, for example, as a splice variant of the full-length sequences.
• the term "substantial identity" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity or more (e.g., 99 percent sequence identity).
• residue positions that are not identical differ by conservative amino acid substitutions.
• Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains.
• a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic- hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.
• Prefened conservative amino acids substitution groups are: valine- leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valme, and asparagine- glutamine.
• "Amplification” is a special case of nucleic acid replication involving template specificity. It is to be contrasted with non-specific template replication (i.e., replication that is template-dependent but not dependent on a specific template). Template specificity is here distinguished from fidelity of replication (i.e., synthesis of the proper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-) specificity. Template specificity is frequently described in terms of "target” specificity.
• Target sequences are "targets" in the sense that they are sought to be sorted out from other nucleic acid.
• Amplification techniques have been designed primarily for this sorting out. Template specificity is achieved in most amplification techniques by the choice of enzyme.
• Amplification enzymes are enzymes that, under conditions they are used, will process only specific sequences of nucleic acid in a heterogeneous mixture of nucleic acid. For example, in the case of Q replicase, MDV-1 RNA is the specific template for the replicase (D.L. Kacian et al, Proc. Natl. Acad. Sci. USA 69:3038 [1972]). Other nucleic acid will not be replicated by this amplification enzyme.
• this amplification enzyme has a stringent specificity for its own promoters (M. Chamberlin et al., Nature 228:227 [1970]).
• T4 DNA ligase the enzyme will not ligate the two oligonucleotides or polynucleotides, where there is a mismatch between the oligonucleotide or polynucleotide substrate and the template at the ligation junction (D.Y. Wu and R. B. Wallace, Genomics 4:560 [1989]).
• Taq and Pfu polymerases by virtue of their ability to function at high temperature, are found to display high specificity for the sequences bounded and thus defined by the primers; the high temperature results in thermodynamic conditions that favor primer hybridization with the target sequences and not hybridization with non-target sequences (H.A. Erlich (ed.), PCR Technology, Stockton Press [1989]).
• amplifiable nucleic acid is used in reference to nucleic acids that may be amplified by any amplification method.
• sample template refers to nucleic acid originating from a sample that is analyzed for the presence of “target” (defined below).
• background template is used in reference to nucleic acid other than sample template that may or may not be present in a sample. Background template is most often inadvertent. It may be the result of carryover, or it may be due to the presence of nucleic acid contaminants sought to be purified away from the sample. For example, nucleic acids from organisms other than those to he detected may be present as background in a test sample.
• the term "primer” refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, ⁇ i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH).
• the primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products.
• the primer is an oUgodeoxyribonucleotide.
• the primer should be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.
• probe or “hybridization probe” refers to an oligonucleotide (i.e., a sequence of nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly or by PCR amplification, that is capable of hybridizing, at least in part, to another oligonucleotide of interest.
• a probe may be single-stranded or double- stranded. Probes are useful in the detection, identification and isolation of particular sequences. In some prefened embodiments, probes used in the present invention will be labeled with a "reporter molecule,” so that is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label. As used herein, the term “target” refers to a nucleic acid sequence or structure to be detected or characterized. As used herein, the term “polymerase chain reaction” (“PCR”) refers to the method of K.B.
• PCR polymerase chain reaction
• Mullis See e.g Berry U.S. Patent Nos. 4,683,195, 4,683,202, and 4,965,188, hereby inco ⁇ orated by reference), which describes a method for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification.
• This process for amplifying the target sequence consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase.
• the two primers are complementary to their respective strands of the double stranded target sequence.
• the mixture is denatured and the primers then annealed to their complementary sequences within the target molecule.
• the primers are extended with a polymerase so as to form a new pair of complementary strands.
• the steps of denaturation, primer annealing, and polymerase extension can be repeated many times (i.e., denaturation, annealing and extension constitute one "cycle”; there can be numerous “cycles") to obtain a high concentration of an amplified segment of the desired target sequence.
• the length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter.
• the method is refened to as the "polymerase chain reaction” (hereinafter "PCR").
• the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be "PCR amplified.”
• PCR it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of 32p_ ⁇ aDe led deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified segment).
• any oligonucleotide or polynucleotide sequence can be amplified with the appropriate set of primer molecules.
• the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications.
• PCR product refers to the resultant mixture of compounds after two or more cycles of the PCR steps of denaturation, annealing and extension are complete. These terms encompass the case where there has been amplification of one or more segments of one or more target sequences.
• amplification reagents refers to those reagents (deoxyribonucleotide triphosphates, buffer, etc.), needed for amplification except for primers, nucleic acid template, and the amplification enzyme.
• reaction vessel refers to a system in which a reaction may be conducted, including but not limited to test tubes, wells, microwells (e.g., wells in microtitre assay plates such as, 96-well, 384-well and 1536-well assay plates), capillary tubes, ends of fibers such as optical fibers, microfluidic devices such as fluidic chips, cartridges and cards (including but not limited to those described, e.g., in US Patent No. 6,126,899, to Woudenberg, et al., US. Patent Nos.
• recombinant DNA molecule refers to a DNA molecule that is comprised of segments of DNA joined together by means of molecular biological techniques.
• antisense is used in reference to RNA sequences that are complementary to a specific RNA sequence (e.g., mRNA).
• antisense strand is used in reference to a nucleic acid strand that is complementary to the "sense” strand.
• the designation (-) i.e., “negative” is sometimes used in reference to the antisense strand, with the designation (+) sometimes used in reference to the sense (i.e., "positive") strand.
• isolated when used in relation to a nucleic acid, as in “an isolated oligonucleotide” or “isolated polynucleotide” refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. Isolated nucleic acid is present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids are nucleic acids such as DNA and RNA found in the state they exist in nature.
• a given DNA sequence e.g., a gene
• RNA sequences such as a specific mRNA sequence encoding a specific protein
• isolated nucleic acids encoding a polypeptide include, by way of example, such nucleic acid in cells ordinarily expressing the polypeptide where the nucleic acid is in a chromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature.
• the isolated nucleic acid, ohgonucleotide, or polynucleotide may be present in single-stranded or double-stranded form.
• the oligonucleotide or polynucleotide will contain at a minimum the sense or coding strand (i.e., the ohgonucleotide or polynucleotide may single-stranded), but may contain both the sense and anti-sense strands (i.e., the oligonucleotide or polynucleotide may be double-stranded).
• portion when in reference to a nucleotide sequence (as in “a portion of a given nucleotide sequence”) refers to fragments of that sequence. The fragments may range in size from four nucleotides to the entire nucleotide sequence minus one nucleotide (e.g., 10 nucleotides, 11, . . ., 20, . . .).
• purified or “to purify” refers to the removal of contaminants from a sample.
• purified refers to molecules (e.g., nucleic or amino acid sequences) that are removed from their natural environment, isolated or separated.
• an “isolated nucleic acid sequence” is therefore a purified nucleic acid sequence.
• “Substantially purified” molecules are at least 60% free, preferably at least 75% free, and more preferably at least 90% free from other components with which they are naturally associated.
• the term "recombinant protein” or “recombinant polypeptide” as used herein refers to a protein molecule that is expressed from a recombinant DNA molecule.
• native protein as used herein to indicate that a protein does not contain amino acid residues encoded by vector sequences; that is the native protein contains only those amino acids found in the protein as it occurs in nature.
• a native protein may be produced by recombinant means or may be isolated from a naturally occurring source.
• portion when in reference to a protein (as in “a portion of a given protein”) refers to fragments of that protein. The fragments may range in size from four consecutive amino acid residues to the entire amino acid sequence minus one amino acid.
• Southern blot refers to the analysis of DNA on agarose or acrylamide gels to fractionate the DNA according to size followed by transfer of the DNA from the gel to a solid support, such as nitrocellulose or a nylon membrane. The immobilized DNA is then probed with a labeled probe to detect DNA species complementary to the probe used. The DNA may be cleaved with restriction enzymes prior to electrophoresis.
• Southern blots are a standard tool of molecular biologists (J. Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, pp 9.31-9.58 [1989]).
• the term "Western blot” refers to the analysis of protein(s) (or polypeptides) immobilized onto a support such as nitrocellulose or a membrane. The proteins are run on acrylamide gels to separate the proteins, followed by transfer of the protein from the gel to a solid support, such as nitrocellulose or a nylon membrane. The immobilized proteins are then exposed to antibodies with reactivity against an antigen of interest.
• test compound refers to any chemical entity, pharmaceutical, drug, and the like that are tested in an assay (e.g., a drug screening assay) for any desired activity (e.g., including but not limited to, the ability to treat or prevent a disease, illness, sickness, or disorder of bodily function, or otherwise alter the physiological or cellular status of a sample).
• test compounds comprise both known and potential therapeutic compounds.
• a test compound can be determined to be therapeutic by screening using the screening methods of the present invention.
• known therapeutic compound refers to a therapeutic compound that has been shown (e.g., through animal trials or prior experience with administration to humans) to be effective in such treatment or prevention.
• sample as used herein is used in its broadest sense.
• a sample suspected of containing a human chromosome or sequences associated with a human chromosome may comprise a cell, chromosomes isolated from a cell (e.g., a spread of metaphase chromosomes), genomic DNA (in solution or bound to a solid support such as for Southern blot analysis), RNA (in solution or bound to a solid support such as for Northern blot analysis), cDNA (in solution or bound to a solid support) and tlie like.
• a sample suspected of containing a protein may comprise a cell, a portion of a tissue, an extract containing one or more proteins and the like.
• Samples include, but are not limited to, tissue sections, blood, blood fractions (e.g. serum, plasma, cells) saliva, cerebral spinal fluid, pleural fluid, milk, lymph, sputum, semen, urine, feces, aminotic fluid, chorionic villus samples (CVS), cervical swabs and buccal swabs.
• label refers to any atom or molecule that can be used to provide a detectable (preferably quantifiable) effect, and that can be attached to a nucleic acid or protein.
• Labels include but are not limited to dyes; radiolabels such as 32 P; binding moieties such as biotin; haptens such as digoxgenin; luminogenic, phosphorescent or fluorogenic moieties; and fluorescent dyes alone or in combination with moieties that can suppress or shift emission spectra by fluorescence resonance energy transfer (FRET). Labels may provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like. A label may be a charged moiety (positive or negative charge) or alternatively, may be charge neutral. Labels can include or consist of nucleic acid or protein sequence, so long as the sequence comprising the label is detectable.
• signal refers to any detectable effect, such as would be caused or provided by a label or an assay reaction.
• the term “detector” refers to a system or component of a system, e.g., an instrument (e.g. a camera, fluorimeter, charge-coupled device, scintillation counter, etc) or a reactive medium (X-ray or camera film, pH indicator, etc.), that can convey to a user or to another component of a system (e.g., a computer or controller) the presence of a signal or effect.
• an instrument e.g. a camera, fluorimeter, charge-coupled device, scintillation counter, etc
• a reactive medium X-ray or camera film, pH indicator, etc.
• a detector can be a photometric or spectrophotometric system, which can detect ultraviolet, visible or infrared light, including fluorescence or chemiluminescence; a radiation detection system; a spectroscopic system such as nuclear magnetic resonance spectroscopy, mass spectrometry or surface enhanced Raman spectrometry; a system such as gel or capillary electrophoresis or gel exclusion chromatography; or other detection system known in the art, or combinations thereof.
• the term “distribution system” refers to systems capable of transferring and/or delivering materials from one entity to another or one location to another.
• a distribution system for transferring detection panels from a manufacturer or distributor to a user may comprise, but is not limited to, a packaging department, a mail room, and a mail delivery system.
• the distribution system may comprise, but is not limited to, one or more delivery vehicles and associated delivery personnel, a display stand, and a distribution center.
• interested parties e.g., detection panel manufactures
• a distribution system to transfer detection panels to users at no cost, at a subsidized cost, or at a reduced cost.
• the term "at a reduced cost” refers to the transfer of goods or services at a reduced direct cost to the recipient (e.g. user).
• "at a reduced cost” refers to transfer of goods or services at no cost to the recipient.
• the term “at a subsidized cost” refers to the transfer of goods or services, wherein at least a portion of the recipient's cost is defened or paid by another party.
• "at a subsidized cost” refers to transfer of goods or services at no cost to the recipient.
• the term “at no cost” refers to the transfer of goods or services with no direct financial expense to the recipient. For example, when detection panels are provided by a manufacturer or distributor to a user (e.g. research scientist) at no cost, the user does not directly pay for the tests.
• detection refers to quantitatively or qualitatively identifying an analyte (e.g., DNA, RNA or a protein) within a sample.
• detection assay refers to a kit, test, or procedure performed for the purpose of detecting an analyte nucleic acid within a sample.
• Detection assays produce a detectable signal or effect when performed in the presence of the target analyte, and include but are not limited to assays incorporating the processes of hybridization, nucleic acid cleavage (e.g., exo- or endonuclease), nucleic acid amplification, nucleotide sequencing, primer extension, or nucleic acid ligation.
• nucleic acid cleavage e.g., exo- or endonuclease
• nucleic acid amplification e.g., exo- or endonuclease
• nucleotide sequencing e.g., primer extension, or nucleic acid ligation.
• the tenn "functional detection oligonucleotide” refers to an oligonucleotide that is used as a component of a detection assay, wherein the detection assay is capable of successfully detecting (i.e., producing a detectable signal) an intended target nucleic acid when the functional detection oligonucleotide provides the oligonucleotide component of the detection assay. This is in contrast to a non-functional detection oligonucleotides, which fail to produce a detectable signal in a detection assay for the particular target nucleic acid when the non-functional detection oligonucleotide is provided as the oligonucleotide component of the detection assay.
• Determining if an oligonucleotide is a functional oligonucleotide can be carried out experimentally by testing the oligonucleotide in the presence of the particular target nucleic acid using the detection assay.
• the term "derived from a different subject," such as samples or nucleic acids derived from a different subjects refers to a samples derived from multiple different individuals. For example, a blood sample comprising genomic DNA from a first person and a blood sample comprising genomic DNA from a second person are considered blood samples and genomic DNA samples that are derived from different subjects.
• a sample comprising five target nucleic acids derived from different subjects is a sample that includes at least five samples from five different individuals.
• the sample may further contain multiple samples from a given individual.
• the term "treating together”, when used in reference to experiments or assays, refers to conducting experiments concu ⁇ ently or sequentially, wherein the results of the experiments are produced, collected, or analyzed together (i.e., during the same time period). For example, a plurality of different target sequences located in separate wells of a multiwell plate or in different portions of a microa ⁇ ay are treated together in a detection assay where detection reactions are carried out on the samples simultaneously or sequentially and where the data collected from the assays is analyzed together.
• test result data refers to data collected from performance of an assay (e.g., to detect or quantitate a gene, SNP or an RNA).
• Test result data may be in any form, i.e., it may be raw assay data or analyzed assay data (e.g., previously analyzed by a different process).
• Collected data that has not been further processed or analyzed is refened to herein as "raw” assay data (e.g., a number conesponding to a measurement of signal, such as a fluorescence signal from a spot on a chip or a reaction vessel, or a number conesponding to measurement of a peak, such as peak height or area, as from, for example, a mass spectrometer, HPLC or capillary separation device), while assay data that has been processed through a further step or analysis (e.g., normalized, compared, or otherwise processed by a calculation) is refened to as "analyzed assay data” or "output assay data".
• raw assay data e.g., a number conesponding to a measurement of signal, such as a fluorescence signal from a spot on a chip or a reaction vessel, or a number conesponding to measurement of a peak, such as peak height or area, as from, for example, a mass spectrometer, HPLC or ca
• genomic information database refers to collections of information (e.g., data) ananged for ease of retrieval, for example, stored in a computer memory.
• genomic information database is a database comprising genomic information, including, but not limited to, polymorphism information (i.e., information pertaining to genetic polymorphisms), genome information (i.e., genomic information), linkage information (i.e., information pertaining to the physical location of a nucleic acid sequence with respect to another nucleic acid sequence, e.g., in a chromosome), and disease association information (i.e., information conelating the presence of or susceptibility to a disease to a physical trait of a subject, e.g., an allele of a subject).
• polymorphism information i.e., information pertaining to genetic polymorphisms
• genome information i.e., genomic information
• linkage information i.e., information pertaining to the physical location of a nucleic acid sequence with respect to another nucle
• Database information refers to information to be sent to a databases, stored in a database, processed in a database, or retrieved from a database.
• Sequence database information refers to database information pertaining to nucleic acid sequences.
• distinct sequence databases refers to two or more databases that contain different information than one another. For example, the dbSNP and GenBank databases are distinct sequence databases because each contains information not found in the other.
• processor and "central processing unit” or “CPU” are used interchangeably and refer to a device that is able to read a program from a computer memory (e.g., ROM or other computer memory) and perform a set of steps according to the program.
• computer memory and “computer memory device” refer to any storage media readable by a computer processor. Examples of computer memory include, but are not limited to, RAM, ROM, computer chips, digital video disc (DVDs), compact discs (CDs), hard disk drives (HDD), and magnetic tape.
• computer readable medium refers to any device or system for storing and providing information (e.g. , data and instructions) to a computer processor.
• hyperlink refers to a navigational link from one document to another, or from one portion (or component) of a document to another. Typically, a hyperlink is displayed as a highlighted word or phrase that can be selected by clicking on it using a mouse to jump to the associated document or documented portion.
• hypertext system refers to a computer-based informational system in which documents (and possibly other types of data entities) are linked together via hyperlinks to form a user-navigable "web.”
• Internet refers to any collection of networks using standard protocols.
• the term includes a collection of interconnected (public and/or private) networks that are linked together by a set of standard protocols (such as TCP/IP, HTTP, and FTP) to form a global, distributed network. While this term is intended to refer to what is now commonly known as the Internet, it is also intended to encompass variations that may be made in the future, including changes and additions to existing standard protocols or integration with other media (e.g., television, radio, etc). The term is also intended to encompass non-public networks such as private (e.g., corporate) Intranets.
• standard protocols such as TCP/IP, HTTP, and FTP
• World Wide Web or “web” refer generally to both (i) a distributed collection of interlinked, user-viewable hypertext documents (commonly refened to as Web documents or Web pages) that are accessible via the Internet, and (ii) the client and server software components which provide user access to such documents using standardized Internet protocols.
• the primary standard protocol for allowing applications to locate and acquire Web documents is HTTP, and the Web pages are encoded using HTML.
• Web and World Wide Web are intended to encompass future markup languages and transport protocols that may be used in place of (or in addition to) HTML and HTTP.
• the term "web site” refers to a computer system that serves informational content over a network using the standard protocols of the World Wide Web. Typically, a Web site conesponds to a particular Internet domain name and includes the content associated with a particular organization. As used herein, the term is generally intended to encompass both (i) the hardware/software server components that serve the informational content over the network, and (ii) the "back end” hardware/software components, including any non-standard or specialized components, that interact with the server components to perform services for Web site users.
• HTML refers to HyperText Markup Language that is a standard coding convention and set of codes for attaching presentation and linking attributes to informational content within documents.
• HTML is based on SGML, the Standard Generalized Markup Language.
• HTML codes (refened to as "tags") are embedded within the informational content of the document.
• the codes are interpreted by the browser and used to parse and display the document.
• HTML tags can be used to create links to other Web documents (commonly refened to as "hyperlinks").
• XML refers to Extensible Markup Language, an application profile that, like HTML, is based on SGML.
• XML differs from HTML in that: information providers can define new tag and attribute names at will; document structures can be nested to any level of complexity; any XML document can contain an optional description of its grammar for use by applications that need to perform structural validation.
• XML documents are made up of storage units called entities, which contain either parsed or unparsed data. Parsed data is made up of characters, some of which form character data, and some of which form markup. Markup encodes a description of the document's storage layout and logical structure.
• XML provides a mechanism to impose constraints on the storage layout and logical structure, to define constraints on the logical structure and to support the use of predefined storage units.
• a software module called an XML processor is used to read XML documents and provide access to their content and structure.
• HTTP refers to HyperText Transport Protocol that is the standard World Wide Web client-server protocol used for the exchange of information (such as HTML documents, and client requests for such documents) between a browser and a Web server.
• HTTP includes a number of different types of messages that can be sent from the client to the server to request different types of server actions. For example, a "GET" message, which has the fonnat GET, causes the server to return the document or file located at the specified URL.
• URL refers to Uniform Resource Locator that is a unique address that fully specifies the location of a file or other resource on the Internet. The general format of a URL is protocol ://machine address:port/ ⁇ ath filename.
• the port specification is optional, and if none is entered by the user, the browser defaults to the standard port for whatever service is specified as the protocol. For example, if HTTP is specified as the protocol, the browser will use the HTTP default port of 80.
• PUSH technology refers to an information dissemination technology used to send data to users over a network. In contrast to the World Wide Web (a "pull" technology), in which the client browser should request a Web page before it is sent, PUSH protocols send the informational content to the user computer automatically, typically based on information pre-specified by the user.
• the term "communication network” refers to any network that allows information to be transmitted from one location to another.
• a communication network for the transfer of information from one computer to another includes any public or private network that transfers information using electrical, optical, satellite transmission, and the like.
• Two or more devices that are part of a communication network such that they can directly or indirectly transmit information from one to the other are considered to be "in electronic communication" with one another.
• a computer network containing multiple computers may have a central computer (“central node") that processes information to one or more sub- computers that carry out specific tasks (“sub-nodes").
• Some networks comprises computers that are in "different geographic locations" from one another, meaning that the computers are located in different physical locations (i.e., aren't physically the same computer, e.g., are located in different countries, states, cities, rooms, etc.).
• the term “detection assay component” refers to a component of a system capable of performing a detection assay. Detection assay components include, but are not limited to, hybridization probes, buffers, and the like.
• a detection assays configured for target detection refers to a collection of assay components that are capable of producing a detectable signal when carried out using the target nucleic acid. For example, a detection assay that has empirically been demonstrated to detect a particular single nucleotide polymorphism is considered a detection assay configured for target detection.
• the phrase "unique detection assay” refers to a detection assay that has a different collection of detection assay components in relation to other detection assays located on the same detection panel.
• a unique assay doesn't necessarily detect a different target (e.g. SNP) than other assays on the same detection panel, but it does have a least one difference in the collection of components used to detect a given target (e.g. a unique detection assay may employ a probe sequences that is shorter or longer in length than other assays on the same detection panel).
• the term “candidate” refers to an assay or analyte, e.g. , a nucleic acid, suspected of having a particular feature or property.
• a “candidate sequence” refers to a nucleic acid suspected of comprising a particular sequence
• a “ candidate oligonucleotide” refers to an oligonucleotide suspected of having a property such as comprising a particular sequence, or having the capability to hybridize to a target nucleic acid or to perform in a detection assay.
• a “candidate detection assay” refers to a detection assay that is suspected of being a valid detection assay.
• the term “detection panel” refers to a substrate or device containing at least two unique candidate detection assays configured for target detection.
• valid detection assay refers to a detection assay that has been shown to accurately predict an association between the detection of a target and a phenotype (e.g. medical condition).
• valid detection assays include, but are not limited to, detection assays that, when a target is detected, accurately predict the phenotype medical 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 99.9% of the time.
• Other examples of valid detection assays include, but are not limited to, detection assays that quality as and/or are marketed as Analyte-Specific Reagents (i.e. as defined by FDA regulations) or In- Vitro Diagnostics (i.e. approved by the FDA).
• kits refers to any delivery system for delivering materials.
• delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. in the appropriate containers) and/or supporting materials (e.g., buffers, written instructions for performing the assay etc.) from one location to another.
• reaction reagents e.g., oligonucleotides, enzymes, etc. in the appropriate containers
• supporting materials e.g., buffers, written instructions for performing the assay etc.
• kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and/or supporting materials.
• fragment kit refers to a delivery systems comprising two or more separate containers that each contain a subportion of the total kit components. The containers may be delivered to the intended recipient together or separately.
• a first container may contain an enzyme for use in an assay, while a second container contains oligonucleotides.
• fragment kit is intended to encompass kits containing Analyte specific reagents (ASR's) regulated under section 520(e) of the Federal Food, Drug, and Cosmetic Act, but are not limited thereto. Indeed, any delivery system comprising two or more separate containers that each contains a subportion of the total kit components are included in the term “fragmented kit.”
• a “combined kit” refers to a delivery system containing all of the components of a reaction assay in a single container (e.g., in a single box housing each of the desired components).
• kit includes both fragmented and combined kits.
• the term "information” refers to any collection of facts or data. In reference to information stored or processed using a computer system(s), including but not limited to internets, the term refers to any data stored in any format (e.g., analog, digital, optical, etc.).
• information related to a subject refers to facts or data pertaining to a subject (e.g., a human, plant, or animal).
• genomic information refers to information pertaining to a genome including, but not limited to, nucleic acid sequences, genes, allele frequencies, RNA expression levels, protein expression, phenotypes conelating to genotypes, etc.
• Allele frequency information refers to facts or data pertaining allele frequencies, including, but not limited to, allele identities, statistical conelations between the presence of an allele and a characteristic of a subject (e.g., a human subject), the presence or absence of an allele in a individual or population, the percentage likelihood of an allele being present in an individual having one or more particular characteristics, etc.
• assay validation information refers to genomic information and/or allele frequency information resulting from processing of test result data (e.g. processing with the aid of a computer). Assay validation information may be used, for example, to identify a particular candidate detection assay as a valid detection assay.
• Detection in biological samples A goal in molecular diagnostics has been to achieve accurate, sensitive detection of analytes in as little time as possible with the least amount of labor and steps as possible.
• One manner in which this is achieved is the multiplex detection of analytes in samples, allowing multiple detection events in a single reaction vessel or solution.
• many of the existing diagnostic methods, including multiplex reaction still require many steps, including sample preparation steps that add to the time, complexity, and cost of conducting reactions.
• the present invention in some embodiments, provides solutions to these problems by providing assay that can be conducted directly in unpurified or untreated biological samples (e.g., blood).
• nucleic acid detection technologies employ enzymes or other reagents that are sensitive to specific salt and pH conditions or that are subject to proteolysis or inhibition by natural factors.
• the present invention provides systems and methods for use of the INVADER assay, alone or in combination with PCR or related technologies, for the direct detection of nucleic acid target sequences in unpurified bodily fluids.
• Example 12 below provides one such example. Such methods may be employed as individual reactions or may be employed as multiplex reactions. Several multiplex embodiments are described in detail below.
• the present invention provides systems, compositions, kits, and methods for detecting one or more target nucleic acids in unpurified (or partially purified) bodily fluids comprising the step of exposing an unpurified bodily fluid to detection assay reagents under conditions such that the target nucleic acid is detected, if present.
• the method is carried out in a single step reaction. For example, once the sample is exposed to the reagents, there is not need to add additional reagents prior to the detection step.
• the method can be carried out in a reaction vessel (e.g., a closed reaction vessel) without the need for addition human or other intervention.
• the method involves an invasive cleavage reaction with or without the polymerase chain reaction. Because of the signal amplification, sensitivity, and ability to quantitate signal using an invasive cleavage reaction, where the polymerase chain reaction is used, limited cycles need only be used (e.g., 20, 15, 12, 10, or fewer).
• the kits for conducting or assisting in such methods may comprise any one or more of the reagents useful in the methods.
• the kits comprise a polymerase, a 5' nuclease (e.g., a FEN-1 endonuclease), and a buffer that permits detectable amplification of the target nucleic acid in an unpurified bodily fluid.
• multiplex PCR Since its introduction in 1988 (Chamberlain, et al. Nucleic Acids Res., 16:11141 (1988)), multiplex PCR has become a routine means of ampUfying multiple genetic loci in a single reaction. This approach has found utility in a number of research, as well as clinical, applications. Multiplex PCR has been described for use in diagnostic virology (Elnifro, et al. Clinical Microbiology Reviews, 13: 559 (2000)), paternity testing (Hidding and Schmitt, Forensic Sci. Int., 113: 47 (2000); Bauer et al, Int. J. Legal Med. 116: 39 (2002)), preimplantation genetic diagnosis (Ouhibi, et al., Cun Womens Health Rep.
• PCR drift is ascribed to stochastic variation in such steps as primer annealing during the early stages of the reaction (Polz and Cavanaugh, Applied and Environmental Microbiology, 64: 3724 (1998)), is not reproducible, and may be more prevalent when very small amounts of target molecules are being amplified (Walsh et al., PCR Methods and Applications, 1: 241 (1992)).
• the other, refened to as PCR selection pertains to the preferential amphfication of some loci based on primer characteristics, amplicon length, G-C content, and other properties of the genome (Polz, supra). Another factor affecting the extent to which PCR reactions can be multiplexed is the inherent tendency of PCR reactions to reach a plateau phase.
• the plateau phase is seen in later PCR cycles and reflects the observation that amplicon generation moves from exponential to pseudo-linear accumulation and then eventually stops increasing. This effect appears to be due to non-specific interactions between the DNA polymerase and the double stranded products themselves.
• the molar ratio of product to enzyme in the plateau phase is typically consistent for several DNA polymerases, even when different amounts of enzyme are included in the reaction, and is approximately 30:1 product: enzyme. This effect thus limits the total amount of double- stranded product that can be generated in a PCR reaction such that the number of different loci amplified must be balanced against the total amount of each amplicon desired for subsequent analysis, e.g. by gel electrophoresis, primer extension, etc.
• the present invention provides methods for substantial multiplexing of PCR reactions by, for example, combining the INVADER assay with multiplex PCR amplification.
• the INVADER assay provides a detection step and signal amplification that allows very large numbers of targets to be detected in a multiplex reaction.
• Direct genotyping by the INVADER assay typically uses from 5 to 100 ng of human genomic DNA per SNP, depending on detection platform. For a small number of assays, the reactions can be performed directly with genomic DNA without target pre-amplification, however, with more than 100,000 INVADER assays being developed and even larger number expected for genome-wide association studies, the amount of sample DNA may become a limiting factor. Because the INVADER assay provides from 10 6 to 10 7 fold amplification of signal, multiplexed PCR in combination with the INVADER assay would use only limited target amplification as compared to a typical PCR.
• low target amplification level alleviates interference between individual reactions in the rnixture and reduces the inhibition of PCR by it's the accumulation of its products, thus providing for more extensive multiplexing. Additionally, it is contemplated that low amplification levels decrease a probability of target cross-contamination and decrease the number of PCR-induced mutations. Uneven amplification of different loci presents one of biggest challenges in the development of multiplexed PCR. Difference in amplification factors between two loci may result in a situation where the signal generated by an INVADER reaction with a slow-amplifying locus is below the limit of detection of the assay, while the signal from a fast-amplifying locus is beyond the saturation level of the assay. This problem can be addressed in several ways.
• the INVADER reactions can be read at different time points, e.g., in real-time, thus significantly extending the dynamic range of the detection.
• multiplex PCR can be performed under conditions that allow different loci to reach more similar levels of amplification. For example, primer concentrations can be limited, thereby allowing each locus to reach a more uniform level of amplification. In yet other embodiments, concentrations of PCR primers can be adjusted to balance amplification factors of different loci.
• the present invention provides for the design and characteristics of highly multiplex PCR including hundreds to thousands of products in a single reaction.
• the target pre- amplification provided by hundred-plex PCR reduces the amount of human genomic DNA required for JNVADER-based SNP genotyping to less than 0.1 ng per assay.
• the specifics of highly multiplex PCR optimization and a computer program for the primer design are described below.
• the present invention further provides methods of conducting target and signal amplification reactions in a single reaction vessel with no subsequent manipulations or reagent additions beyond initial reaction set-up. Such combined reactions are suitable for quantitative analysis of limiting target quantities in very short reaction times.
• the following discussion provides a description of certain prefened illustrative embodiments of the present invention and is not intended to limit the scope of the present invention.
• the INVADER assay can be used for the detection of single nucleotide polymorphisms (SNPs) with as little as 100-10 ng of genomic DNA without the need for target pre-amplification.
• SNPs single nucleotide polymorphisms
• the amount of sample DNA becomes a limiting factor for large scale analysis.
• multiplex PCR coupled with the INVADER assay requires only limited target amplification (10 3 -10 4 ) as compared to typical multiplex PCR reactions which require extensive amplification (10 9 -l 0 12 ) for conventional gel detection methods.
• the low level of target amplification used for INVADERTM detection provides for more extensive multiplexing by avoiding amplification inhibition commonly resulting from target accumulation.
• the present invention provides methods and selection criteria that allow primer sets for multiplex PCR to be generated (e.g. that can be coupled with a detection assay, such as the INVADER assay).
• software applications of the present invention automated multiplex PCR primer selection, thus allowing highly multiplexed PCR with the primers designed thereby.
• MAP INVADER Medically Associated Panel
• the methods, software, and selection criteria of the present invention allowed accurate genotyping of 94 of the 101 possible amplicons (-93%) from a single PCR reaction.
• the original PCR reaction used only 10 ng of hgDNA as template, conesponding to less than 150 pg hgDNA per INVADER assay.
• the INVADER assay allows for the simultaneous detection of two distinct alleles in the same reaction using an isothermal, single addition format.
• Allele discrimination takes place by "structure specific" cleavage of the Probe, releasing a 5 ' flap which conesponds to a given polymorphism. In the second reaction, the released 5' flap mediates signal generation by cleavage of the appropriate FRET cassette.
• Creation of one of the primer pairs (both a forward and reverse primer) for a 101 primer sets from sequences available for analysis on the INVADER Medically Associated Panel using one embodiment of the software application of the present invention involves sample input file of a single entry (e.g. target sequence information for a single target sequence containing a SNP that is processed the method and software of the present invention).
• the target sequence information includes Third Wave Technologies's SNP#, short name identifier, and sequence with the SNP location indicated in brackets.
• Sample output file of a the same entry includes the sequence of the footprint region (capital letters flanking SNP site, showing region where INVADER assay probes hybridize to this target sequence in order to detect the SNP in the target sequence), forward and reverse primer sequences (bold), and their conesponding Tms.
• the selection of primers to make a primer set capable of multiplex includes the sequence of the footprint region (capital letters flanking SNP site, showing region where INVADER assay probes hybridize to this target sequence in order to detect the SNP in the target sequence), forward and reverse primer sequences (bold), and their conesponding Tms.
• PCR is performed in automated fashion (e.g. by a software application). Automated primer selection for multiplex PCR may be accomplished employing a software program designed as shown by the flow chart in Figure 8. Multiplex PCR commonly requires extensive optimization to avoid biased amplification of select amplicons and the amplification of spurious products resulting from the formation of primer-dimers.
• the present invention provides methods and software application that provide selection criteria to generate a primer set configured for multiplex PCR, and subsequent use in a detection assay (e.g. INVADER detection assays). In some embodiments, the methods and software applications of the present invention start with user defined sequences and conesponding SNP locations.
• tlie methods and/or software application determines a footprint region within the target sequence (the minimal amplicon required for INVADER detection) for each sequence.
• the footprint region includes the region where assay probes hybridize, as well as any user defined additional bases extending outward therefore (e.g. 5 additional bases included on each side of where the assay probes hybridize).
• primers are designed outward from the footprint region and evaluated against several criteria, including the potential for primer-dimer formation with previously designed primers in the cunent multiplexing set. This process may be continuedthrough multiple iterations of the same set of sequences until primers against all sequences in the cunent multiplexing set can be designed.
• multiplex PCR may be carried out, for example, under standard conditions using only 10 ng of hgDNA as template. After 10 min at 95°C, Taq (2.5 units) may be added to a 50ul reaction and PCR carried out for 50 cycles. The PCR reaction may be diluted and loaded directly onto an INVADER MAP plate (3ul/well). An additional 3ul of 15mM MgCl 2 may be added to each reaction on the INVADER MAP plate and covered with 6ul of mineral oil. The entire plate may then be heated to 95°C for 5 min. and incubated at 63°C for 40 min.
• FAM and RED fluorescence may then be measured on a Cytofluor 4000 fluorescent plate reader and "Fold Over Zero" (FOZ) values calculated for each amplicon.
• Results from each SNP may be color coded in a table as “pass” (green), “mis-call” (pink), or “no-call” (white) (See, Example 2 below).
• the number of PCR reactions is from about 1 to about 10 reactions.
• the number of PCR reactions is from about 10 to about 50 reactions. In further embodiments, the number of PCR reactions is from about 50 to about 100. In additional embodiments, the number of PCR reactions is from about than 100 to 1,000. In still other embodiments, the number of PCR reactions is greater than 1,000.
• the present invention also provides methods to optimize multiplex PCR reactions (e.g. once a primer set is generated, the concentration of each primer or primer pair may be optimized). For example, once a primer set has been generated and used in a multiplex PCR at equal molar concentrations, the primers may be evaluated separately such that the optimum primer concentration is determined such that the multiplex primer set performs better.
• Multiplex PCR reactions are being recognized in the scientific, research, clinical and biotechnology industries as potentially time effective and less expensive means of obtaining nucleic acid information compared to standard, monoplex PCR reactions. Instead of performing only a single amplification reaction per reaction vessel (tube or well of a multi-well plate for example), numerous amplification reactions are performed in a single reaction vessel. The cost per target is theoretically lowered by eliminating technician time in assay set-up and data analysis, and by the substantial reagent savings (especially enzyme cost). Another benefit of the multiplex approach is that far less target sample is required.
• the dimers form at or near the 3' ends of the primers, no amplification or very low levels of amplification will occur, since the 3' end is required for the priming event.
• the methods, systems and applications of the present help prevent primer dimers in large sets of primers, making the set suitable for highly multiplexed PCR.
• the order in which primer pairs are designed can influence the total number of • compatible primer pairs for a reaction. For example, if a first set of primers is designed for a first target region that happens to be an A/T rich target region, these primer will be A T rich.
• the present invention randomizes the order in which primer sets are designed (See, Figure 8). Furthermore, in some embodiments, the present invention re-orders the set of input target sequences in a plurality of different, random orders to maximize the number of compatible primer sets for any given multiplex reaction (See, Figure 8).
• N[l] is an A or C (in alternative embodiments, N[l] is a G or T).
• N[2]-N[l] of each of the forward and reverse primers designed should not be complementary to N[2]-N[l] of any other oligonucleotide.
• N[3]-N[2]-N[l] should not be complementary to N[3]- N[2]-N[l] of any other oligonucleotide.
• the next base in the 5' direction for the forward primer or the next base in the 3 ' direction for the reverse primer may be evaluated as an N[l] site. This process is repeated, in conjunction with the target randomization, until all criteria are met for all, or a large majority of, the targets sequences (e.g. 95% of target sequences can have primer pairs made for the primer set that fulfill these criteria).
• Another challenge to be overcome in a multiplex primer design is the balance between actual, required nucleotide sequence, sequence length, and the oligonucleotide melting temperature (Tm) constraints.
• Tm oligonucleotide melting temperature
• the primers in a multiplex primer set in a reaction should function under the same reaction conditions of buffer, salts and temperature, they need therefore to have substantially similar Tin's, regardless of GC or AT richness of the region of interest.
• the present invention allows for primer design which meet minimum Tm and maximum Tm requirements and minimum and maximum length requirements.
• x is selected such the primer has a predetermined melting temperature (e.g. bases are included in the primer until the primer has a calculated melting temperature of about 50 degrees Celsius).
• a predetermined melting temperature e.g. bases are included in the primer until the primer has a calculated melting temperature of about 50 degrees Celsius.
• the products of a PCR reaction are used as the target material for another nucleic acid detection means, such as a hybridization-type detection assays, or the INVADER reaction assays for example.
• Selection criteria may be employed such that the primers designed for a multiplex primer set do not react (e.g. hybridize with, or trigger reactions) with oligonucleotide components of a detection assay. For example, in order to prevent primers from reacting with the FRET oligonucleotide of a bi-plex INVADER assay, certain homology criteria is employed.
• N[4]-N[3]-N[2]-N[l]-3' is selected such that it is less than 90% homologous with the FRET or INVADER oligonucleotides.
• N[4]-N[3]-N[2]-N[l]-3' is selected for each primer such that it is less than 80% homologous with the FRET or INVADER oligonucleotides.
• N[4]-N[3]-N[2]-N[l]-3' is selected for each primer such that it is less than 70% homologous with the FRET or INVADER oligonucleotides.
• some primer pairs may not meet all of the stated criteria (these may be rejected as enors).
• set 31 fails. In the method of the present invention, set 31 may be flagged as failing, and the method could continue through the list of 100 targets, again flagging those sets which do not meet the criteria (See Figure 8).
• Figure 8 shows a flow chart with the basic flow of certain embodiments of the methods and software application of the present invention. In prefened embodiments, the processes detailed in Figure 8 are inco ⁇ orated into a software application for ease of use (although, the methods may also be performed manually using, for example, Figure 8 as a guide). Target sequences and or primer pairs are entered into the system shown in Figure 8.
• the first set of boxes show how target sequences are added to the list of sequences that have a footprint determined (See “B” in Figure 8), while other sequences are passed immediately into the primer set pool (e.g. PDPass, those sequences that have been previously processed and shown to work together without forming Primer dimers or having reactivity to FRET sequences), as well as DimerTest entries (e.g. pair or primers a user wants to use, but that has not been tested yet for primer dimer or fret reactivity).
• the initial set of boxes leading up to "end of input” sort the sequences so they can be later processed properly.
• the primer pool is basically cleared or "emptied” to start a fresh run.
• Target sequences are then sent to "B” to be processed, and DimerTest pairs are sent to "C” to be processed.
• Target sequences are sent to "B", where a user or software application determines the footprint region for the target sequence (e.g. where the assay probes will hybridize in order to detect the mutation (e.g. SNP) in the target sequence). It is important to design this region (which the user may further expand by defining that additional bases past the hybridization region be added) such that the primers that are designed fully encompass this region.
• the software application INVADER CREATOR is used to design the JNVADER oligonuclotide and downstream probes that will hybridize with the target region (although any type of program of system could be used to create any type of probes a user was interested in designing probes for, and thus detei ⁇ nining the footprint region for on the target sequence).
• the core footprint region is then defined by the location of these two assay probes on the target.
• the system starts from the 5' edge of the footprint and travels in the 5' direction until the first base is reached, or until the first A or C (or G or T) is reached. This is set as the initial starting point for defining the sequence of the forward primer (i.e. this serves as the initial N[l] site).
• the sequence of the primer for the forward primer is the same as those bases encountered on the target region. For example, if the default size of the primer is set as 12 bases, the system starts with the bases selected as N[l] and then adds the next 11 bases found in the target sequences. This 12-mer primer is then tested for a melting temperature (e.g. using JNVADER CREATOR), and additional bases are added from the target sequence until the sequence has a melting temperature that is designated by the user as the default minimum and maximum melting temperatures (e.g. about 50 degrees Celsius, and not more than 55 degrees Celsius).
• a melting temperature e.g. using JNVADER CREATOR
• the system employs the formula 5'-N[x]-N[x-l]- -N[4]-N[3]-N[2]-N[l]-3', and x is initially 12. Then the system adjusts x to a higher number
• a maximum primer size is employed as a default parameter to serve as an upper limit on the length of the primers designed.
• the maximum primer size is about 30 bases (e.g. 29 bases, 30, bases, or 31 bases).
• the default settings e.g. minimum and maximum primer size, and minimum and maximum Tm
• the next box in Figure 8 is used to determine if the primer that has been designed so far will cause primer-dimer and/or fret reactivity (e.g. with the other sequences already in the pool). The criteria used for this determination are explained above.
• the forward primer is added to the primer pool. However, if the forward primer fails this criteria, as shown in Figure 8, the starting point (N[l] is moved) one nucleotide in the 5' direction (or to the next A or C, or next G or T).
• the system first checks to make sure shifting over leaves enough room on the target sequence to successfully make a primer. If yes, the system loops back and check this new primer for melting temperature. However, if no sequence can be designed, then the target sequence is flagged as an enor (e.g. indicating that no forward primer can be made for this target). This same process is then repeated for designing the reverse primer, as shown in Figure 8.
• the DimerTest pair passes the criteria, they are added to the primer set pool, and then the system goes back to "C” if there are more DimerTest pairs to be evaluated, or or goes on to "D” if there are no more DimerTest pairs to be evaluated.
• the pool of primers that has been created is evaluated. The first step in this section is to examine the number of enor (failures) generated by this particular randomized run of sequences. If there were no enors, this set is the best set as maybe ouputted to a user. If there are more than zero enors, the system compares this run to any other previous runs to see what run resulted in the fewest enors.
• the cunent run has fewer enors, it is designated as the cunent best set.
• the system may go back to "A" to start the run over with another randomized set of the same sequences, or the pre-set maximum number of runs (e.g. 5 runs) may have been reached on this run (e.g. this was the 5th run, and the maximum number of runs was set as 5). If the maximum has been reached, then the best set is outputted as the best set.
• This best set of primers may then be used to generate as physical set of oligonucleotides such that a multiplex PCR reaction may be carried out.
• Another challenge to be overcome with multiplex PCR reactions is the unequal amplicon concentrations that result in a standard multiplex reaction.
• the different loci targeted for amplification may each behave differently in the amplification reaction, yielding vastly different concentrations of each of the different amplicon products.
• the present invention provides methods, systems, software applications, computer systems, and a computer data storage medium that may be used to adjust primer concentrations relative to a first detection assay read (e.g. INVADER assay read), and then with balanced primer concentrations come close to substantially equal concentrations of different amplicons.
• the concentrations for various primer pairs may be determined experimentally.
• the yield of the single step process is then used to successfully obtain test result data for, for example, several hundred assays.
• each well on a 384 well plate can have a different detection assay thereon.
• the results of the single step mutliplex PCR reaction has amplified 384 different targets of genomic DNA, and provides you with 384 test results for each plate. Where each well has a plurality of assays even greater efficiencies can be obtained. Therefore, the present invention provides the use of the concentration of each primer set in highly multiplexed PCR as a parameter to achieve an unbiased amplification of each PCR product. Any PCR includes primer annealing and primer extension steps.
• primer annealing kinetics can become a rate hmiting step and PCR amplification factor should strongly depend on primer concentration, association rate constant of the primers, and the annealing time.
• the amplification factor should strongly depend on primer concentration.
• biased loci amplification whether it is caused by individual association rate constants, primer extension steps or any other factors, can be conected by adjusting primer concentration for each primer set in the multiplex PCR.
• the adjusted primer concentrations can be also used to conect biased perfonnance of INVADER assay used for analysis of PCR pre- amplified loci.
• the present invention has demonstrated a linear relationship between amplification efficiency and primer concentration and used this equation to balance primer concentrations of different amplicons, resulting in the equal amplification often different amplicons in Example 1.
• This technique may be employed on any size set of multiplex primer pairs.
• Detection Assay Design The following section describes detection assays that may be employed with the present invention. For example, many different assays may be used to determine the footprint on the target nucleic sequence, and then used as the detection assay run on the output of the multiplex PCR (or the detection assays may be run simultaneously with the multiplex PCR reaction). There are a wide variety of detection technologies available for determining the sequence of a target nucleic acid at one or more locations. For example, there are numerous technologies available for detecting the presence or absence of SNPs. Many of these techniques require the use of an ohgonucleotide to hybridize to the target.
• the oligonucleotide is then cleaved, elongated, ligated, disassociated, or otherwise altered, wherein its behavior in the assay is monitored as a means for characterizing the sequence of the target nucleic acid.
• a number of these technologies are described in detail, in Section IV, below.
• the present invention provides systems and methods for the design of oligonucleotides for use in detection assays.
• the present invention provides systems and methods for the design of oligonucleotides that successfully hybridize to appropriate regions of target nucleic acids (e.g., regions of target nucleic acids that do not contain secondary structure) under the desired reaction conditions (e.g., temperature, buffer conditions, etc.) for the detection assay.
• the systems and methods also allow for the design of multiple different oligonucleotides (e.g., oligonucleotides that hybridize to different portions of a target nucleic acid or that hybridize to two or more different target nucleic acids) that all function in the detection assay under the same or substantially the same reaction conditions. These systems and methods may also be used to design control samples that work under the experimental reaction conditions. While the systems and methods of the present invention are not limited to any particular detection assay, the following description illustrates the invention when used in conjunction with the INVADER assay (Third Wave Technologies, Madison WI; See e.g., U.S. Pat. Nos.
• the JNVADER assay provides means for forming a nucleic acid cleavage structure that is dependent upon the presence of a target nucleic acid and cleaving the nucleic acid cleavage o structure so as to release distinctive cleavage products.
• 5' nuclease activity for example, is used to cleave the target-dependent cleavage structure and the resulting cleavage products are indicative of the presence of specific target nucleic acid sequences in the sample.
• invasive cleavage 5 can occur.
• the cleavage agent e.g., a 5' nuclease
• the upstream oligonucleotide can be made to cleave the downstream oligonucleotide at an internal site in such a way that a distinctive fragment is produced.
• the INVADER assay provides detections assays in which the target nucleic acid is reused or recycled during multiple rounds of hybridization with0 oligonucleotide probes and cleavage of the probes without the need to use temperature cycling (i.e., for periodic denaturation of target nucleic acid strands) or nucleic acid synthesis (i.e., for the polymerization-based displacement of target or probe nucleic acid strands).
• temperature cycling i.e., for periodic denaturation of target nucleic acid strands
• nucleic acid synthesis i.e., for the polymerization-based displacement of target or probe nucleic acid strands.
• probe-probe displacement or through an equilibrium between probe/target5 association and disassociation, or through a combination comprising these mechanisms, (Reynaldo, et al, J. Mol. Biol. 97: 511-520 [2000]), multiple probes can hybridize to the same target, allowing multiple cleavages, and the generation of multiple cleavage products.
• ⁇ B. Oligonucleotide Design for the INVADER assay In some embodiments where an oligonucleotide is designed for use in the INVADER assay to detect a SNP, the sequence(s) of interest are entered into the INVADERCREATOR program (Third Wave Technologies, Madison, WI). As described above, sequences may be input for analysis from any number of sources, either directly into the computer hosting the INVADERCREATOR program, or via a remote computer linked through a communication network (e.g., a LAN, Intranet or Internet network). The program designs probes for both the sense and antisense strand. Strand selection is generally based upon the ease of synthesis, miriimization of secondary structure formation, and manufacturability.
• oligonucleotide probes may be designed to operate at a pre-selected assay temperature (e.g., 63°C). Based on these criteria, a final probe set (e.g., primary probes for 2 alleles and an INVADER oligonucleotide) is selected.
• a pre-selected assay temperature e.g., 63°C
• the INVADERCREATOR system is a web-based program with secure site access that contains a link to BLAST (available at the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health website) and that can be linked to RNAstructure (Mathews et al, RNA 5:1458 [1999]), a software program that inco ⁇ orates mfold (Zuker, Science, 244:48 [1989]).
• RNAstructure tests the proposed oligonucleotide designs generated by INVADERCREATOR for potential uni- and bimolecular complex formation.
• INVADERCREATOR is open database connectivity (ODBC)-compliant and uses the Oracle database for export/integration.
• the INVADERCREATOR system was configured with Oracle to work well with UNIX systems, as most genome centers are UNIX-based.
• the INVADERCREATOR analysis is provided on a separate server (e.g., a Sun server) so it can handle analysis of large batch jobs. For example, a customer can submit up to 2,000 SNP sequences in one email.
• the server passes the batch of sequences on to the INVADERCREATOR software, and, when initiated, the program designs detection assay oligonucleotide sets. In some embodiments, probe set designs are returned to the user within 24 hours of receipt of the sequences.
• Each INVADER reaction includes at least two target sequence-specific, unlabeled oligonucleotides for the primary reaction: an upstream INVADER oligonucleotide and a downstream Probe ohgonucleotide.
• the INVADER oligonucleotide is generally designed to bind stably at the reaction temperature, while the probe is designed to freely associate and disassociate with the target strand, with cleavage occurring only when an uncut probe hybridizes adjacent to an overlapping INVADER ohgonucleotide.
• the probe includes a 5' flap or "arm" that is not complementary to the target, and this flap is released from the probe when cleavage occurs.
• the released flap participates as an JNVADER oligonucleotide in a secondary reaction.
• the following discussion provides one example of how a user interface for an INVADERCREATOR program may be configured.
• the user opens a work screen, e.g., by clicking on an icon on a desktop display of a computer (e.g., a Windows desktop).
• the user enters information related to the target sequence for wliich an assay is to be designed.
• the user enters a target sequence.
• the user enters a code or number that causes retrieval of a sequence from a database.
• additional information may be provided, such as the user's name, an identifying number associated with a target sequence, and/or an order number.
• the user indicates (e.g. via a check box or drop down menu) that the target nucleic acid is DNA or RNA. Jh other prefened embodiments, the user indicates the species from which the nucleic acid is derived. In particularly prefened embodiments, the user indicates whether the design is for monoplex (i.e., one target sequence or allele per reaction) or multiplex (i.e., multiple target sequences or alleles per reaction) detection. When the requisite choices and entries are complete, the user starts the analysis process. In one embodiment, the user clicks a "Go Design It" button to continue.
• the software validates the field entries before proceeding. In some embodiments, the software verifies that any required fields are completed with the appropriate type of information. In other embodiments, the sof ware verifies that the input sequence meets selected requirements (e.g., minimum or maximum length, DNA or RNA content). If entries in any field are not found to be valid, an enor message or dialog box may appear. In prefened embodiments, the enor message indicates which field is incomplete and/or inconect. Once a sequence entry is verified, the software proceeds with the assay design. In some embodiments, the information supplied in the order entry fields specifies what type of design will be created. In prefened embodiments, the target sequence and multiplex check box specify which type of design to create.
• Design options include but are not limited to SNP assay, Multiplexed SNP assay (e.g., wherein probe sets for different alleles are to be combined in a single reaction), Multiple SNP assay (e.g., wherein an input sequence has multiple sites of variation for which probe sets are to be designed), and Multiple Probe Arm assays.
• the INVADERCREATOR software is started via a Web Order
• WebOE WebOE Entry
• applet ⁇ param> tags
• the user chooses two or more designs to work with. In some embodiments, this selection opens a new screen view (e.g., a Multiple SNP Design
• the software creates designs for each locus in the target sequence, scoring each, and presents them to the user in this screen view. The user can then choose any two designs to work with. In some embodiments, the user chooses a first and second design (e.g., via a menu or buttons) and clicks a "Go Design It" button to continue.
• T m melting temperature
• RNA DNA heteroduplex formation parameters appropriate for RNA DNA heteroduplex formation may be used. Because the assay's salt concentrations are often different than the solution conditions in which the nearest-neighbor parameters were obtained (IM NaCl and no divalent metals), and because the presence and concentration of the enzyme influence optimal reaction temperature, an adjustment should be made to the calculated T m to detennine the optimal temperature at which to perform a reaction. One way of compensating for these factors is to vary the value provided for the salt concentration within the melting temperature calculations. This adjustment is termed a 'salt conection 1 .
• salt conection refers to a variation made in the value provided for a salt concentration for the pu ⁇ ose of reflecting the effect on a T m calculation for a nucleic acid duplex of a non-salt parameter or condition affecting said duplex. Variation of the values provided for the strand concentrations will also affect the outcome of these calculations.
• the algorithm for used for calculating probe-target melting temperature has been adapted for use in predicting optimal INVADER assay reaction temperature.
• the average deviation between optimal assay temperatures calculated by this method and those experimentally dete ⁇ nined is about 1.5 °C.
• the length of the downstream probe to a given SNP is defined by the temperature selected for ranning the reaction (e.g., 63°C). Starting from the position of the variant nucleotide on the target DNA (the target base that is paired to the probe nucleotide 5' of the intended cleavage site), and adding on the 3' end, an iterative procedure is used by which the length of the target-binding region of the probe is increased by one base pair at a time until a calculated optimal reaction temperature (T m plus salt conection to compensate for enzyme effect) matching the desired reaction temperature is reached.
• the non-complementary arm of the probe is preferably selected to allow the secondary reaction to cycle at the same reaction temperature.
• the entire probe oligonucleotide is screened using programs such as mfold (Zuker, Science, 244: 48 [1989]) or Oligo 5.0 (Rychlik and Rhoads, Nucleic Acids Res, 17: 8543 [1989]) for the possible formation of dimer complexes or secondary structures that could interfere with the reaction.
• mfold Zauker, Science, 244: 48 [1989]
• Oligo 5.0 Oligo 5.0
• the 3' end of the INVADER oligonucleotide is designed to have a nucleotide not complementary to either allele suspected of being contained in the sample to be tested.
• the mismatch does not adversely affect cleavage (Lyamichev et al, Nature Biotechnology, 17: 292 [1999]), and it can enhance probe cycling, presumably by minimizing coaxial stabilization effects between the two probes.
• Additional residues complementary to the target DNA starting from residue N-l are then added in the 5' direction until the stability of the INVADER oligonucleotide-target hybrid exceeds that of the probe (and therefore the planned assay reaction temperature), generally by 15-20 °C.
• the all of the probe sequences may be selected to allow the primary and secondary reactions to occur at the same optimal temperature, so that the reaction steps can run simultaneously.
• the probes may be designed to operate at different optimal temperatures, so that the reaction steps are not simultaneously at their temperature optima.
• the software provides the user an opportunity to change various aspects of the design including but not limited to: probe, target and INVADER oligonucleotide temperature optima and concentrations; blocking groups; probe arms; dyes, capping groups and other adducts; individual bases of the probes and targets (e.g., adding or deleting bases from the end of targets and/or probes, or changing internal bases in the INVADER and/or probe and/or target oligonucleotides).
• changes are made by selection from a menu.
• changes are entered into text or dialog boxes. In prefened embodiments, this option opens a new screen (e.g., a Designer Worksheet view).
• the software provides a scoring system to indicate the quality (e.g., the likelihood of performance) of the assay designs.
• the scoring system includes a starting score of points (e.g., 100 points) wherein the starting score is indicative of an ideal design, and wherein design features known or suspected to have an adverse affect on assay performance are assigned penalty values. Penalty values may vary depending on assay parameters other than the sequences, including but not limited to the type of assay for which the design is intended (e.g., monoplex, multiplex) and the temperature at which the assay reaction will be performed.
• the following example provides an illustrative scoring criteria for use with some embodiments of the INVADER assay based on an intelligence defined by experimentation.
• design features that may incur score penalties include but are not limited to the following [penalty values are indicated in brackets, first number is for lower temperature assays (e.g., 62-64 °C), second is for higher temperature assays (e.g., 65-66 °C)]:
• a probe has 5-base stretch (i.e., 5 of the same base in a row) containing the polymo ⁇ hism;
• a probe has 5-base stretch adjacent to the polymo ⁇ hism; 4. [50:50] a probe has 5-base stretch one base from the polymo ⁇ hism;
• a probe has 5-base stretch two bases from the polymo ⁇ hism
• probe hybridizing region is short (13 bases or less for designs 65-67°C; 12 bases or less for designs 62-64°C)
• probe hybridizing region is long (29 bases or more for designs 65-67°C, 28 bases or more for designs 62-64°C) 12. [5:5] probe hybridizing region length — per base additional penalty
• INVADER oligonucleotide 6-base stretch is of Gs - additional penalty 20.
• probe hybridizing region is 14, 15 or 24-28 bases long (65-67°C) or 13,14 or 26,27 bases long (62-64°C) 21.
• a probe has a 4-base stretch of Gs containing the polymo ⁇ hism
• temperatures for each of the oligonucleotides in the designs are recomputed and scores are recomputed as changes are made.
• score descriptions can be seen by clicking a "descriptions" button.
• a BLAST search option is provided.
• a BLAST search is done by clicking a "BLAST Design” button. In some embodiments, this action brings up a dialog box describing the BLAST process.
• the BLAST search results are displayed as a highlighted design on a Designer Worksheet.
• a user accepts a design by clicking an "Accept" button.
• the program approves a design without user intervention.
• the program sends the approved design to a next process step (e.g., into production; into a file or database).
• the program provides a screen view (e.g.. an Output Page), allowing review of the final designs created and allowing notes to be attached to the design.
• the user can return to the Designer Worksheet (e.g., by clicking a "Go Back” button) or can save the design (e.g., by clicking a "Save It” button) and continue (e.g., to submit the designed oligonucleotides for production).
• the program provides an option to create a screen view of a design optimized for printing (e.g., a text-only view) or other export (e.g., an Output view).
• a design optimized for printing e.g., a text-only view
• other export e.g., an Output view
• the Output view provides a description of the design particularly suitable for printing, or for exporting into another application (e.g., by copying and pasting into another application).
• the Output view opens in a separate window.
• the present invention is not limited to the use of the INVADERCREATOR software.
• GCG Wisconsin Package Genetics computer Group, Madison, WI
• Vector NTI Vector NTI
• Other detection assays may be used in the present invention.
• variant sequences are detected using a direct sequencing technique.
• DNA samples are first isolated from a subject using any suitable method.
• the region of interest is cloned into a suitable vector and amplified by growth in a host cell (e.g., a bacteria).
• DNA in the region of interest is amplified using PCR.
• DNA in the region of interest is sequenced using any suitable method, including but not limited to manual sequencing using radioactive marker nucleotides, or automated sequencing.
• the results of the sequencing are displayed using any suitable method. The sequence is examined and the presence or absence of a given SNP or mutation is determined.
• variant sequences are detected using a
• the PCR assay comprises the use of oligonucleotide primers that hybridize only to the variant or wild type allele (e.g., to the region of polymo ⁇ hism or mutation). Both sets of primers are used to amplify a sample of DNA. If only the mutant primers result in a PCR product, then the patient has the mutant allele. If only the wild-type primers result in a PCR product, then the patient has the wild type allele.
• variant sequences are detected using a fragment length polymo ⁇ hism assay.
• a fragment length polymo ⁇ hism assay a unique DNA banding pattern based on cleaving the DNA at a series of positions is generated using an enzyme (e.g. , a restriction enzyme or a CLEAVASE I [Third Wave Technologies, Madison, WI] enzyme).
• an enzyme e.g. , a restriction enzyme or a CLEAVASE I [Third Wave Technologies, Madison, WI] enzyme.
• DNA fragments from a sample containing a SNP or a mutation will have a different banding pattern than wild type.
• RFLP restriction fragment length polymo ⁇ hism assay
• the region of interest is first isolated using PCR.
• the PCR products are then cleaved with restriction enzymes known to give a unique length fragment for a given polymo ⁇ hism.
• the restriction-enzyme digested PCR products are generally separated by gel electrophoresis and may be visualized by ethidium bromide staining.
• the length of the fragments is compared to molecular weight markers and fragments generated from wild-type and mutant controls.
• variant sequences are detected using a CLEAVASE fragment length polymo ⁇ hism assay (CFLP; Third Wave Technologies, Madison, WI; See e.g., U.S. Patent Nos.
• one or both strands are labeled. Then, DNA strands are separated by heating. Next, the reactions are cooled to allow intrastrand secondary structure to form.
• the PCR products are then treated with the CLEAVASE I enzyme to generate a series of fragments that are unique to a given SNP or mutation.
• the CLEAVASE enzyme treated PCR products are separated and detected (e.g., by denaturing gel electrophoresis) and visualized (e.g., by autoradiography, fluorescence imaging or staining). The length of the fragments is compared to molecular weight markers and fragments generated from wild-type and mutant controls.
• hybridization assays In prefened embodiments of the present invention, variant sequences are detected a hybridization assay.
• a hybridization assay the presence of absence of a given SNP or mutation is determined based on the ability of the DNA from the sample to hybridize to a complementary DNA molecule (e.g., a oligonucleotide probe).
• a complementary DNA molecule e.g., a oligonucleotide probe.
• a variety of hybridization assays using a variety of technologies for hybridization and detection are available. A description of a selection of assays is provided below.
• a. Direct Detection of Hybridization In some embodiments, hybridization of a probe to the sequence of interest (e.g., a SNP or mutation) is detected directly by visualizing a bound probe (e.g.
• RNA Northern
• DNA or RNA is then cleaved with a series of restriction enzymes that cleave infrequently in the genome and not near any of the markers being assayed.
• the DNA or RNA is then separated (e.g., on an agarose gel) and transfened to a membrane.
• a labeled (e.g., by inco ⁇ orating a radionucleotide) probe or probes specific for the SNP or mutation being detected is allowed to contact the membrane under a condition or low, medium, or high stringency conditions. Unbound probe is removed and the presence of binding is detected by visualizing the labeled probe.
• variant sequences are detected using a labeled (e.g., by inco ⁇ orating a radionucleotide) probe or probes specific for the SNP or mutation being detected is allowed to contact the membrane under a condition or low, medium, or high stringency conditions. Unbound probe is removed and the presence of binding is detected by visualizing the labeled probe.
• variant sequences are detected using a labeled (e.g., by inco ⁇ orating a radionucleotide) probe or probes specific for the SNP or mutation being detected is allowed to contact the membrane under a condition or low, medium, or high stringency conditions. Unbound probe is removed and the presence of binding is detected by visual
• DNA chip hybridization assay In this assay, a series of oligonucleotide probes are affixed to a solid support. The ohgonucleotide probes are designed to be unique to a given SNP or mutation.
• the DNA sample of interest is contacted with the DNA "chip” and hybridization is detected.
• the DNA chip assay is a GeneChip (Affymetrix, Santa Clara, CA; See e.g., U.S. Patent Nos. 6,045,996; 5,925,525; and 5,858,659; each of which is herein inco ⁇ orated by reference) assay.
• Probe anays are manufactured by Affymetrix's light-directed chemical synthesis process, which combines solid-phase chemical synthesis with, photolithographic fabrication techniques employed in the semiconductor industry. Using a series of photolithographic masks to define chip exposure sites, followed by specific chemical synthesis steps, the process constructs high-density anays of oligonucleotides, with each probe in a predefined position in the anay. Multiple probe anays are synthesized simultaneously on a large glass wafer.
• the wafers are then diced, and individual probe anays are packaged in injection-molded plastic cartridges, which protect them from the environment and serve as chambers for hybridization.
• the nucleic acid to be analyzed is isolated, amplified by PCR, and labeled with a fluorescent reporter group.
• the labeled DNA is then incubated with the anay using a fluidics station.
• the anay is then inserted into the scanner, where patterns of hybridization are detected.
• the hybridization data are collected as light emitted from tlie fluorescent reporter groups already incorporated into the target, which is bound to the probe anay. Probes that perfectly match the target generally produce stronger signals than those that have mismatches.
• a DNA microchip containing electronically captured probes (Nanogen, San Diego, CA) is utilized (See e.g., U.S. Patent Nos. 6,017,696; 6,068,818; and 6,051,380; each of which are herein inco ⁇ orated by reference).
• Nanogen's technology enables the active movement and concentration of charged molecules to and from designated test sites on its semiconductor microchip.
• DNA capture probes unique to a given SNP or mutation are electronically placed at, or "addressed" to, specific sites on tire microchip.
• DNA Since DNA has a strong negative charge, it can be electronically moved to an area of positive charge. First, a test site or a row of test sites on the microchip is electronically activated with a positive charge. Next, a solution containing the DNA probes is introduced onto the microchip. The negatively charged probes rapidly move to the positively charged sites, where they concentrate and are chemically bound to a site on the microchip. The microchip is then washed and another solution of distinct DNA probes is added until the anay of specifically bound DNA probes is complete. A test sample is then analyzed for the presence of target DNA molecules by determining which of the DNA capture probes hybridize, with complementary DNA in the test sample (e.g., a PCR amplified gene of interest).
• complementary DNA in the test sample e.g., a PCR amplified gene of interest
• An electronic charge is also used to move and concentrate target molecules to one or more test sites on the microchip.
• the electronic concentration of sample DNA at each test site promotes rapid hybridization of sample DNA with complementary capture probes (hybridization may occur in minutes).
• To remove any unbound or nonspecifically bound DNA from each site the polarity or charge of the site is reversed to negative, thereby forcing any unbound or nonspecifically bound DNA back into solution away from the capture probes.
• a laser-based fluorescence scanner is used to detect binding,
• an anay technology based upon the segregation of fluids on a flat surface (chip) by differences in surface tension (ProtoGene, Palo Alto, CA) is utilized (See e.g., U.S. Patent Nos.
• Protogene's technology is based on the fact that fluids can be segregated on a flat surface by differences in surface tension that have been imparted by chemical coatings. Once so segregated, oligonucleotide probes are synthesized directly on the chip by ink-jet printing of reagents.
• the array with its reaction sites defined by surface tension is mounted on a X/Y translation stage under a set of four piezoelectric nozzles, one for each of the four standard DNA bases. The translation stage moves along each of the rows of the anay and the appropriate reagent is delivered to each of the reaction site.
• the A amidite is delivered only to the sites where amidite A is to be coupled during that synthesis step and so on.
• Common reagents and washes are delivered by flooding the entire surface and then removing them by spinning.
• DNA probes unique for the SNP or mutation of interest are affixed to the chip using Protogene's technology.
• the chip is then contacted with the PCR-amplified genes of interest.
• unbound DNA is removed and hybridization is detected using any suitable method (e.g., by fluorescence de-quenching of an inco ⁇ orated fluorescent group).
• a "bead anay" is used for the detection of polymo ⁇ hisms
• Illumina uses a BEAD ARRAY technology that combines fiber optic bundles and beads that self-assemble into an anay. Each fiber optic bundle contains thousands to millions of individual fibers depending on the diameter of the bundle. The beads are coated with an oligonucleotide specific for the detection of a given SNP or mutation. Batches of beads are combined to form a pool specific to the anay. To perform an assay, the BEAD ARRAY is contacted with a prepared subject sample (e.g., DNA). Hybridization is detected using any suitable method. c.
• a prepared subject sample e.g., DNA
• hybridization is detected by enzymatic cleavage of specific structures (INVADER assay, Third Wave Technologies; See e.g., U.S. Patent Nos. 5,846,717, 6,090,543; 6,001,567; 5,985,557; and 5,994,069; each of which is herein inco ⁇ orated by reference).
• the INVADER assay detects specific DNA and RNA sequences by using structure-specific enzymes to cleave a complex formed by the hybridization of overlapping oligonucleotide probes. Elevated temperature and an excess of one of the probes enable multiple probes to be cleaved for each target sequence present without temperature cycling.
• the secondary probe oligonucleotide can be 5 '-end labeled with a fluorescent dye that is quenched by a second dye or other quenching moiety.
• the de-quenched dye-labeled product may be detected using a standard fluorescence plate reader, or an instrument configured to collect fluorescence data during the course of the reaction (i.e., a "real-time" fluorescence detector, such as an ABI 7700 Sequence Detection System, Applied Biosystems, Foster City, CA).
• the INVADER assay detects specific mutations and SNPs in unamplified genomic DNA.
• two oligonucleotides hybridize in tandem to the genomic DNA to form an overlapping structure.
• a structure-specific nuclease enzyme recognizes this overlapping structure and cleaves the primary probe.
• cleaved primary probe combines with a fluorescence-labeled secondary probe to create another overlapping structure that is cleaved by the enzyme.
• the initial and secondary reactions can run concunently in the same vessel. Cleavage of the secondary probe is detected by using a fluorescence detector, as described above.
• the signal of the test sample may be compared to known positive and negative controls.
• hybridization of a bound probe is detected using a TaqMan assay
• the assay is performed during a PCR reaction.
• the TaqMan assay exploits the 5 -3' exonuclease activity of DNA polymerases such as AMPLITAQ DNA polymerase.
• a probe, specific for a given allele or mutation, is included in the PCR reaction.
• the probe consists of an oligonucleotide with a 5'-reporter dye (e.g., a fluorescent dye) and a 3'-quencher dye.
• polymo ⁇ hisms are detected using the SNP-IT primer extension assay (Orchid Biosciences, Princeton, NJ; See e.g., U.S. Patent Nos. 5,952,174 and 5,919,626, each of which is herein inco ⁇ orated by reference).
• SNPs are identified by using a specially synthesized DNA primer and a DNA polymerase to selectively extend the DNA chain by one base at the suspected SNP location.
• DNA in the region of interest is amplified and denatured.
• Polymerase reactions are then performed using miniaturized systems called microfluidics. Detection is accomplished by adding a label to the nucleotide suspected of being at the SNP or mutation location. Inco ⁇ oration of the label into the DNA can be detected by any suitable method (e.g., if the nucleotide contains a biotin label, detection is via a fluorescently labelled antibody specific for biotin).
• Sequences may be input for analysis from any number of sources.
• sequence information is entered into a computer.
• the computer need not be the same computer system that carries out in silico analysis.
• candidate target sequences may be entered into a computer linked to a communication network (e.g., a local area network, Internet or Intranet).
• a communication network e.g., a local area network, Internet or Intranet.
• users anywhere in the world with access to a communication network may enter candidate sequences at their own locale.
• a user interface is provided to the user over a communication network (e.g., a World Wide Web-based user interface), containing entry fields for the information required by the in silico analysis (e.g., the sequence of the candidate target sequence).
• the user interface can ensure that the user inputs the requisite amount of information in the conect format.
• the user interface requires that the sequence information for a target sequence be of a minimum length (e.g., 20 or more, 50 or more, 100 or more nucleotides) and be in a single format (e.g., FASTA).
• the information can be input in any format and the systems and methods of the present invention edit or alter the input information into a suitable form for analysis.
• the systems and methods of the present invention search public databases for the short sequence, and if a unique sequence is identified, convert the short sequence into a suitably long sequence by adding nucleotides on one or both of the ends of the input target sequence.
• sequence information is entered in an undesirable format or contains extraneous, non-sequence characters, the sequence can be modified to a standard format (e.g., FASTA) prior to further in silico analysis.
• the user interface may also collect information about tlie user, including, but not limited to, the name and address of the user.
• target sequence entries are associated with a user identification code.
• sequences are input directly from assay design software (e.g., the INVADERCREATOR software.
• assay design software e.g., the INVADERCREATOR software.
• each sequence is given an ID number.
• the JD number is linked to the target sequence being analyzed to avoid duplicate analyses. For example, if the in silico analysis determines that a target sequence conesponding to the input sequence has already been analyzed, the user is informed and given the option of by-passing in silico analysis and simply receiving previously obtained results.
• Web-ordering systems and methods Users who wish to order detection assays, have detection assay designed, or gain access to databases or other information of the present invention may employ a electronic communication system (e.g., the Internet).
• an ordering and information system of the present invention is connected to a public network to allow any user access to the information.
• private electronic communication networks are provided.
• a customer or user is a repeat customer (e.g., a distributor or large diagnostic laboratory)
• the full-time dedicated private connection may be provided between a computer system of the customer and a computer system of the systems of the present invention.
• the system may be ananged to minimize human interaction.
• inventory control software is used to monitor the number and type of detection assays in possession of the customer.
• a query is sent at defined intervals to determine if the customer has the appropriate number and type of detection assay, and if shortages are detected, instructions are sent to design, produce, and/or deliver additional assays to the customer.
• the system also monitors inventory levels of the seller and in prefened embodiments, is integrated with production systems to manage production capacity and timing.
• a user-friendly interface is provided to facilitate selection and ordering of detection assays. Because of the hundreds of thousands of detection assays available and/or polymo ⁇ hisms that the user may wish to intenogate, the user-friendly interface allows navigation through the complex set of option. For example, in some embodiments, a series of stacked databases are used to guide users to the desired products.
• the first layer provides a display of all of the chromosomes of an organism.
• the user selects the chromosome or chromosomes of interest.
• Selection of the chromosome provides a more detailed map of the chromosome, indicating banding regions on the chromosome. Selection of the desired band leads to a map showing gene locations.
• One or more additional layers of detail provide base positions of polymo ⁇ hisms, gene names, genome database identification tags, annotations, regions of the chromosome with pre-existing developed detection assays that are available for purchase, regions where no pre-existing developed assays exist but that are available for design and production, etc.
• Selecting a region, polymo ⁇ hism, or detection assay takes the user to an ordering interface, where information is collected to initiate detection assay design and/or ordering.
• a search engine is provided, where a gene name, sequence range, polymo ⁇ hism or other query is entered to more immediately direct the user to the appropriate layer of information.
• the ordering, design, and production systems are integrated with a finance system, where the pricing of the detection assay is determined by one or more factors: whether or not design is required, cost of goods based on the components in the detection assay, special discounts for certain customers, discounts for bulk orders, discounts for re-orders, price increases where the product is covered by intellectual property or contractual payment obligations to third parties, and price selection based on usage.
• pricing is increased.
• the pricing increase for clinical products occurs automatically.
• the systems of the present invention are linked to FDA, public publication, or other databases to determine if a product has been certified for clinical diagnostic or ASR use.
• EXAMPLE 1 DESIGNING A 10-PLEX (MANUAL): TEST FOR INVADER ASSAYS
• the following experimental example describes the manual design of amplification primers for a multiplex amplification reaction, and the subsequent detection of the amplicons by the INVADER assay.
• Ten target sequences were selected from a set of pre-validated SNP-containing sequences, available in a TWT in-house oligonucleotide order entry database. Each target contains a single nucleotide polymo ⁇ hism (SNP) to which an INVADER assay had been previously designed.
• the INVADER assay oligonucleotides were designed by the INVADER CREATOR software (Third Wave Technologies, Inc.
• the footprint region in this example is defined as the INVADER "footprint", or the bases covered by the INVADER and the probe oligonucleotides, optimally positioned for the detection of the base of interest, in this case, a single nucleotide polymo ⁇ hism.
• About 200 nucleotides of each of the 10 target sequences were analyzed for the amplification primer design analysis, with the SNP base residing about in the center of the sequence. . Criteria of maximum and minimum probe length (defaults of 30 nucleotides and 12 nucleotides, respectively) were defined, as was a range for the probe melting temperature Tm of 50- 60°C.
• the melting temperature (T m ) of the oligonucleotide is calculated using the nearest-neighbor model and published parameters for DNA duplex formation (Allawi and SantaLucia, Biochemistry, 36:10581 [1997], herein inco ⁇ orated by reference). Because the assay's salt concentrations are often different than the solution conditions in which the nearest-neighbor parameters were obtained (IM NaCl and no divalent metals), and because the presence and concentration of the enzyme influence optimal reaction temperature, an adjustment should be made to the calculated T m to determine the optimal temperature at which to perform a reaction.
• salt conection refers to a variation made in the value provided for a salt concentration for the pu ⁇ ose of reflecting the effect on a T m calculation for a nucleic acid duplex of a non-salt parameter or condition affecting said duplex. Variation of the values provided for the strand concentrations will also affect the outcome of these calculations.
• the algorithm for used for calculating probe-target melting temperature has been adapted for use in predicting optimal primer design sequences.
• sequence adjacent to the footprint region, both upstream and downstream were scanned and the first A or C was chosen for design start such that for primers described as 5'- N[x]-N[x-1]- -N[4]-N[3]-N[2]-N[l]-3', where N[l] should be an A or C.
• N[2]-N[l] of a given oligonucleotide primer should not be complementary to N[2]-N[l] of any other oligonucleotide
• N[3]-N[2] ⁇ N[l] should not be complementary to N[3]-N[2]-N[l] of any other oligonucleotide. If these criteria were not met at a given N[l], the next base in the 5' direction for the forward primer or the next base in the 3 ' direction for the reverse primer will be evaluated as an N[l] site. In the case of manual analysis, A/C rich regions were targeted in order to minimize the complementarity of 3' ends.
• an INVADER assay was performed following the multiplex amplification reaction. Therefore, a section of the secondary JNVADER reaction oligonucleotide (the FRET oligonucleotide sequence) was also inco ⁇ orated as criteria for primer design; the amplification primer sequence should be less than 80% homologous to the specified region of the FRET oligonucleotide. All primers were synthesized according to standard oligonucleotide chemistry, desalted (by standard methods) and quantified by absorbance at A260 and diluted to 50 ⁇ M concentrated stock.
• Multiplex PCR was then carried out using 10-plex PCR using equimolar amounts of primer (O.OluM/primer) under the following conditions; lOOmM KC1, 3mM MgCl 2 , lOmM Tris pH8.0, 200uM dNTPs, 2.5U Taq DNA polymerase, and lOng of human genomic DNA (hgDNA) template in a 50ul reaction.
• primer O.OluM/primer
• lOOmM KC1 3mM MgCl 2
• lOmM Tris pH8.0 200uM dNTPs
• 2.5U Taq DNA polymerase 2.5U Taq DNA polymerase
• lOng of human genomic DNA (hgDNA) template in a 50ul reaction.
• the reaction was incubated for (94C/30sec, 50C/44sec.) for 30 cycles.
• the multiplex PCR reaction was diluted 1:10 with water and subjected to INVADER analysis using INVADER Assay FRET Detection Plates, 96 well genomic biplex, lOOng CLEAVASE VIII enzyme, INVADER assays were assembled as 15ul reactions as follows; lul of the 1:10 dilution of the PCR reaction, 3ul of PPI mix, 5ul of 22.5 mM MgC12, 6ul of dH20, covered with 15ul of CHJXLOUT liquid wax. Samples were denatured in the INVADER biplex by incubation at 95C for 5min., followed by incubation at 63C and fluorescence measured on a Cytofluor 4000 at various timepoints.
• FOZ values generated by different INVADER assays are directly comparable to one another and can reliably be used as indicators of the efficiency of amplification.
• the FOZ values of the INVADER assay can be used to estimate amplicon abundance.
• FOZm represents the sum of RED FOZ and FAM_FOZ of an unknown concentration of target incubated in an INVADER assay for a given amount of time (m).
• FO 24 o fa& average of FAM_FOZ 240 +RED_FOZ 24 o over the entire INVADER MAP plate using hgDNA as target and the dilution factor D is set to 0.125.
• F ((FOZ,,, - 2) * 500 f(FOZi4o - 2) * D) * (240 / m) ⁇ 2 (equation lb)
• FOZ values should be within the dynamic range of the instrument on which the reading are taken. In the case of the Cytofluor 4000 used in this study, the dynamic range was between about 1.5 and about 12 FOZ.
• Section 3 Linear Relationship between Amplification Factor and Primer Concentration.
• primer concentration and amplification factor F
• four distinct uniplex PCR reactions were run at using primers 1117-70-17 and 1117- 70-18 at concentrations of O.OluM, 0.012 uM, 0.014 uM, 0.020 uM respectively.
• the four independent PCR reactions were carried out under the following conditions; lOOmM KC1, 3mM MgCl, lOmM Tris pH 8.0, 200uM dNTPs using lOng of hgDNA as template. Incubation was carried out at (94C/30 sec, 50C/20 sec.) for 30 cycles.
• amplification bias observed under conditions of equimolar primer concentrations in multiplex PCR could be measured as the "apparent" primer concentration (X) based on the amplification factor F.
• Section 4.Calculation of Apparent Primer Concentrations from a Balanced Multiplex Mix As described in a previous section, primer concentration can directly influence the amplification factor of given amplicon. Under conditions of equimolar amounts of primers, FOZm readings can be used to calculate the "apparent" primer concentration of each amplicon using equation 2. Replacing Y in equation 2 with log(F) of a given amplification factor and solving for X, gives an "apparent" primer concentration based on the relative abundance of a given amplicon in a multiplex reaction. Using equation 2 to calculate the "apparent" primer concentration of all primers (provided in equimolar concentration) in a multiplex reaction provides a means of normalizing primer sets against each other.
• R[n] Xmax X[n]
• the values of R[n] are multiplied by a constant primer concentration to provide working concentrations for each primer in a given multiplex reaction.
• the amplicon conesponding to SNP assay 41646 has an R[n] value equal to 1.
• Multiplex PCR was carried out under conditions identical to those used in with equimolar primer mix; lOOmMKCl, 3mMMgCl, lOmM Tris ⁇ H8.0, 200uM dNTPs, 2.5U taq, and lOng of hgDNA template in a 50ul reaction.
• the reaction was incubated for (94C/30sec, 50C/44sec.) for 30 cycles. After incubation, the multiplex PCR reaction was diluted 1:10 with water and subjected to INVADER analysis.
• EXAMPLE 2 DESIGN OF 101-PLEX PCR USING THE SOFTWARE APPLICATION Using the TWT Oligo Order Entry Database, 144 sequences of less than 200 nucleotides in length were obtained, with SNPs annotated using brackets to indicate the SNP position for each sequence (e.g. In order to expand sequence data flanking tlie SNP of interest, sequences were expanded to approximately IkB in length (500 nts flanking each side of the SNP) using BLAST analysis. Of the 144 starting sequences, 16 could not expanded by BLAST, resulting in a final set of 128 sequences expanded to approximately IkB length.
• the output file contained 128 primer sets (256 primers), four of which were thrown out due to excessively long primer sequences (SNP # 47854, 47889, 54874, 67396), leaving 124 primers sets (248 primers) available for synthesis.
• the remaining primers were synthesized using standard procedures at the 200nmol scale and purified by desalting. After synthesis failures, 107 primer sets were available for assembly of an equimolar 107-plex primer mix (214 primers). Of the 107 primer sets available for amplification, only 101 were present on the INVADER MAP plate to evaluate amplification factor.
• Multiplex PCR was carried out using 101-plex PCR using equimolar amounts of primer (0.025uM primer) under the following conditions; 1 OOmMKCl, 3mM MgCl, 1 OmM Tris pH8.0, 200uM dNTPs, and lOng of human genomic DNA (hgDNA) template in a 50ul reaction. After denaturation at 95C for lOmin, 2.5 units of Taq was added and the reaction incubated for (94C/30sec, 50C/44sec.) for 50 cycles. After incubation, tlie multiplex PCR reaction was diluted 1:24 with water and subjected to INVADER assay analysis using JNVADER MAP detection platform.
• Optimized primer concentrations of the 101-plex were calculated using the basic principles outlined in the 10-plex example and equation lb, with an R[n] of 1 conesponding to 0.025uM primer (see Fig.15 for various primer concentrations).
• Multiplex PCR was under the following conditions;100mMKCl, 3mM MgCl, lOmM Tris pH8.0, 200uM dNTPs, and lOng of human genomic DNA (hgDNA) template in a 50ul reaction. After denaturation at 95C for lOmin, 2.5 units of Taq was added and the reaction incubated for (94C/30sec, 50C/44sec.) for 50 cycles.
• the multiplex PCR reaction was diluted 1 :24 with water and subjected to INVADER analysis using INVADER MAP detection platform.
• EXAMPLE 3 USE OF THE INVADER ASSAY TO DETERMINE AMPLIFICATION FACTOR OF PCR
• the INVADER assay can be used to monitor the progress of amplification during PCR reactions, i. e. , to determine the amplification factor F that reflects efficiency of amplification of a particular amplicon in a reaction.
• the INVADER assay can be used to determine the number of molecules present at any point of a PCR reaction by reference to a standard curve generated from quantified reference DNA molecules.
• the amplification factor F is measured as a ratio of PCR product concentration after amplification to initial target concentration.
• This example demonstrates the effect of varying primer concentration on the measured amplification factor.
• PCR reactions were conducted for variable numbers of cycles in increments of 5, i.e., 5, 10, 15, 20, 25, 30, so that the progress of the reaction could be assessed using the INVADER assay to measure accumulated product.
• the reactions were diluted serially to assure that the target amounts did not saturate the INVADER assay, i.e., so that the measurements could be made in the linear range of the assay.
• INVADER assay standard curves were generated using a dilution series containing known amounts of the amplicon.
• PCR Reactions PCR reactions were set up using equimolar amounts of primers (e.g., 0.02 ⁇ M or 0.1 ⁇ M primers, final concentration). Reactions at each primer concentration were set up in triplicate for each level of amplification tested, i.e., 5, 10, 15, 20, 25, and 30 PCR cycles.
• amplification products generated with the same primers used in the tests of different numbers of cycles were isolated from non-denaturing polyacrylimide gels using standard methods and quantified using the PICOGREEN assay.
• a working stock of 200 pM was created, and serial dilutions of these concentration standards were created in dH 0 containing tRNA at 30 ng/ ⁇ l to yield a series with final amplicon concentrations of 0.5, 1, 2.5, 6.25, 15.62, 39, and 100 fM.
• oligonucleotides were included in the PPI mix.
• 0.25 ⁇ M INVADER for assay 2 (GAAGCGGCGCCGGTTACCACCA) 2.5 ⁇ M
• a Probe for assay 2 (CGCGCCGAGGTGGTTGAGCAATTCCAA) 2.5 ⁇ M
• G Probe for assay 2 (ATGACGTGGCAGACCGGTTGAGCAATTCCA) All wells were overlaid with 15 ⁇ l mineral oil, incubated at 95 °C 5 min, then at 63 °C read at various intervals, eg. 20, 40, 80, or 160 min, depending on the level of signal generated.
• the reaction plate was read on a CytoFluor ® Series 4000 Fluorescence Multi-Well Plate Reader.
• the settings used were: 485/20 nm excitation bandwidth and 530/25 nm emission bandwidth for F dye detection, and 560/20 nm excitation/bandwidth and 620/40 nm emission/bandwidth for R dye detection.
• the instrument gain was set for each dye so that the No Target Blank produced between 100 - 200 Absolute Fluorescence Units (AFUs).
• PCR bias and (b) how amplification of different genomic regions depends on primer concentration.
• F was measured by generating a standard curve for each locus using a dilution series of purified, quantified reference amplicon preparations. In this case, 12 different reference amplicons were generated: one for each allele of the SNPs contained in the 6 genomic regions amplified by the primer pairs. Each reference amplicon concentration was tested in an INVADER assay, and a standard curve of fluorescence counts versus amplicon concentration was created. PCR reactions were also run on genomic DNA samples, the products diluted, and then tested in an INVADER assay to determine the extent of amplification, in terms of number of molecules, by comparison to the standard curve.
• amplified DNA was gel isolated using standard methods and previously quantified using the PICOGREEN assay. Serial dilutions of these concentration standards were created as follows: Each purified amplicon was diluted to create a working stock at a concentration of 200 pM. These stocks were then serially diluted as follows. A working stock solution of each amplicon was prepared with a concentration of 1.25 pM in dH 2 0 containing tRNA at 30 ng ⁇ .1. The working stock was diluted in 96-well microtiter plates and then serially diluted to yield the following final concentrations in the INVADER assay: 1, 2.5, 6.25, 15.6, 39, 100, and 250 fM.
• One plate was prepared for the amplicons to be detected in the INVADER assay using probe oligonucleotides reporting to FAM dye and one plate for those to be tested with probe oligonucleotides reporting to RED dye. All amplicon dilutions were analyzed in duplicate. Aliquots of 100 ⁇ l were transfened, in this layout, to 96 well MJ Research plates and denatured for 5 min at 95 °C prior to addition to INVADER assays.
• PCR amplification of genomic samples at different primer concentrations were set up for individual amplification of the 6 genomic regions described in the previous example on each of 2 alleles at 4 different primer concentrations, for a total of 48 PCR reactions. All PCRs were nm for 20 cycles. The following primer concentrations were tested: 0.01 ⁇ M, 0.025 ⁇ M, 0.05 ⁇ M, and 0.1 ⁇ M.
• a master mix for all 48 reactions was prepared according to standard procedures, with the exception of the modified primer concentrations, plus overage for an additional 23 reactions (16 reactions were prepared but not used, and overage of 7 additional reactions was prepared).
• INVADER assay analysis of PCR dilutions and reference amplicons INVADER analysis was carried out on all dilutions of the products of each PCR reaction as well as the indicated dilutions of each quantified reference amplicon (to generate a standard curve for each amplicon) in standard biplex JNVADER assays. All wells were overlaid with 15 ⁇ l of mineral oil. Samples were heated to 95 °C for 5 min to denature and then incubated at 64°C. Fluorescence measurements were taken at 40 and 80 minutes in a CytoFluor ® 4000 fluorescence plate reader (Applied Biosystems, Foster City, CA).
• the settings used were: 485/20 nm excitation/bandwidth and 530/25 nm emission/bandwidth for F dye detection, and 560/20 nm excitation/bandwidth and 620/40 nm emission/bandwidth for R dye detection.
• the instrument gain was set for each dye so that the No Target Blank produced between 100 - 200 Absolute Fluorescence Units (AFUs).
• AFUs Absolute Fluorescence Units
• the raw data is that generated by the device/instrument used to measure the assay performance (real-time or endpoint mode).
• the amplification factor strongly depends on c at low primer concentrations with a trend to plateau at higher primer concentrations. This phenomenon can be explained in terms of the kinetics of primer annealing. At high primer concentrations, fast annealing kinetics ensures that primers are bound to all targets and maximum amplification rate is achieved, on the contrary, at low primer concentrations the primer annealing kinetics become a rate limiting step decreasing F. This analysis suggests that plotting amplification factor as a function of primer concentration in ln (2 - F n ) vs. c coordinates should produce a straight line with a slope -k a t a . _ Re-plotting of the data in the In (2 - F" ) vs.
• c coordinates demonstrates the expected linear dependence for low primer concentrations (low amplification factor) which deviates from the linearity at 0.1 ⁇ M primer concentration (Fis 10 5 or larger) due to lower than expected S3 amplification factor.
• the k a t recipe. values can be calculated for each PCR using the following equation.
• Genomic DNA extraction Genomic DNA was isolated from 5 mis of whole bl od and purified using the Autopure, manufactured by Gentra Systems, Inc. (Minneapolis, MN). The purified DNA was in 500 ⁇ l of dH 2 0.
• Primer design Forward and reverse primer sets for the 192 loci were designed using Primer Designer, version 1.3.4 (See Primer Design section above, including Figure 8).
• Target sequences used for INVADER designs were converted into a comma-delimited text file for use as an input file for PrirnerDesigner.
• PrimerDesigner was run using default parameters, with the exception of oligo T m , ⁇ which was set at 60 °C.
• Primer synthesis Oligonucleotide primers were synthesized using standard procedures in a Polyplex (GeneMachines, San Carlos, CA). The scale was 0.2 ⁇ rnole, desalted only (not purified) on NAP-10 and not dried down.
• Master mix 1 contained primers to amplify loci 1-96; master mix 2, 97-192.
• the mixes were made according to standard procedures and contained standard components. All primers were present at a final concentration of 0.025 ⁇ M, with KCl at 100 mM, and MgCl at 3 mM.
• PCR cycling conditions were as follows in a MJ PTC-100 thermocycler (MJ Research, Waltham, MA): 95 °C for 15 min; 94°C for 30 sec, then 55°C 44 sec X 50 cycles Following cycling, all 4 PCR reactions were combined and aliquots of 3 ⁇ l were distributed into a 384 deep-well plate using a CYBI- ell 2000 automated pipetting station (CyBio AG, Jena, Germany). This instrument makes individual reagent additions to each well of a 384-well microplate. The reagents to be added are themselves anayed in 384-well deep half plates.
• INVADER assay reactions were set up using the CYBI-well 2000. Aliquots of 3 ⁇ l of the genomic DNA target were added to the appropriate wells. No target controls were comprised of 3 ⁇ l of Te (10 mM Tris, pH 8.0, 0.1 mM EDTA). The reagents for use in the INVADER assays were standard PPI mixes, buffer, FRET oligonucleotides, and Cleavase VIII enzyme and were added individually to each well by the CYBI-well 20O0. Following the reagent additions, 6 ⁇ l of mineral oil were overlaid in each well.
• the plates were heated in a MJ PTC-200 DNA ENGINE thermocycler (MJ Research) to 95 °C for 5 minutes then cooled to the incubation temperature of 63 °C. Fluorescence was read after 20 minutes and 40 minutes using the Satire microplate reader (Tecan, Zurich, Switzerland) using the following settings. 495/5 nm excitation/bandwidth and 520/5 nm emission/bandwidth for F dye detection; and 600/5 nm emission/bandwidth, 575/5 nm excitation/bandwidth Z position, 5600 ⁇ s; number of flashes, 10; lag time, 0; integration time, 40 ⁇ sec for R dye detection. Gain was set for F dye at 90 nm and R dye at 120. The raw data is that generated by the device/instrument used to measure the assay performance (real-time or endpoint mode). Of the 192 reactions, genotype calls could be made for 157 after 20 minutes and 158 after
• genotyping results were available for comparison from data obtained previously using either monoplex PCR followed by INVADER analysis or INVADER results obtained directly from analysis of genomic DNA. For 69 results, no conoborating genotype results were available.
• This example shows that it is possible to amplify more than 150 loci in a single multiplexed PCR reaction. This example further shows that the amount of each amplified fragment generated in such a multiplexed PCR reaction is sufficient to produce discernable genotype calls when used as a target in an INVADER assay.
• variable levels of signal produced from the different loci amplified in the 192-plex PCR of the previous example taken with the results from Example 3 that show the effect of primer concentration on amplification factor, further suggest that it may be possible to improve the percentage of PCR reactions that generate sufficient target for use in the INVADER assay by modulating primer concentrations.
• one particular sample analyzed in Example 5 yielded FOZ results, after a 40 minute incubation in the INVADER assay, of 29.54 FAM and 66.98 RED, while another sample gave FOZ results after 40 min of 1.09 and 1.22, respectively, prompting a determination that there was insufficient signal to generate a genotype call.
• Modulation of primer concentrations should make it possible to bring the amplification factors of the two samples closer to the same value. It is envisioned that this sort of modulation may be an iterative process, requiring more than one modification to bring the amplification factors sufficiently close to one another to enable most or all loci in a multiplex PCR reaction to be amplified with approximately equivalent efficiency.
• EXAMPLE 7 MULTIPLEX EXAMPLE
• PCR amplification can be carried out in a multiplex format in which multiple loci are amphfied in the same tube. In practice, however, this approach can result in highly variable yields of individual amplified products due to PCR bias.
• This Example describes the optimization of multiplex reaction conditions to minimize amplification bias.
• Amplification bias is caused by the variable amplification rate among individual reactions which leads to a significant difference in PCR product yields over a large number of cycles.
• PCR target amplification was analyzed across the full range of the reaction and parameters affecting PCR yield were investigated by using the quantitative INVADER assay. From this work, a model describing the dependence of the target amplification factor on primer concentration and primer annealing time was developed that elucidates a mechanism underlying amplification bias. Using 6-plex PCR as a model system to test different conditions minimizing bias, two approaches were identified.
• the first relies on adjusting primer concentrations to balance the amplification factors of different loci, ha the second approach, the primer concentration was kept the same for all the individual reactions, but tlie primer annealing time and the number of amplification cycles were optimized to minimize amplification bias.
• the optimized PCR conditions were used to cany out a 192-plex PCR amplification of 8 genomic DNA samples and for use in genotyping using INVADER assays.
• Genomic DNA samples GI, G2, G3, G4, G5, G6, G7 and G8 were prepared from 10 ml of leukocytes using an AutoPure LS instrument (Gentra Systems, Minneapolis, MN). The purified DNA was diluted to 13.3ng/ ⁇ l in Te buffer containing 10 mM Tris HCl pH 8.0, 0.1 mM EDTA. Oligonucleotide synthesis.
• Oligonucleotides used in the INVADER assay with the monoplex and 6- ⁇ lex PCR reactions were synthesized using a PerSeptive Biosystems instrument and standard phosphoramidite chemistries including A, G, C, T, 6-carboxyfluorescein dye (FAM) (Glen Research), Redmond REDTM dye (RED) (Epoch Biosciences, Redmond, WA), and EclipseTM Dark Quencher (Z) (Epoch Biosciences).
• FAM 6-carboxyfluorescein dye
• RED Redmond REDTM dye
• Z EclipseTM Dark Quencher
• the primary probes and FRET cassettes were purified by ion exchange HPLC using a Resource Q column (Amersham-Pharmacia Biotech, Newark, NJ), and the invasive probes were purified by desalting over NAP-10 columns (Amersham 17-0854-02).
• Tlie primary probes used in the 192-plex PCR assays were synthesized by Biosearch Technologies using C16 CPG columns (Biosearch Technologies, Novato, CA, BG1-SD14-1), and purified using SuperPure Plus Purification columns (Biosearch, SP-2000-96).
• the invasive probes for the 192-plex assays were synthesized and purified by Biosearch Technologies using trityl-on 5' capture purification.
• PCR primers were synthesized by Integrated DNA Technologies, Chicago, IL. Oligonucleotide concentrations were determined using the absorption at 260 nm (A 26 o) and extinction coefficients of 15,400, 7,400, 11,500, and 8,700 A 26 o M "1 for A, C, G, and T, respectively.
• Primer design for multiplex PCR A computer program, PrimerDesigner software (Third Wave Technologies; Madison, WI, See Figure 8 and discussion of Primer Design above), has been developed to assist in designing primers for multiplexed PCR and to reduce the probability of primer-dimer formation.
• PCR primers for the multiplex format were designed with the PrimerDesigner software using the following parameters in conjunction with the primer design discussion above and in Figure 8. For each of the loci to be amplified, 500 nucleotides were included on either side of the SNP for a total of 1001 bases per locus.
• primers were chosen based on the following criteria: (1) primers must have an A or C at the 3' end to avoid primer-dimer formation; (2) T m of the primers was 60°C (11,12); (3) primers should be between 12 and 30 nucleotides in length; (4) the two and three 3' terminal bases of any primer should not be complementary to the two and three 3' terminal bases of any other primer of the multiplex PCR mixture; (5) no primer should have more than 80% sequence similarity to the cleaved 5' arm sequence of either INVADER primary probe.
• the algorithm is initiated by the design of the first two primers for a randomly selected locus and proceeds by iteration adding more primers to the pool. If no primers can be designed for one of the loci, the algorithm starts from the beginning using a new randomly selected locus.
• INVADER assay design The primary and invasive probes for the INVADER assays were designed with the INVADERCreator algorithm as described elsewhere (Lyamichev, V. and Neri, B. (2003) INVADER assay for SNP genotyping. Methods Mol Biol, 212, 229-40, herein incorporated by reference). The probe sequences for INVADER assays 1-6 conesponding to the PCRs 1-6, respectively.
• PCRs 1-6 in uniplex or 6- plex format were carried out in 50 ⁇ l GeneAmp PCR buffer (PE Biosystems, Foster City, CA) containing primers at concentration specified in the text, 0.2 mM dNTPs, 1 ⁇ l (5U/ ⁇ l) Amplitaq DNA polymerase (PE Biosystems, N808-0171), 1 ⁇ l (l.l ⁇ g/ ⁇ l) TaqStart Antibody (Clontech, catalog number 5400-2, Palo Alto, CA) and 50 ng of human genomic DNA or 3.8 ⁇ l Te buffer for the no target control. To prevent evaporation, each well was covered with 15 ⁇ l of clear PCR buffer (PE Biosystems, Foster City, CA) containing primers at concentration specified in the text, 0.2 mM dNTPs, 1 ⁇ l (5U/ ⁇ l) Amplitaq DNA polymerase (PE Biosystems, N808-0171), 1 ⁇ l (l.l ⁇ g/ ⁇ l) Taq
• PCR included an initial sample denaturing step of 15 min at 95°C and a final incubation step of 10 min at 99°C. Each reaction was performed in triplicate in a 96-well plate. The PCR products were serially diluted 20-fold in the first step followed by 5-fold subsequent dilution in Te buffer containing 30 ⁇ g/ml tRNA (Boehringer Mannheim, cat. no.
• INVADER reactions with the diluted PCR products were carried out In 15 ⁇ L containing 0.05 ⁇ M invasive oligonucleotide, 0.5 ⁇ M of each primary probe, 0.33 ⁇ M of each FRET cassette, 5.3 ng/ ⁇ L Cleavase XI enzyme, 12 mM MOPS (pH 7.5), 15.3 mM MgCl 2 , 2.5% PEG 8000, 0.02% NP40, 0.02% Tween 20 overlaid with 15 ⁇ l mineral oil (Sigma) in 96-well plate.
• the PCR products constituted 7.5 ⁇ L of the 15 ⁇ L reactions.
• PCR standards for the assays 1-6 were prepared by PCR amplification of DNA samples GI, G2, G6, or G8.
• the amplified products were concentrated by ethanol precipitation, purified using electrophoresis in 8% polyacrylamide non-denaturing gel and quantitated using a
• Picogreen dsDNA quantitation kit (Molecular Probes, Eugene, OR, catalog no. P7589).
• the INVADER reactions for the standard curves were carried out with 0 to 100 fM of the PCR standards in duplicate in the same microtiter plate as the analyzed PCR products.
• the concentration of the analyzed PCR products was determined from the fluorescence signal by a linear regression using the three data points of the standard curve closest to the value of the fluorescence signal of the PCR samples.
• the PCR product concentration and the variance were estimated for each of the PCR replicates from the triplicate INVADER assay measurements.
• PCR product concentration for the triplicate PCRs was estimated by using the average values for each of the replicates weighted by the variance of the triplicate INVADER assay analysis.
• the initial concentration of tlie genomic DNA samples used in the PCR was determined by the triplicate INVADER assay using the same standard curves.
• the amplification factor F was determined as the estimated PCR product concentration multiplied by the dilution factor and divided by the genomic DNA concentration used for the PCR.
• the 192-plex PCR was carried out in a single replicate under the conditions described for PCRs 1-6 for 17 cycles with the DNA samples G1-G8, each primer concentration of 0.2 ⁇ M, primer annealing time 1.5 min, primer extension time 2.5 min and the initial sample denaturing step of 2.5 min at 95 D C.
• Te buffer was used instead of genomic DNA.
• the 192-plex PCR reactions were diluted 30-fold in Te buffer containing 30 ⁇ g/ml tRNA (Boehringer Mannheim, 109 525) and heated at 95°C for 5 min prior to addition to the INVADER reactions.
• the INVADER reactions were performed as described for assays 1-6 except that the invasive probe was at 0.07 ⁇ M, and each primary probe was at 0.7 ⁇ M.
• the FAM and RED fluorescence signals were collected after 15, 30 and 60 min or as specified in the text for the genomic PCR samples and no-target PCR controls. The net fluorescence signal was determined by subtracting the no-target signal from the sample signal for each of the 192 INVADER assays. The following algorithm was applied to the analysis by the genotyping software.
• Fold-over-zero values for the FAM (FOZF) and RED (FOZR) signals were determined for each INVADER assay by dividing the sample signal by the no-target control signal.
• a ratio value H was determined as (FOZF-l)/(TOZR-l).
• a sample was defined as heterozygous (HET) if 0.25 ⁇ H ⁇ 4 and both FOZF and FOZR >1.3; a sample was defined as homozygous FAM if H >4 and FOZF >1.6; and a sample was defined as homozygous RED if H ⁇ 0.25 and FOZR >1.6 (4). In all other cases a sample was called an "equivocal".
• the F factor was defined as a ratio of concentrations of the amplified product and the initial genomic DNA, both measured with the INVADER assay using standard curves obtained with known amounts of the PCR products as described in "Materials and Methods".
• F was analyzed as a function of the number of PCR cycles n.
• the uniplex PCR 5 was performed with a primer concentration c of 0.1 ⁇ M using DNA G2, and F was determined after n of 5, 10, 15, 20, 25, 30 and 35 ( Figure 2).
• PCR 5 reveals a linear dependence of IgF on n for the first 25 cycles with a slope of 0.296 ⁇ 0.0016, demonstrating that target amplification is exponential over 7 orders of magnitude.
• the average amplification factor per PCR cycle determined from slope of the linear dependence is equal to 1.98 ⁇ 0.0O7, indicating that the amount of the target almost doubles after each cycle.
• the inset in Figure 2 sho ⁇ VS the dependence of IgF on n for cycles 1 , 2, 3, and 5 of PCR 5 under the same conditions except a larger amount of DNA G2 is used as a target. This dependence can also be approximated by a linear function with the IgF vs n slope of 0.283.
• Figure 3 A shows the effect of primer concentration c on IgF for the PCR 1 (•), PCR 2(o), PCR 4 ( ⁇ ), and PCR 5 ( ⁇ ).
• PCR amplification was performed in 50 mL with c of 0.01, 0.025, 0.04, 0.05 or 0.1 mM and with 50 ng of the genomic DNA G2 for the PCRs 1, 4, and 5 or the genomic DNA G6 for the PCR 2.
• Each PCR was performed for 20 cycles using template denaturing step for 30 s at 95°C, primer annealing step for 44 s at 55°C and primer extension step for 60 s at 72°C for each cycle.
• the IgF value for the PCR 1 with c of 0.01 mM was too low for reliable measurements.
• PCRs 3 and 6 performed very similarly to PCRs 5 and 2, respectively, and are not shown for brevity. There is a significant difference in F between PCRs performed under the same reaction conditions. The difference is most pronounced at low c; however it becomes less significant at higher c where IgF approaches the theoretical maximal value of lg(2 20 ) or 6.0. As shown in the previous section, PCR can be considered to be an exponential reaction at 20 cycles, and F can be used to determine the target 1 amplification factor z in a single PCR cycle as » .
• ln(2-F») should be a linear function of c with a slope equal to —k a t a .
• Transformation of the data shown in Figure 3 A using ln(2 — F" ) vs. c coordinates demonstrates the expected linear dependence for each of the PCRs ( Figure 3 B) providing a strong support for the model.
• the straight lines show the least-squares fit for each of the PCRs.
• the data points for the PCRs 2 and 5 at c of 0.1 mM were not used because of high standard enor.
• c can be used to determine an apparent association rate constant k° pp of the primer annealing step which is mostly defined by the primer with the lowest k a .
• the k_f p values for PCRs 1, 2, 4 and 5 determined from Figure 3 B using t a of 44 s are 0.34 10 6 , 0.73 10 ⁇ , 0.45 10 6 , and 1.2 10 6 s "1 M “1 , respectively. These values are close to the k a values of 1.5 10 6 s "1 M "1 and 2.6 10 6 s "1 M "1 obtained for short oligonucleotides under similar buffer conditions.
• a Multiplexed PCRs 1, 2, 3, 4, 5, and 6 were performed in 50 ⁇ L with 50 ng of the genomic DNA G2 or G6 for 20 cycles using denaturing step for 30 s at 95°C, p ⁇ mer annealing step for 44 s at 55°C and p ⁇ mer extension step for 60 s at 72°C for each cycle.
• b c a ⁇ was determined for each of the PCRs 1, 2, 3, 4, 5, and 6 from Figure 2 to provide expected IgF value of 4.
• the 6-plex PCRs 1-6 were performed with either the adjusted concentrations c aa _, or a fixed c 0 25 of 0.025 ⁇ M for each of the PCRs under the same conditions as in Figure 3 using as a target DNAs G2 or G6. As shown in Table 1, under the c a ⁇ conditions, all six targets were amplified approximately 10 4 -fold with an average IgF of 4.15 ⁇ 0.17 and a 2.75-fold difference in F between the fastest (PCR 3) and the slowest (PCR 1).
• PCRs 1-6 were also carried out in a uniplex format with c a ⁇ or c 0 0 2 5 under the conditions of the 6-plex format and demonstrated F values very similar to the conesponding F values shown in Table 1. This result suggests that there is no significant interference between the individual PCRs in the 6-plex format.
• Balancing PCR by adjusting c is a powerful approach minimizing the amplification bias; however it uses a known dependence of F on c for each of the PCRs or an iterative optimization of primer concentration.
• An alternative approach is to use a fixed c value, but to perform PCR under conditions minimizing the bias.
• Both the experimental data ( Figure 3) and the theoretical analysis (Eq. 3) suggest that z should asymptotically approach 2 as value of the ct a term increases. Therefore, multiplex PCRs were performed with a fixed c of 0.1 ⁇ M, the maximal concentration used under the conditions shown in Figure 3, or 0.2 ⁇ M and the primer annealing step of 90 s instead of 44 s.
• the 6-plex PCRs 1-6 were carried out for 17 cycles to provide the theoretically maximal tg value of 5.1 using as a target the DNAs GI, G2, G6, or G8. Quantitative analysis of J 7 with INVADER assays 1-6 was performed by using both FAM and RED signals (for the genotypes of the genomic DNAs see Table S3) and the IgF values are shown in Table 2.
• Multiplexed PCRs 1, 2, 3, 4, 5, and 6 were performed in 50 ⁇ L with e of 0.1 or 0.2 ⁇ M, 50 ng of the genomic DNA GI, G2, G6, or G8 for 17 cycles using denaturing step for 30 s at 95°C, primer annealing step for 90 s at 55°C and primer extension step for 150 s at 72°C for each cycle.
• the standard error was determined from triplicate PCR reactions each analyzed by the corresponding INVADER assay also in triplicate. b Reporting fluorescent dye of the INVADER assay.
• 192 SNPs representing chromosomes 5,11,14,15,16,17 and 19 were randomly selected and an INVADER assay was designed for each of the SNPs. During the selection process, no discrimination against SNPs in repetitive regions was carried out. Therefore some of the 192 SNPs were likely to be amplified at multiple loci. PCR conditions developed for balanced amplification were used with a fixed primer concentration because of simplicity and short development time. Genomic DNA samples G1-G8 were amplified with the 192-plex PCR for 17 cycles with fixed c of 0.2 ⁇ M, primer annealing time of 1.5 min, primer extension time of 2.5 min, and then analyzed with the 192 biplex INVADER assays as described in "Materials and Methods".
• the RED and FAM net signals were obtained by subtracting the no-target control signal from the sample signal.
• One way to identify genotypes from the net signals is to use universal calling criteria for each of the assays as described in the "Materials and Methods". These criteria assume that the homozygous samples have only signal from one of the alleles with no or very little cross-reactivity signal from the other one, and that heterozygous samples produce approximately equal signals for both alleles. Such rigid criteria can often lead to equivocal calls in otherwise functional INVADER assays.
• genotypes were called by plotting the FAM and RED net signals for all eight DNA samples as a scatter plot for each of the INVADER assays and visually identifying clusters corresponding to the homozygous and heterozygous samples. Scatter plot analysis cannot be performed if too few samples are included; this analysis also contains an element of subjectivity, since this type of visual analysis depends on the judgment of the operator. In this work, it was determined that eight samples are sufficient to make visual calls for the majority of the 192 INVADER assays. Examples of both successful and failed scatter plot analyses are shown in Figure 4. Figure 4 shows scatter plots of the net FAM and RED INVADER assay signals for eight genomic DNA samples.
• the INVADER assay net FAM and RED signals were plotted for the DNA samples GI, G2, G3, G4, G5, G6, G7 and G8 amplified with the 196-plex PCR.
• A-C successful genotyping with the assays 7, 9, and 25 assigning all samples to distinctive clusters identified as homozygous FAM (o), homozygous RED (G) or heterozygous (x).
• D-F failed genotyping.
• the sample closest to the origin of coordinates cannot be assigned to any of the clusters; in the assay 47 (E), the samples form three distinctive clusters but there is no FAM signal for any of the samples; in the assay 54 (F), the samples cannot be distinguished between homozygous RED with high FAM signal cross-reactivity and heterozygous with skewed RED/FAM ratio. RFU - relative fluorescence units. Conservative criteria were used for the visual analysis, excluding a whole set of samples if just one of the samples could not be assigned to a cluster.
• the PCR target sequences were analyzed using BLAT to determine if any of the individual PCRs amplified more than one locus. Eight of 31 assays apparently failed because, for each of them, multiple loci were likely amplified by the PCR and each of the loci could be detected by the INVADER assay. The remaining 23 assays were assumed to fail because of one or a combination of the following reasons: poor PCR amplification, flaw in oligonucleotide design and manufacturing , or unrecognized repeat sequences not included in the April 2003 human genome assembly. Excluding the 8 assays that failed because of repeat sequences in the genome, the efficiency of the 192-plex PCR with INVADER assay genotyping was estimated as 161/184 or 87.5%.
• the RED net fluorescence signal normalized per allele was plotted for the 161 successful INVADER assays performed on the eight DNA samples versus PCR target length as shown in Figure 5.
• Figure 5 shows the net RED fluorescence signal normalized per allele for the 161 successful INVADER assays as a function of PCR target length.
• the INVADER reactions were perfonned for 60 min with the eight DNA samples each amplified with the 196-plex PCR.
• the line shows a linear regression of the net signal as a function of PCR target length.
• FIG. 6 shows scatter plots of the net FAM and RED signals for the eight DNA samples.
• the INVADER assay 110 was performed with the DNA samples amplified with 196-plex PCR and signal was measured after 15 (A), 30 (B) and 60 min (C).
• the samples were identified as homozygous FAM (o), homozygous RED ( ⁇ ) or heterozygous (x) by tlie scatter plot analysis. RFU - relative fluorescence units.
• the scatter plots demonstrate that INVADER genotyping by cluster analysis is not affected by a strong net signal and can be interpreted even for the 60 min reaction, where both the FAM and RED net signal reach saturation. As a result of this effect, more calls can be made with longer INVADER reactions, because more signal is generated for slow PCRs, improving genotype identification, but at the same time the higher signal for the fast PCRs does not affect sample clustering.
• PCR AMPLIFICATION AND INVADER ASSAY ANALYSIS IN A SINGLE REACTION VESSEL This example describes a method for using PCR to amplify small amounts of a target followed by INVADER assay analysis is a single reaction vessel. In particular, this example describes conducting these two reactions without the need for mampulations or reagent additions after a single reaction set-up. Unless otherwise stated, the following examples were carried out with the indicated reagents for assays to detect sequences in the DLEU gene (chromosome 13) and ⁇ -actin gene (chromosome 1):
• CLEAVASE enzyme (VIII or X) 100 ng Stoffel or native Taq DNA polymerase 1 u
• PCR primers for DLEU Forward primer 1716-14-1 (SEQ ID NO: 1):
• Reverse primer 1716-14-4 (SEQ ID NO: 4): 5'-GCGTGAGGGTGGAAGGAGATGCCCATGG-3', Tm 74.7 °C
• Probes, INVADER oligos, FRET cassettes (underlined bases indicate flap sequences; bold bases indicate position 1 in the INVADER assay) ⁇ -actin probe 1734-57 ACGGACGCGGAGAGGAACCCTGTGACAT-hex (SEQ ID NO: 5) ⁇ -actin INVADER oligo 1734-57 CCATCCAGGGAAGAGTGGCCTGTTT (SEQ ID NO: 6) DLEU probe CGCGCCGAGGTTCTGCGCATGTGC-hex (SEQ ID NO: 7) DLEU INVADER oligo AGGGAGAGCCGTGCACCACGATGAC (SEQ ID NO: 8) DLEU FAM FRET 23-428 Fam-TCT-Z28-AGCCGGTTTTCCGGCTGAGACCTCGGCGCG- hex (SEQ ID NO: 9) ⁇ -actin RED FRET Red-TCT-Z28-TCGGCCTTTTGGCCGAGAGACTCCGCGTCCGT-hex (SEQ ID NO: 10).
• A. Configuration of combined PCR-INVADER reactions it may be desirable to separate the PCR and INVADER reactions temporally, e.g. by carrying out the PCR reaction under conditions that disfavor the INVADER reaction and then modifying the reaction conditions to permit the INVADER reaction to proceed.
• One such means of creating differential reaction conditions is via the use of antibodies to the enzymes used in the reaction, such as the Light Cycler TaqBlock antibody (Roche Applied Sciences). Another such means is via temperature.
• PCR primers were designed with annealing temperatures ⁇ 70°C while the probe oligonucleotides for use in the INVADER assay were designed with Tm of approximately 63°C, such that the probes should not be capable of reacting with target molecules during the annealing, extension, or denaturation phases of the PCR cycle.
• Tm approximately 63°C
• Reactions were carried out in which all reagents were combined in a final volume of 10 ⁇ l using the components described above and overlaid with mineral oil. PCR was allowed to proceed for 11-20 cycles (95 °C for 30 seconds; 72 °C for 30 seconds to 2 minutes). Following these cycling reactions, mixtures were heated to 99 °C for 10 minutes to inactivate the Taq DNA polymerase. The reaction mixtures were then incubated at 63 °C for 30 minutes to 3 hours to allow the INVADER reactions to proceed.
• Exemplary data obtained using native Taq polymerase are presented below and indicate that FAM signal generation is dependent on the presence of the DLEU INVADER oligo and that both INVADER reactions generate signal following 17 cycles of PCR followed by 10 min at 99 °C to denature the native Taq DNA polymerase followed by a 30 minute INVADER reaction at 61 °C.
• the Coriel samples were numbered as follows (e.g. "C” n) Coriell # Genotype 1 NAI 1277 1507 del HET 2 NAI 1280 711+1G>T/621+1G>T HET 3 NA01531 delF508 HOM 4 NA04539 delF508 HOM 5 NA07381 delF508/3849+10kb HET 6 NA07441 3120+1G>A/621+1G>T HET 7 NA07469 delF508/R553X 8 NAI 1283 A455E/delF508 HET 9 NAI 1284 R560T/delF508 HET
• PCR primers were selected from the following. cftr exon 3 TGGTCCCAC I I I H " ATTCTTTTGCAGA cftr exon 4 AAGTCACCAAAGCAGTACAGCC cftr exon 5 GCTGTCAAGCCGTGTTCTAGATAAA cftr exon 7 CGGAAGGCAGCCTATGTGAGA cftr exon 9 CATGGGCCATGTGCTTTTCAAAC cftr exon 9-1 CATGGGCCATGTGCTTTTCAAAC cftr exon 9-2 CTTCTTGGTACTCCTGTCCTGAAAGA cftr exon 10 ATTATGGGAGAACTGGAGCCTTCA cftr exon 11 GATTACATTAGAAGGAAGATGTGCCTTTCAA cftr exon 12 TAAGGCAAATCATCTACACTAGATGACCA cftr exon 13 TAACTGAGACCTTACACCGTTTCTCA cftr exon 14B
• PCR reactions were run as described above with a 2.5 minute extension at 72°C and a 45 sec denaturation at 95 °C for 14 cycles. Mixtures were heated to 99 °C for 10 minutes and then cooled to 63 °C for 1 hour. The results are presented in Figure 9.
• the delF508 sample was Coriel #3; the G85E/621 +1 G>T was Coriel 21; 1717-1G>A, Coriel 28; delF508/R117H was Coriel 30; delF508/3849 + lOkb, Coriel 5; A455E/delF508, Coriel 8, and R560T/delF508 Coriel 9.
• INVADER reactions that can be run and analyzed in a single reaction or reaction vessel.
• the present example describes the implementation of a 4-plex INVADER assay in which four sets of oligonucleotides are included in a single reaction.
• the reaction also included four distinct target sequences: wild type and variant versions of two different SNPs.
• Alternative configurations are also contemplated, including four distinct loci, tliree distinct loci and one internal control, etc.
• One variable in configuring the INVADER assay for multiplex FRET analysis is related to the choice of dyes for inclusion on the FRET probes. Numerous combinations of dyes and quenchers are known in the art (see, e.g., U.S.
• Another consideration affecting the choice of dyes relates to their spectral characteristics. In some embodiments, e.g., for assays detected in a fluorescence plate reader, it is preferred that the fluorescent signals from each dye be spectrally resolvable from one another by the instrument. If they are not sufficiently spectrally distinct, the fluorescence output from one dye could interfere or "bleed over" into the signal attributed to another dye.
• the fluorescence output of a given dye from a fluorescence plate reader scan is proportional to its concentration as follows:
• ⁇ is a constant that varies with the excitation and emission wavelengths and the gain settings of the plate reader and b is background. If multiple dyes are present, then each dye contributes to the total fluorescence as
• Fluorescence — background ⁇ • [dye + ⁇ • [dye + ⁇ • [dye c ] +... n • [dyej (3)
• F Ad (4)
• the elements of the linear matrix F are the background subtracted fluorescence readings
• A is the two-dimensional coefficient matrix
• d is the linear matrix whose elements are the amounts of each free dye released from the INVADER assay.
• the elements of the F and A matrices can be determined providing that there is some sort of calibration using pure dyes and blanks for each different scan. Therefore, the solution for d can be found by left multiplying both sides of equation (4) by the inverse of A.
• Such a matrix was derived for the 4-plex dye set as follows.
• Different ratios of these dTIO oligos were combined with FRET probes comprising the corresponding dye and an appropriate quencher to mimic signal generation from the INVADER assay over time.
• Working stocks of 500 nM were made of each dTIO and each FRET probe, respectively. Total sample volumes were 15 ⁇ l, and each sample was overlaid with 15 ⁇ l mineral oil. Ratios tested were 0% dT10/100% FRET probe; 25% dT10/75% FRET probe; 50% dT10/50% FRET probe; 75% dT10/25% FRET probe; and 100% dT10/0% FRET probe.
• the dyes tested were fluorescein (FAM), Cal-Gold and Cal-Orange (Biosearch Technologies, Inc., Novato, CA), and REDMOND RED (Synthetic Genetics). Tubes were read in a Tecan Safire XFLUOR 4 at excitation and emission wavelengths appropriate for each dye. In each case, the fluorescence observed from each dye increased linearly with increasing proportions of dTIO oligo, and the signals were additive. The slopes from the linear regressions were entered into the coefficient matrix as follows.
• a corresponding matrix was generated by taking the inverse of each value to obtain A '1 , as described above and thus derive d, the percentage of free dye in each case.
• INVADER assays were run as follows. Standard reactions were set up in a 15 ⁇ l final volume as described above with CLEAVASE VIII enzyme and 5 pM (final) synthetic target. Four different synthetic targets were used in the present example: wild-type and mutant for SNPs 1 and 2. The FRET probes used were as follows:
• EXAMPLE 10 MICROFLUIDIC CARD PRE-LOADED WITH INVADER ASSAY REAGENTS FOR TARGET DETECTION
• the following example described the use of a microfluidics card containing the INVADER assay reagents for interrogation of DNA samples.
• the target material has been prepared by prepared separately by PCR.
• the 3M microfluidic card has 8 loading ports, each of which is configured to supply liquid reagent to 48 individual reaction chambers upon centrifugation of the card.
• the reaction chambers contain pre-dispensed and dried INVADER assay reaction components for detection of one or more particular alleles (e.g. as shown in Example 11 , below).
• Multiplex PCR reaction mixtures were prepared using the following components (concentrations shown are at their final concentration in the PCR reaction): Genomic DNA at 2 ng/uL, multiplex PCR primer mix at 0.2 uM, PCR Buffer plus MgCl 2 at IX, dNTPs at 0.2 mM, and native Taq polymerase at 0.2 U/rxn. The final reaction volume was 20 uL. These mixtures were heated for 2.5 min at 95C , then were cycled 20 times through a 30 sec 95C step, a 1.5 min 55C step, and a 2.5 min 72C step.
• the samples were incubated at 99C for 10 min to destroy the polymerase activity.
• the amplicons were diluted 1:125 with dH 2 0, and 50 uL of this sample was mixed with 50 ⁇ l of a solution containing 28mM MgCl 2 and CLEAVASE X enzyme at 4ng/ ⁇ l. This mixture was then added to one of the 8 individual ports of the 3M CF microfluidics card described in the previous example.
• the INVADER assay was performed at 63C for 20 min, and fluorescence from the assay was detected on a microplate fluorimeter. The results are shown in Figures 11A-11G. The genotype of the genomic sample DNA is indicated at the top of each panel, and each of the mutations tested is indicated along the X-axis.
• EXAMPLE 11 INVADER PLUS PCR ON 3M CF MICROFLUIDICS CARD
• the following example described the use of a microfluidics card containing the INVADER assay reagents for interrogation of DNA samples.
• the target material is amplified and detected hi a single reaction.
• the reactions were performed on a 3M microfluidic card, as described above.
• the reaction chambers of the microfluidic card contain INVADER assay reaction components (i.e., the INVADER ohgonucleotide, primary probe, and FRET cassettes) for running the 48 different INVADER assays dried down onto the card.
• INVADER assay reaction components i.e., the INVADER ohgonucleotide, primary probe, and FRET cassettes
• IX PPIFF-MOPS mix (0.25 ⁇ M each Primary Probe Oligonucleotide, 0.125 ⁇ M each FRET oligonucleotide, 0.025 ⁇ M INVADER oligonucleotide, in 10 mM MOPS buffer) is dispensed into the wells of the microfluidics card.
• the cards are then allowed to dry in an air box through which HEPA filtered air is forced. It generally not necessary to control temperature or relative humidity of the air.
• the volume of each reaction chamber in the assembled microfluidic card is about 1.7 uL, so the final concentrations of these components during the reaction are about 1.18 times those of the IX PPIFF-MOPS mix).
• allelic variants detected by these INVADER assay oligonucleotide sets were as follows:
• the final concentration of components in these mixtures was as follows: 7.5 mM MgCl 2 , 6.67 ng/uL Cleavase VIII, .033 U/uL Native Taq-pol, 25 uM dNTP mix, 0.2 uM multiplex PCR primers.
• the combined PCR and INVADER assay reactions were incubated as follows: 95C for
• this example similar to the Examples above, describes the combination of PCR amplification and INVADER assay detection in a single reaction vessel to detect a genomic DNA.
• This Example further extends the above Examples by applying the method of single reaction vessel, combined PCR-INVADER analysis, to an un-purified whole blood sample.
• the PCR/INVADER assay reaction mixture in a total volume of 20 ul, is prepared as follows.
• For the buffer about 4 ul of either 0.5X AMPDIRECT-A from Shimadzu (without 5X Amp Addition-1) or 10 mM TAPS biological buffer (3- [[tris(Hydroxymethyl)methyl]amino]propanesulfonic acid) approximately pH 9 are employed.
• TAPS pH 9 rather than just AMPDIRECT- A, will serve as the buffer for direct PCR and INVADER detection in whole blood.
• additional details on the AMPDIRECT-A buffer and PCR in whole blood may be found, for example, in U.S. Patent Pubs 20020102660 and 20020142402, as well as Nishimura et al., Clin. Lab., 2002, 48:377-84, and Nishimura et al., Ann. Clin Biochem, 2000, 37:674-80, all of which are herein incorporated by reference for all purposes).
• the following additional reagents are used: 6.25 uM dNTPs each dNTP, 0.2 uM each PCR primer, 0.3 units of Taq polymerase (native), 40 ng of CLEAVASE VIII, 3 mM MgCl 2 (in addition to any MgCl 2 in the
• AMPDIRECT buffer if this buffer is used), 0.5 uM Primary Probe for each target to be detected (e.g., for targeted genomic DNA and for internal control), 0.05 uM INVADER oligonucleotide for each allele to be detected (for use with multiple Primary Probes, if a SNP is to be detected) or 0.05 uM INVADER oligonucleotide for each target to be detected (for use, e.g., when quantitating a variable target against an internal control target) 0.25 uM each FRET probe (for target and control reactions), and distilled water for a total reaction volume of 20 uL.
• 0.5 uM Primary Probe for each target to be detected e.g., for targeted genomic DNA and for internal control
• 0.05 uM INVADER oligonucleotide for each allele to be detected for use with multiple Primary Probes, if a SNP is to be detected
• the liquid whole human blood sample to be tested is first treated with an anticoagulant, such as sodium citrate, dipotassium EDTA, or sodium heparinate. About 0.4 ul (or less) of this treated whole human blood is added to the PCR/INVADER reaction mixture by loading it to the bottom ofthe reaction tube without mixing. Mineral oil can be overlayed if needed.
• PCR is carried out on the sample for a total of 28 cycles. PCR can be carried out, for example, using the following temperature profile, which is suitable for whole human blood: preheating at 80 C for 15 min, then 94 C for 4.5 min, followed by 28 cycles of 94 C for 30 seconds, annealing temperature for 1 minute, 72 C for 1 minute, and 72 C for 7 minutes.
• the mixture is heated to 99 °C for 10 minutes to inactivate the Taq DNA polymerase.
• the reaction mixture is then incubated at 63 °C for about 30 minutes to about 3 hours to allow the INVADER assay reactions to proceed.
• Results from the INVADER assay are collected (see, e.g., the Examples described above).
• the results of this example show successful PCR amplification of a target sequence in genomic DNA within the whole blood, as wells as successful INVADER assay detection ofthe target sequence of interest. Success in detecting the target nucleic acids of interest from this whole blood is possible whether AMPDIRECT or TAPS Ph 9 is used as the buffer.
• Direct DNA detection with combined PCR and INVDADER assays may also be performed using blood-spot cards, such as those from WHATMAN.
• the PCR-INVADER reaction buffer similar to the above, can be prepared as follows: 10 mM TAPS pH 9 buffer, 3 mM MgCl 2j 0.2 uM of each PCR primer, 6.25 uM each dNTP, 0.5 uM Primary Probe for each target to be detected (e.g., for targeted genomic DNA and for internal control), 0.05 uM INVADER oligonucleotide for each allele to be detected (for use with multiple Primary Probes, if a SNP is to be detected) or 0.05 uM INVADER oligonucleotide for each target to be detected (for use, e.g., when quantitating a variable target against an internal control target) 0.25 uM each FRET probe (for target and control reactions), 0.06 ul of TaqPol (native, 5 u/ul), 0.2 ul of

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