JP2007306918A - Amplification primer and detection of mutation in pooled sample - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for designing and utilizing an oligonucleotide primer used in a multiplex amplification reaction. <P>SOLUTION: This highly multiplexed PCR method is provided by optimizing the multiplex amplification reaction and combining with an invader assay method related to the judgment of gene type by using <150 pg DNA per one reaction. The method can be utilized as a method for detecting a mutation in a pooled nucleic acid sample. Especially, by using the invader detecting assay method, it can be utilized for detecting the mutation in the pooled nucleic acid sample and for measuring an allele. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  This application is a U.S. Provisional Application No. 60 / 329,113 filed on October 12, 2001, a U.S. Provisional Application No. 60 / 360,489 filed on October 19, 2001, May 9, 2001. Filed on Nov. 30, 2001, claiming priority of U.S. Provisional Application No. 60 / 289,764, filed on Oct. 2, and U.S. Provisional Application No. 60 / 326,549, filed Oct. 2, 2001. It is in the transitional stage in Japan from International Application No. PCT / US01 / 45705.

The present invention relates to a method for designing oligonucleotide primers used in multiplex amplification reactions. The present invention also provides a method for optimizing a multiplex amplification reaction. The present invention also provides a method for performing highly multiplexed PCR methods in combination with INVADER assays for genotyping using less than 150 pg of DNA per reaction. The invention also relates to detecting mutations in pooled nucleic acid samples. In particular, the invention relates to compositions and methods for detecting mutations and measuring alleles in pooled nucleic acid samples using invader detection assays.

Background As the human genome project is nearing completion and the amount of gene sequence information available is increasing, genome analysis studies and subsequent drug design attempts are likewise increasing. There is a need for systems and methods that enable efficient ordering, development, production and sale of detection assays that can be used in genomic analysis studies, drug design and personalized medicine. A number of institutions are actively examining available gene sequence information to identify correlations between genes, gene expression and phenotypes (eg, disease state, metabolic response, etc.). These analyzes include attempts to characterize the effects of gene mutations and genetic and gene expression heterogeneity in individuals and populations. However, despite the large amount of available sequence information, information about the frequency and clinical relevance of numerous polymorphisms and other mutations still has to be obtained and confirmed. For example, human reference sequences used in recent genome sequencing attempts do not show exact matches to any person's genome. In the Human Genome Project (HGP), researchers collected blood (female) or semen (male) samples from a number of donors. However, only a small number of samples are treated as DNA sources, and their source names are protected, neither the donor nor the scientist knows which DNA is being sequenced. Human genome sequences generated by the private genome analysis company Celera were based on DNA samples collected from five donors identified as Hispanic, Asian, Caucasian or African American. The few human samples used to generate that reference sequence do not reflect genetic diversity among population groups and individuals. Attempts to analyze individuals based on genomic sequence information often fail. For example, many gene detection assays are based on hybridization of probe oligonucleotides to target regions in genomic DNA or mRNA. Probes made based on that reference sequence often fail because the target sequence for multiple individuals is different from the reference sequence (e.g., a sequence at a specific location on the target that does not hybridize correctly). Not accurately characterized). Differences may be based on individuals, but many follow a regional population pattern (eg, many are highly correlated with race, ethnicity, geographic land, age, environmental exposure, etc.). Due to the limited availability of currently available information, the art aims to provide an array of one or more types of detection assay technologies for the study and clinical analysis of biological samples. What is needed is a system and method that can be selectively used in one or more production devices to acquire, analyze, accumulate, and apply large amounts of genetic information. It is an object of the present invention to satisfy these various requirements.

SUMMARY OF THE INVENTION The present invention provides a method for designing oligonucleotide primers used in multiplex amplification reactions. The present invention also provides a method for optimizing a multiplex amplification reaction. The present invention also provides a method for performing highly multiplexed PCR methods in combination with invader assays for genotyping using less than 150 pg of DNA per reaction. The present invention also provides a method for detecting mutations in a pooled nucleic acid sample. In particular, the present invention provides compositions and methods for detecting mutations and measuring alleles in pooled nucleic acid samples using invader detection assays.

In some embodiments, the present invention provides a method comprising the following steps:
For at least Y target sequences where each target sequence comprises i) a footprint region, ii) a 5 ′ region adjacent upstream of the footprint region, and iii) a 3 ′ region adjacent downstream of the footprint region Providing target sequence information of; and
b) includes a forward primer sequence and a reverse primer sequence for each of at least Y target sequences, each forward primer sequence and reverse primer sequence being 5'-N [x] -N [x-1] -....- N [4] -N [3] -N [2] -N [1] -3 ′ comprising a nucleic acid sequence, wherein N represents a nucleotide base, x is at least 6, and N [1] is a nucleotide N [2] -N [1] -3 'for each forward and reverse primer is A or C, and N [2] -N [1] -3' for any forward and reverse primer in the primer set Processing the target sequence information so that a primer set is produced that is also not complementary. In one variation, it is also recognized that customer-provided sequences are automatically increased upstream and downstream to allow for proper primer design using the methods and systems described herein.

In another embodiment, the present invention provides a method comprising the following steps:
For at least Y target sequences, each comprising a) a footprint region, ii) a 5 ′ region adjacent upstream of the footprint region, and iii) a 3 ′ region adjacent downstream of the footprint region Providing target sequence information of; and
b) includes a forward primer sequence and a reverse primer sequence for each of at least Y target sequences, each forward primer sequence and reverse primer sequence being 5'-N [x] -N [x-1] -....- N [4] -N [3] -N [2] -N [1] -3 ′ comprising a nucleic acid sequence, wherein N represents a nucleotide base, x is at least 6, and N [1] is a nucleotide N [2] -N [1] -3 'for each forward primer and reverse primer to N [2] -N [1] -3' for any forward primer and reverse primer in the primer set Processing the target sequence information so that a primer set is produced that is also not complementary.

In certain embodiments, the methods (including computer programs and routines that provide the following functionality) include:
For at least Y target sequences, each comprising a) a footprint region, ii) a 5 ′ region adjacent upstream of the footprint region, and iii) a 3 ′ region adjacent downstream of the footprint region Providing target sequence information of; and
b) i) a forward primer sequence that is identical to at least a portion of the 5 ′ region for each of the Y target sequences, and ii) at least a portion of the complementary sequence of the 3 ′ region for each of the at least Y target sequences. Each forward primer sequence and reverse primer sequence is 5'-N [x] -N [x-1] -....- N [4] -N [3] -N [2] -N [1] -3 ', wherein N represents a nucleotide base, x is at least 6, N [1] is nucleotide A or C, and each forward primer And the reverse primer N [2] -N [1] -3 'is not complementary to any forward primer or reverse primer N [2] -N [1] -3' in the primer set. Processing the target sequence information to

In other embodiments, the present invention provides methods (including computer programs and routines that provide the following functionality) comprising the following steps:
For at least Y target sequences where each target sequence comprises i) a footprint region, ii) a 5 ′ region adjacent upstream of the footprint region, and iii) a 3 ′ region adjacent downstream of the footprint region Providing target sequence information of; and
b) i) a forward primer sequence that is identical to at least a portion of the 5 ′ region for each of the Y target sequences, and ii) at least a portion of the complementary sequence of the 3 ′ region for each of the at least Y target sequences. Each forward primer sequence and reverse primer sequence is 5'-N [x] -N [x-1] -....- N [4] -N [3] -N [2] -N [1] -3 ′ comprising a nucleic acid sequence, wherein N represents a nucleotide base, x is at least 6, N [1] is a nucleotide G or T, and each forward primer And the reverse primer N [2] -N [1] -3 'is not complementary to any forward primer or reverse primer N [2] -N [1] -3' in the primer set. Processing the target sequence information to

In certain embodiments, the present invention provides methods (and routines that provide the following functionality) comprising the following steps:
a) providing target sequence information for at least Y target sequences, each target sequence comprising a single nucleotide polymorphism;
b) determining where one or more assay probes hybridize on each target sequence to detect single nucleotide polymorphisms such that the footprint region is located on each target sequence And
c) i) a forward primer sequence that is identical to at least part of the target sequence 5 ′ adjacent to the footprint region for each of the Y target sequences, and ii) a footprint region for each of the at least Y target sequences. A reverse primer sequence that is identical to at least part of the complementary sequence of the target sequence adjacent to 3 ′ of each of the 5′-N [x] -N [x-1]- ....- N [4] -N [3] -N [2] -N [1] -3 'comprises a nucleic acid sequence, N represents a nucleotide base, x is at least 6, N [1] is nucleotide A or C, and each forward primer and reverse primer N [2] -N [1] -3 ′ is any forward primer and reverse primer N [2] -N [ 1] -3 'not complementary to primer set Processing the target sequence information.

In some embodiments, the present invention provides a method (and a routine that provides the following functionality) comprising the following steps:
a) providing target sequence information for at least Y target sequences, each target sequence comprising a single nucleotide polymorphism;
b) Determine where on the target sequence one or more assay probes will hybridize to detect single nucleotide polymorphisms such that the footprint region is located on each target sequence A stage of; and
c) the primer set is i) a forward primer sequence that is identical to at least part of the target sequence 5 ′ adjacent to the footprint region for each of Y target sequences, and ii) at least Y target sequences for each A reverse primer sequence that is identical to at least part of the complementary sequence of the target sequence 3 ′ adjacent to the footprint region, wherein each forward primer sequence and reverse primer sequence is 5′-N [x] -N [x− 1] -....- N [4] -N [3] -N [2] -N [1] -3 ′ comprising a nucleic acid sequence, where N represents a nucleotide base and x is at least 6 N [1] is nucleotide T or G, and N [2] -N [1] -3 ′ for each forward primer and reverse primer is N [2] for which forward primer and reverse primer in the primer set Primer set that is not complementary to -N [1] -3 ' Processing the target sequence information such that a target is created.

  In some embodiments, the primer set is configured to perform a multiplex PCR reaction that amplifies at least Y amplicons, each amplicon being limited by the position of the forward primer sequence and the reverse primer sequence. In other embodiments, the primer set is made as digital or printed sequence information. In some embodiments, the primer set is made as a physical primer oligonucleotide. Using the methods, routines and components herein, it is possible to generate PCR primer reactions of 100 and higher.

  In some embodiments, each forward primer and reverse primer N [3] -N [2] -N [1] -3 'is the same as any forward primer and reverse primer N [3] -N [2]- It is not complementary to N [1] -3 '. In other embodiments, the treatment comprises first selecting N [1] for each forward primer as the 3 ′ most A or C in the 5 ′ region. In certain embodiments, initially selecting N [1] for each forward primer as the 3 ′ most G or T in the 5 ′ region. In some embodiments, the treatment comprises first selecting N [1] for each forward primer as the 3 ′ most A or C in the 5 ′ region, and the treatment comprises N [ For forward primer sequences lacking the requirement that 2] -N [1] -3 'is not complementary to any forward and reverse primer N [2] -N [1] -3' in the primer set, N [1 Is further changed to A or C on the 3 ′ side in the 5 ′ region.

  In other embodiments, the (preferably electronic) processing step comprises first selecting N [1] for each reverse primer as the 3'-most A or C in the complement of the 3 'region. In some embodiments, the treatment comprises first selecting N [1] for each reverse primer as the 3 ′ most G or T in the complement of the 3 ′ region. In a further embodiment, the method comprises first selecting N [1] for each reverse primer as the 3 ′ most A or C in the 3 ′ region and the treatment comprises N [2] -N [1 of the reverse primer ] -3 'N [1] 3' region for reverse primer sequences that lack the requirement that each is not complementary to any forward and reverse primer N [2] -N [1] -3 'in the primer set And then further changing to the most 3 ′ A or C.

  In certain embodiments, the footprint region comprises a single nucleotide polymorphism. In some embodiments, the footprint includes a mutation. In some embodiments, the footprint region for each target sequence includes a portion of the target sequence that hybridizes to one or more assay probes configured to detect single nucleotide polymorphisms. In certain embodiments, the footprint is this region to which the probe hybridizes. In other embodiments, the footprint further comprises additional nucleotides at either end.

  In some embodiments, the processing step (electronic in one of the variants of the present invention) is N [for each forward and reverse primer such that there is less than 80 percent homology with the assay component sequence. 5] -N [4] -N [3] -N [2] -N [1] -3 'is further included. In a preferred embodiment, the assay component is a FRET probe sequence. In certain embodiments, the target sequence is about 300 base pairs to 500 base pairs long, or about 200 base pairs to 600 base pairs long. In certain embodiments, Y is an integer between 2 and 500, or between 2 and 10,000.

  In some embodiments, the processing step (electronic in one of the variants of the present invention) is performed by each forward primer and reverse primer at about 50 degrees Celsius (e.g., 50 degrees Celsius, or at least 50 degrees Celsius, and 55 degrees Celsius). Selecting x for each forward primer and reverse primer to have a melting temperature for the target sequence (not exceeding degrees). In a preferred embodiment, the melting temperature of the primer (when hybridizing to the target sequence) is at least 50 degrees Celsius, but differs by at least 10 degrees from the optimal reaction temperature of the selected detection assay.

  In some embodiments, the optimized concentration of a forward primer and reverse primer pair is determined for that primer set. In other embodiments, the process is automated. In a further aspect, the process is automated with a processing device.

  In other embodiments, the invention relates to primer sets made by the methods of the invention, and at least one other component (e.g., cleaving agent, polymerase, invader oligonucleotide, or another variation of the invention). Other detection assays or detection assay components) are provided. In certain embodiments, the present invention provides compositions comprising primers and primer sets made by the methods of the present invention.

  In certain embodiments, the present invention provides methods (and routines that utilize methodologies) that include the following steps: a) i) a user interface configured to receive sequence data, ii) multiplex PCR therein Providing a computer system storing a primer software application; and b) transmitting sequence data from the user interface to the computer system, the sequence data including target sequence information for at least Y target sequences; Each target sequence comprises i) a footprint region, ii) a 5 ′ region adjacent upstream of the footprint region, and iii) a 3 ′ region adjacent downstream of the footprint region, and c) creating a primer set To process target sequence information in a multiplex PCR primer pair software application A primer set is i) a forward primer sequence that is identical to at least a portion of the target sequence 5 'adjacent to the footprint region for each of Y target sequences, and ii) a foot for each of at least Y target sequences A reverse primer sequence that is identical to at least part of the complementary sequence of the target sequence 3 ′ adjacent to the print region, each forward primer sequence and reverse primer sequence 5′-N [x] -N [x−1 ] -....- N [4] -N [3] -N [2] -N [1] -3 ′ comprising a nucleic acid sequence, wherein N represents a nucleotide base and x is at least 6 Yes, N [1] is nucleotide A or C, and N [2] -N [1] -3 'for each forward primer and reverse primer is N [2]-for which forward primer and reverse primer in the primer set N [1] -3 'also includes those that are not complementary .

  In some embodiments, the present invention provides methods (and routines used in methodologies) comprising the following steps: a) i) a user interface configured to receive sequence data, ii) multiplex PCR therein Providing a computer system storing a primer software application; and b) transmitting sequence data from the user interface to the computer system, the sequence data including target sequence information for at least Y target sequences; Each target sequence comprises i) a footprint region, ii) a 5 ′ region adjacent upstream of the footprint region, and iii) a 3 ′ region adjacent downstream of the footprint region, and c) creating a primer set Process target sequence information in a multiplex PCR primer pair software application for The primer set is i) a forward primer sequence that is identical to at least a portion of the target sequence 5 ′ adjacent to the footprint region for each of the Y target sequences, and ii) for each of the at least Y target sequences. A reverse primer sequence that is identical to at least part of the complementary sequence of the target sequence 3 ′ adjacent to the footprint region, wherein each forward primer sequence and reverse primer sequence is 5′-N [x] -N [x− 1] -....- N [4] -N [3] -N [2] -N [1] -3 ′ comprising a nucleic acid sequence, where N represents a nucleotide base and x is at least 6 N [1] is nucleotide G or T, and N [2] -N [1] -3 ′ for each forward primer and reverse primer is N [2] for which forward primer and reverse primer in the primer set -N [1] -3 'also includes non-complementary ones Stage.

  In some embodiments, the present invention provides a system that includes: a) a computer system configured to receive data from a user interface (and routines used in methodology) such that the user interface receives sequence data. Set and the sequence data includes target sequence information for at least Y target sequences, each target sequence i) footprint region, ii) 5 ′ region adjacent upstream of the footprint region, and iii) foot B) a multiplex PCR primer pair software application that includes an adjacent 3 ′ region downstream of the print region, b) operably linked to the user interface, and the multiplex PCR primer software application uses the target sequence information to create a primer set. Set to process The primer set is i) a forward primer sequence that is identical to at least part of the target sequence 5 ′ adjacent to the footprint region for each of Y target sequences, and ii) a foot for each of at least Y target sequences. A reverse primer sequence that is identical to at least part of the complementary sequence of the target sequence 3 ′ adjacent to the print region, each forward primer sequence and reverse primer sequence 5′-N [x] -N [x−1 ] -....- N [4] -N [3] -N [2] -N [1] -3 ′ comprising a nucleic acid sequence, wherein N represents a nucleotide base and x is at least 6 Yes, N [1] is nucleotide A or C, and N [2] -N [1] -3 'for each forward primer and reverse primer is N [2]-for which forward primer and reverse primer in the primer set N [1] -3 'also includes those that are not complementary, Beauty c) a computer system which stores the multiplex PCR primer pair software application therein, the computer system includes a computer memory and computer processor.

  In another aspect, the present invention provides a system comprising: a) a computer system or computer configured to receive data from a user interface, wherein the user interface is configured to receive sequence data; The sequence data includes target sequence information for at least Y target sequences, each target sequence i) a footprint region, ii) a 5 ′ region adjacent upstream of the footprint region, and iii) downstream of the footprint region. B) A multiplex PCR primer pair software application that includes an adjacent 3 'region and is operably linked to a user interface, where the multiplex PCR primer software application is configured to process target sequence information to create primer sets The primer I) a forward primer sequence that is identical to at least a portion of the target sequence 5 ′ adjacent to the footprint region for each of the Y target sequences, and ii) a footprint for each of the at least Y target sequences. A reverse primer sequence that is identical to at least part of the complementary sequence of the target sequence 3 ′ adjacent to the region, wherein each forward primer sequence and reverse primer sequence is 5′-N [x] -N [x-1] -.... includes the nucleic acid sequence represented by -N [4] -N [3] -N [2] -N [1] -3 ', where N represents a nucleotide base and x is at least 6 , N [1] is nucleotide G or T, and N [2] -N [1] -3 ′ for each forward and reverse primer is N [2] -N for which forward and reverse primer in the primer set [1] -3 'also includes those that are not complementary, and c) A computer system which stores the multiplex PCR primer pair software application, the computer system includes a computer memory and computer processor. In certain embodiments, the computer system is configured to return the primer set to the user interface.

In some embodiments, the present invention relates to detecting mutations in pooled nucleic acid samples. In particular, the present invention relates to compositions for detecting mutations or measuring allelic frequency in pooled nucleic acid samples using invader detection assays or other detection assays described herein. Regarding the method. In some embodiments, the present invention provides a method for detecting polymorphic allele frequencies comprising the following steps:
a) i) a target nucleic acid sequence from which the pooled sample is derived from at least 10 individuals (or at least 50, or at least 100, or at least 250, or at least 500, or at least 1000 individuals, etc.) Including pooled samples; and
ii) Invader detection reagents set to detect the presence or absence of polymorphisms (e.g., primary probes, invader oligonucleotides, FRET cassettes, structure-specific enzymes, etc.)
Providing a stage; and
b) contacting the pooled sample with an invader detection reagent that produces a detectable signal; and
c) measuring a detectable signal, thereby determining the number of target nucleic acid sequences comprising the polymorphism (eg, determining the quantitative number of molecules or the allelic frequency for the polymorphism in the population). In some embodiments, signals from two or more alleles for a particular target nucleic acid locus are measured and the numbers are compared. In preferred embodiments, measurements for two or more different alleles of a particular target nucleic acid locus are measured in a single reaction. In other embodiments, measurements from one or more alleles of a particular target nucleic acid locus are compared to measurements from one or more reference target nucleic acid loci. In preferred embodiments, measurements from one or more alleles of a particular target nucleic acid locus are compared to measurements from one or more reference target nucleic acid loci in the same reaction mixture. Further methods include a specific number of molecules found to have a particular allelic frequency (i.e., frequency of mutations between multiple copies of a sequence within an individual) or polymorphisms in a single individual ( (E.g., measured by an invader assay) can be compared to the allelic frequency (or expected number) of a population to determine if the single individual is susceptible to the disease and how far the disease has progressed. (Eg, diseases such as cancer that can be diagnosed by identifying loss of heterozygosity) and the like. In some embodiments, the individuals are from the same racial or ethnic class (eg, European, African, Asian, Mexican, etc.).

  In certain embodiments, the present invention provides a method for detecting a rare mutation comprising the following steps: a) i) a sample comprising at least 10,000 target nucleic acid sequences (eg, from 10,000 cells) Or a sample from a single subject containing at least 20,000 target nucleic acid sequences, or at least 100,000 target nucleic acid sequences), ii) present at an allele frequency of 1: 1000 or less compared to a wild-type allele Providing a detection assay (eg, an invader assay) capable of detecting mutations in the target nucleic acid sequence population to be; and b) a rare mutation (eg, 1: 100, or 1: 500 compared to the wild type) Assaying the sample in a detection assay under conditions such that the presence or absence of an allele frequency of 1: 1000 or less is detected. In some embodiments, the target nucleic acid sequence is a genome (eg, polymerase chain reaction, ie not PCR amplified, directly from the cell). In other embodiments, the target nucleic acid sequence is amplified (eg, by PCR).

  In some embodiments, the present invention provides a method for detecting a rare mutation comprising the following steps: a) i) from a single subject, wherein the sample comprises at least 10,000 target nucleic acid sequences Providing a sample, ii) a detection assay capable of detecting mutations in a target nucleic acid sequence population present at an allele frequency of 1: 1000 or less compared to a wild-type allele; and b) a rare mutation Assaying the sample in a detection assay under conditions such that the allele frequency in the sample is determined. In some embodiments, the allele frequency of a subject is known relative to a known reference allele frequency so that a diagnosis can be made (e.g., the extent of the disease, the likelihood of having the disease or transmitting it to offspring, etc.). Be compared statistically.

  The present invention also provides a method for determining the number of one or more polymorphic molecules present in a sample, for example by using invader assays (e.g., like SNPs associated with disease). Polymorph). This assay measures the number of a particular polymorphism in the first sample and then determines if there is a statistically significant difference between that number and the number of the same polymorphism in the second sample Can be used to Preferably, one sample presents the number of polymorphisms expected to occur in a healthy individual or in a sample obtained from a healthy population if a pooled sample is used. The statistically significant difference between the number of polymorphisms expected to be at a single nucleotide locus in healthy individuals and the number measured in samples from patients is clinically suggestive .

  The accompanying drawings form part of the present specification and are included to further demonstrate certain aspects and embodiments of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the description of specific embodiments presented herein.

Definitions In order to facilitate understanding of the invention, several terms and phrases are defined as follows.

  The terms “computer memory” and “computer memory device” as used herein refer to any storage medium readable by a computer processing device. Examples of computer memory include, but are not limited to, RAM, ROM, computer chip, digital video disc (DVD), compact disc (CD), hard disk drive (HDD), and magnetic tape.

  The term “computer readable medium” as used herein refers to any device or system for storing and supplying information (eg, data and instructions) to a computer processing device. Examples of computer readable media include, but are not limited to, a DVD, CD, hard disk drive, magnetic tape, and server for streaming media over a network.

  As used herein, the terms "processor" and "central processor" or "CPU" can be used interchangeably and can read programs from computer memory (eg, ROM or other computer memory). Refers to the device and executes a set of steps according to a program.

  As used herein, the term “SNP”, “SNPs” or “single nucleotide polymorphism” refers to a single base change at a particular position in the genome of an organism (eg, human). “SNPs” can be located in portions of the genome that are not encoded as genes. Alternatively, “SNP” may be located in the coding region of a gene. In this case, the “SNP” may alter the structure and function of the RNA or protein with which it is associated.

  As used herein, the term “allele” refers to a variant of a given sequence (eg, but not limited to a gene comprising one or more SNPs). In a population, there are a large number of genes as multiallelic types. A homozygote has two copies of the same allele, whereas a diploid organism with two different alleles of a gene is said to be heterozygous for that gene.

  As used herein, the term “linkage” refers to the proximity of two or more markers (eg, genes) on a chromosome.

  As used herein, the term `` allele frequency '' refers to the frequency of occurrence of a given allele (e.g., a sequence that includes a SNP) in a given population (e.g., a particular sex, race, or ethnic population). Point to. A particular population may contain a given allele within a higher percentage of its members than other populations. For example, a special mutation in the breast cancer gene called BRCA1 was found to be present in 1 percent of the general Jewish population. By comparison, the percentage of people in the general US population with any mutation in BRCA1 is estimated to be between 0.1 percent and 0.6 percent. Two additional mutations (one in the BRCA1 gene and one in another breast cancer gene called BRCA2) have a greater prevalence in the Ashkenazi Jewish population, and these three mutations The overall risk of having one of these is 2.3%.

  As used herein, the term “in silico analysis” refers to an analysis performed using a computer processor and computer memory. For example, “in silico SNP analysis” refers to analysis of SNP data using a computer processor and memory.

  As used herein, the term “genotype” refers to the actual genetic composition of an organism (eg, with respect to a particular allele at a locus). Genotypic expression gives rise to the physical appearance and characteristics ("phenotype") of the organism.

  As used herein, the term “locus” refers to the location of a gene or any other characterized sequence on a chromosome.

  The term “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. Wild-type genes are those that are most frequently observed in a population, and thus are intended to be “normal” or “wild-type” forms of the gene. In contrast, the terms `` modified '', `` mutant '' and `` variant '' refer to alterations in sequence and / or functional properties when compared to a wild-type gene or gene product ( That is, it refers to a gene or gene product that exhibits altered characteristics). It is noted that naturally occurring variants can be isolated, but these are identified by the fact that they have altered characteristics when compared to the wild type gene or gene product.

  As used herein, the terms “complementary” or “complementarity” are used in reference to polynucleotides (ie, sequences of nucleotides) that are related by base pairing rules. For example, the sequence “5′-A-G-T-3 ′” is complementary to the sequence “3′-T-C-A-5 ′”. Complementarity can be “partial” in which only some of the nucleic acid bases are aligned 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 a significant effect on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions as well as in detection methods that rely on binding between nucleic acids.

As used herein, the term “hybridization” is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (ie, the strength of binding between nucleic acids) is the degree of complementarity between nucleic acids, the stringency of the conditions involved, the T _{m of the} constructed hybrid, and the G within the nucleic acid: It is strongly influenced by factors such as C ratio.

As used herein, the term “T _m ” is used in reference to “melting temperature”. The melting temperature is a temperature at which a group of double-stranded nucleic acid molecules is half-dissociated into a single strand. The equation for calculating the T _m of nucleic acids is well known in the art. As shown by standard references, a simple estimate of the T _m value can be calculated by the equation: T _m = 81.5 + 0.41 (% G + C) when the nucleic acid is in an aqueous solution of 1 M NaCl ( (See (1985) in Anderson and Young, “Quantitative Filter Hybridization”, “Nucleic Acid Hybridization”). Other references include more complex calculation methods that take into account the sequence and structural properties of T _m calculations.

  As used herein, the term “stringency” is used in reference to the conditions of the presence of other compounds such as temperature, ionic strength, and organic solvents when nucleic acid hybridization is performed. One of ordinary skill in the art will recognize that “stringency” conditions can be changed by changing the parameters just described, either separately or simultaneously. Under "high stringency" conditions, nucleobase pairing will occur only between nucleic acid fragments with a high frequency of complementary base sequences (e.g., hybridization under "high stringency" conditions is about 85% Can occur between homologues with ˜100% identity, preferably about 70% to 100% identity). At moderate stringency conditions, nucleobase pairing will occur between nucleic acids with intermediate frequencies of complementary base sequences (e.g., hybridization under `` moderate stringency '' conditions is approximately Can occur between homologues with 50% to 70% identity). Thus, for nucleic acids from genetically diverse organisms, conditions of “weak” or “low” stringency are often required because the frequency of complementary sequences is usually lower.

`` High stringency conditions '' when used in reference to nucleic acid hybridization means that 5 × SSPE (43.8 g / l NaCl, 6.9 g / l NaH ₂ PO ₄ H ₂ O and 1.85 g / l EDTA, adjusted to pH 7.4 with NaOH), binding or hybridization at 42 ° C in a solution consisting of 0.5% SDS, 5x Denhardt's reagent and 100 μg / ml denatured salmon sperm DNA, followed by 0.1x Includes conditions equivalent to washing at 42 ° C in a solution containing SSPE, 1.0% SDS.

`` Medium stringency conditions '' when used in connection with nucleic acid hybridization means 5 × SSPE (43.8 g / l NaCl, 6.9 g / l NaH ₂ PO ₄ H ₂ when using a probe of about 500 nucleotides in length. O and 1.85 g / l EDTA, adjusted to pH 7.4 with NaOH), binding or hybridization at 42 ° C. in a solution consisting of 0.5% SDS, 5 × Denhardt's reagent and 100 μg / ml denatured salmon sperm DNA, followed by Includes conditions equivalent to washing at 42 ° C. in a solution containing 1.0 × SSPE and 1.0% SDS.

`` Low stringency conditions '' refer to 5 x SSPE (43.8 g / l NaCl, 6.9 g / l NaH ₂ PO ₄ H ₂ O and 1.85 g / l EDTA, NaOH when using a probe of approximately 500 nucleotides in length. pH 7.4), 0.1% SDS, 5 × Denhardt's reagent (50 × Denhardt's reagent 500 ml, 5 g Ficoll (type 400, Pharmacia), 5 g BSA (fraction V; Sigma)) And binding or hybridization at 42 ° C in a solution consisting of 100 g / ml denatured salmon sperm DNA, followed by washing at 42 ° C in a solution containing 5xSSPE, 0.1% SDS. Including.

  “Amplification” is a special case of nucleic acid replication associated with template specificity. It is in contrast to non-specific template replication (ie template dependent but not dependent on a specific template). Template specificity is here distinguished from replication fidelity (ie synthesis of the correct polynucleotide sequence) and nucleotide (ribonucleotide or deoxyribonucleotide) specificity. Template specificity is often described in terms of “target” specificity. Target sequences are “targets” in the sense that they are sought to be distinguished from other nucleic acids. Amplification techniques were initially designed for this distinction.

  Template specificity is achieved in most amplification techniques by the choice of enzyme. Amplifying enzymes seek to process only specific sequences of nucleic acids in a heterogeneous nucleic acid mixture. For example, in the case of Q replicase, MDV-1 RNA is a specific template for the replicase (D. L. Kacian et al., Proc. Natl. Acad. Sci. USA 69: 303038 [1972]). Other nucleic acids are not replicated by this amplification enzyme. Similarly, in the case of T7 RNA polymerase, this amplifying enzyme has stringent specificity for its own promoter (M. Chamberlin et al., Nature 228: 227 [1970]). In the case of T4 DNA ligase, the enzyme does not attempt to ligate the two oligonucleotides or polynucleotides where there is a mismatch between the oligonucleotide or polynucleotide substrate and the template at the ligation junction (DY Wu and RB Wallace, Genomics 4: 560 [1989]). Finally, Taq and Pfu polymerases have been found to exhibit high specificity for sequences bound by primers and thus limited due to their ability to function at high temperatures. Results in a thermodynamic state that favors hybridization of the primer with its target sequence and not favors hybridization with target-free sequences (HA Erlich (ed.), "PCR Technology ) ", Stockton Press [1989]).

  As used herein, the term “amplifiable nucleic acid” is used in reference to a nucleic acid that can be amplified by any amplification method. “Amplifiable nucleic acids” are generally intended to include “sample templates”.

  As used herein, the term “sample template” refers to a nucleic acid derived from a sample that is analyzed for the presence of a “target” (defined below). In contrast, “background template” is used in reference to nucleic acid excluding sample templates that may or may not be present in a sample. Background templates are often due to carelessness. It may be the result of carry-over contamination or may be due to the presence of nucleic acid contaminants that are required to be purified and removed from the sample. For example, nucleic acid derived from an organism other than the nucleic acid to be detected may be present as background in the test sample.

  As used herein, the term “primer” refers to the synthesis of a primer extension product that is complementary to a nucleic acid strand, whether it occurs naturally or synthetically in the form of a purified restriction enzyme digest. When placed under induced conditions (i.e., in the presence of an inducer such as nucleotides and DNA polymerase, at a suitable temperature and pH), an oligonucleotide capable of functioning as a starting point for synthesis Point to. 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. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be long enough to initiate the synthesis of the extension product in the presence of an inducer. The exact length of the primer will depend on many factors, including temperature, primer source and method application.

  As used herein, the term “probe” or “hybridization probe” may be made synthetically, recombinantly, or by PCR amplification, whether naturally occurring in the form of a purified restriction enzyme digest. Refers to an oligonucleotide (ie, a sequence of nucleotides) that is capable of at least partially hybridizing to another oligonucleotide of interest. The probe can be single-stranded or double-stranded. Probes are useful for the detection, identification and isolation of specific sequences. In some preferred embodiments, probes used in the present invention include, but are not limited to, enzymes (e.g., ELISA, and enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. Labeled with a “reporter molecule” so that it can be detected in any detection system. The present invention is not intended to 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.

  The term “polymerase chain reaction” (“PCR”), as used herein, describes a method for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. Refers to methods (see, eg, US Pat. Nos. 4,683,195, 4,683,202, and 4,965,188, incorporated herein by reference). This step for amplifying the target sequence introduces a large excess of two oligonucleotide primers into the DNA mixture containing the desired target sequence, followed by an accurate sequence of thermal cycling in the presence of DNA polymerase. Become. The two primers are complementary to each strand of the double stranded target sequence. To effect amplification, the mixture is denatured and then the primers are annealed to their complementary sequences within the target molecule. After annealing, the primer is extended with a polymerase to form a new pair of complementary strands. The steps of denaturation, primer annealing, and polymerase extension can be repeated many times to obtain a high concentration of amplified segment of the desired target sequence (i.e., denaturation, annealing, and extension take one `` cycle ''). And there can be multiple “cycles”). The length of the amplified segment of the desired target sequence is determined by the relative position of the primers with respect to each other, and thus this length is a controllable parameter. Depending on the repetitive aspect of the steps, the method is referred to as the “polymerase chain reaction” (hereinafter “PCR”). They are said to be “PCR amplified” because the segments of the desired amplified target sequence become the dominant sequences (in terms of concentration) in the mixture.

Using PCR, a single copy of a particular target sequence in genomic DNA can be made in several different ways (eg, hybridization with a labeled probe; incorporation of a biotinylated primer followed by avidin-enzyme conjugate detection; Incorporation of ³² P-labeled deoxynucleotide triphosphates into the amplified segment, such as dCTP or dATP, can be amplified to detectable levels. In addition to genomic DNA, any oligonucleotide or polynucleotide sequence can be amplified with an appropriate set of primer molecules. In particular, the amplified segments produced by the PCR step itself are themselves effective templates for subsequent PCR amplification.

  As used herein, the terms “PCR product”, “PCR fragment” and “amplification product” refer to a mixture of compounds that results after two or more cycles of the PCR steps of denaturation, annealing, and extension are completed. Point to. These terms encompass the case where there has been amplification of one or more segments of one or more target sequences.

  As used herein, the term “amplification reagent” refers to those reagents (deoxyribonucleotide triphosphates, buffers, etc.) required for amplification except for primers, nucleic acid templates and amplification enzymes. Typically, amplification reagents are placed with other reaction components and placed in reaction vessels (test tubes, microwells, etc.).

  As used herein, the term “sample” is used in its broadest sense. Samples suspected of containing human chromosomes or sequences related to human chromosomes are cells, chromosomes isolated from cells (e.g., metaphase chromosome spreads), such as in solution or for Southern blot analysis It may include genomic DNA (bound to a solid support), RNA (in solution or bound to a solid support for Northern blot analysis), cDNA (in solution or bound to a solid support), and the like. Samples suspected of containing proteins can include cells, tissue parts, extracts containing one or more proteins, and the like.

As used herein, the term “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; radioactive labels such as ³² P; binding moieties such as biotin; haptens such as digoxigenin; luminescent, phosphorescent or fluorogenic moieties; and fluorescence Includes dyes alone or in combination with moieties capable of suppressing or converting the emission spectrum by fluorescence resonance energy transfer (FRET). The label can provide a detectable signal by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or adsorption, magnetism, enzyme activity, and the like. The label can be a charged moiety (positive or negative charge) or can be charge neutral. The label may comprise or consist of a nucleic acid or protein sequence so long as the sequence containing the label is detectable.

  As used herein, the term “signal” refers to any detectable effect as caused or provided by a label or assay reaction.

  As used herein, 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.

  As used herein, the term “kit” refers to any delivery system for delivering materials. In the context of reaction assays, such delivery systems can be used from one location to another, such as reaction reagents (e.g., oligonucleotides, enzymes, etc. in appropriate containers) and / or auxiliary materials (e.g., buffers, Including a system that allows storage, transport or delivery of written instructions for performing the assay. For example, the kit includes one or more enclosures (eg, boxes) that contain relevant reaction reagents and / or auxiliary materials. As used herein, the term “split kit” refers to a delivery system that includes two or more separate containers, each containing a portion of all kit components. The containers can be delivered together or separately to the intended recipient. For example, a first container contains an assay enzyme, while a second container contains an oligonucleotide. The term “segmented kit” is intended to encompass kits containing Analyte specific reagents (ASR's) controlled under section 520 (e) of the US Food, Drug, and Cosmetic Act. It is not limited. Indeed, any delivery system that includes two or more separate containers, each containing a portion of all kit components, is included in the term “split kit”. In contrast, an “integrated kit” refers to a delivery system that contains all of the components of a reaction assay in a single container (eg, in a single box containing each of the desired components). The term “kit” includes both segmented and integrated kits.

  As used herein, the term “information” refers to any collection of facts or data. With respect to information stored or processed using a computer system, including but not limited to the Internet, the term is stored in any format (eg, analog, digital, optical, etc.) Refers to arbitrary data. As used herein, the term “information related to a subject” refers to facts or data relating to a subject (eg, a human, plant or animal). The term “genomic information” refers to information related to the genome including, but not limited to, nucleic acid sequences, genes, allelic frequencies, RNA expression levels, protein expression, phenotypes corresponding to genotypes, etc. Point to. “Allele frequency information” includes, but is not limited to, allele identification, statistical correlation between the presence of an allele and the characteristics of an object (eg, a human object), an individual or a population Refers to facts or data related to allelic frequency, including the presence or absence of alleles, the percentage of alleles that are likely to be present in individuals with one or more special characteristics, and the like.

  As used herein, the term “assay validation information” refers to genomic information and / or allelic frequency information that results from processing test data (eg, processing using a computer). The assay validation information can be used, for example, to identify a particular candidate detection assay as an effective detection assay.

  As used herein, the term `` rare mutation '' refers to 20% or less (preferably 10% or less, more preferably 5% or less, and even more) of a population of nucleic acid molecules in a sample. Refers to mutations that are preferably present at 1% or less) (ie, the remaining 80% or more of the nucleic acid molecule has a wild-type sequence or a different mutation in the corresponding region of the nucleic acid molecule).

  The term “unique” as used herein with respect to signal refers to spectral characteristics such as, for example, fluorescence emission wavelength, color, absorbance, mass, size, fluorescence polarization properties, charge, etc., or chemical reagents, enzymes Refers to a signal that can be differentiated from another by virtue of its ability to interact with another moiety, such as an antibody.

DETAILED DESCRIPTION OF THE INVENTION The following discussion provides a description of certain preferred exemplary embodiments of the invention and does not limit the scope of the invention. For convenience, the discussion focuses on the detection of DNA targets in the present application, but the method and system are directed to use in the development of tools for the analysis of any nucleic acid analyte, eg, DNA or RNA. It should be understood that Also, for illustration purposes, the discussion often focuses on characterization of SNPs using invader assay technology. It should be understood that the methods and systems of the present invention are directed to use in detecting other biologically relevant factors using a wide variety of detection assay technologies.

I. Detection assays
There are a wide variety of detection technologies that can be used to determine the sequence of a target nucleic acid at one or more positions. For example, there are a number of technologies that can be used to detect the presence or absence of SNPs. Many of these techniques require the use of oligonucleotides that hybridize to the target. Depending on the assay used, the oligonucleotide is then cleaved, extended, bound, dissociated, or otherwise altered, and its behavior in the assay depends on the target nucleic acid As a means to characterize the sequence of Some of these technologies are described in detail in Section V below.

  The present invention provides systems and methods for the design of oligonucleotides used in detection assays. In particular, the present invention successfully works on appropriate regions of the target nucleic acid (e.g., regions of the target nucleic acid that do not contain secondary structure) under the reaction conditions desired for the detection assay (e.g., temperature, buffer conditions, etc.). Systems and methods are provided for the design of hybridizing oligonucleotides. The systems and methods also include a plurality of different oligonucleotides that function all under the same or substantially the same reaction conditions in the detection assay (e.g., hybridize to different portions of the target nucleic acid, or two or more Allows the design of oligonucleotides that hybridize to different target nucleic acids. These systems and methods can also be used to design control samples that operate under experimental reaction conditions. The present invention also provides a method for designing a sequence to amplify the target sequence to be detected (eg, designing PCR primers for multiplex PCR).

  Although the systems and methods of the present invention are not limited to any particular detection assay, the following description is provided to illustrate preferred features of the present invention for detecting SNPs or other sequences of interest. Invader assay (Third Wave Technologies, Madison, WI; eg, US Pat. Nos. 5,846,717; 6,090,543; 6,001,567; 5,985,557, each incorporated herein by reference in its entirety for all purposes. No. 5,994,069, 6,214,545, 6,210,880, and 6,194,880; Lyamichev et al., Nat. Biotech., 17: 292 (1999), Hall et al., PNAS, USA, 97: 8272 (2000), Agarwal et al. , Diagn. Mol. Pathol. 9: 158 [2000], Cooksey et al., Antimicrob. Agents Chemother. 44: 1296 [2000], Griffin and Smith, Trends Biotechnol., 18: 77 [2000], Griffin and Smith, Analytical Chemistry. 72: 3298 [2000], Hessner et al., Clin. Chem. 46: 1051 [2000], Ledford et al. J. Molec. Diagnostics 2: 97 [2000], Lyamichev et al., Biochemistry 39: 9523 [2000], Mein et al., Genome Res., 10: 330 [2000], Neri et al., Advances in Nucleic Acid and Protein Analysis 3826: 117 [2000], Fors et al., Pharmacogenomics 1: 219 [2000], Griffin et al., Proc. Natl. Acad. Sci. USA 96: 6301 [1999], Kwiatkowski et al., Mol. Diagn. 4: 353 [1999], and Ryan. Mol. Diagn. 4: 135 [1999], Ma et al., J. Biol. Chem., 275: 24693 [2000], Reynaldo et al., J. Mol. Biol., 297: 511 [2000], and Kaiser et al. , J. Biol. Chem., 274: 21387 [1999]; and WO 97/27214, WO 98/42873, and WO 98/50403). The present invention when used together is illustrated. Invader assays, when used in conjunction with the systems and methods of the present invention, provide simple use and sensitivity levels for use in detection panels, ASRs and clinical diagnostics. Those skilled in the art will recognize that the specific and general features of the present exemplary embodiments are generally applicable to other detection assays.

A. Invader Assay The Invader Assay provides a means for cleaving a nucleic acid cleavage structure to construct a nucleic acid cleavage structure that depends on the presence of the target nucleic acid and to release a specific cleavage product (Figure 1). reference). For example, 5 ′ nuclease activity is used to cleave target-dependent cleavage structures, and the resulting cleavage product indicates the presence of a particular target nucleic acid sequence in the sample. When both nucleic acid or oligonucleotide strands are hybridized to a target nucleic acid strand to form overlapping invasive cleavage structures, as described below, invasive cleavage can occur. Through interaction of the cleaving agent (eg, 5 ′ nuclease) with the upstream oligonucleotide, the downstream oligonucleotide can be cleaved at an internal site in such a way that the cleaving agent produces a unique fragment.

  Invader assays do not require the use of temperature cycling (i.e. due to intermittent denaturation of the target nucleic acid strand) or nucleic acid synthesis (i.e. due to substitution based on polymerization of the nucleic acid strand of the target or probe), A detection assay is provided that is reused or recycled during multiple iterations of hybridization with an oligonucleotide probe and cleavage of the probe. Under conditions in which the probe is continuously displaced on the target strand (through probe-probe displacement, or through equilibrium between probe / target binding and dissociation, or through combinations involving these mechanisms (Reynaldo et al., J. Mol. Biol. 97: 511-520 [2000])), when a cleavage reaction is performed, multiple probes can hybridize to the same target, allowing multiple cleavages and generation of multiple cleavage products become.

  As with other assays, invader assays can also use degenerate oligonucleotides (eg, degenerate invaders and probe oligonucleotides). For example, standard invader oligonucleotides and probes can be randomly varied at another position so that a set of degenerate invader and / or probe oligonucleotides is created. Degenerate sets of invader and probe oligonucleotides are particularly useful for use in connection with target sequences that tend to be heavily mutated (eg, the HIV-1 pol gene). Using a degenerate set of such invader and probe oligonucleotides allows the target sequence to be detected at a particular position to be detected, even if its surrounding sequence no longer represents a wild-type or expected sequence. Admit existence.

  Invader assay technology can be used to quantify mRNA (eg, without target amplification). Low variability (3% to 10% coefficient of variation) provides accurate quantification of less than 2-fold change in mRNA levels. A dual detection format based on FRET allows simultaneous quantification of expression from two genes in the same sample. One of these genes can be an invariant housekeeping gene used as an internal standard. Normalizing the signal from the gene of interest with an internal standard gives accurate results and eliminates the need for duplicate samples. A simple and rapid method for preparing cell lysate samples can be used with mRNA invader assays. The combined features of the dual detection method and easy sample preparation make this assay readily adaptable for use in high throughput applications.

  In certain embodiments, invader assays (and other detection assays such as the TAQMAN method) use E-TAG labels (eg, as part of an invader oligonucleotide, probe oligonucleotide, or FRET oligonucleotide). E-TAG labeling is particularly useful in multiplex analysis. E-TAG labeling does not require surface immobilization of the affinity agent. US Pat. Nos. 5,858,188; 5,883,211; 5,935,401; 6,007,690; 6,043,036; 6,054,034; 6,056,860; 6,074,827; 6,093,296; 6,103,199; 6,103,537; 6,176,962; and 6,284,113, all of which are incorporated herein by reference.

II. Multiplex PCR Primer Design Invader assays can be used to detect single nucleotide polymorphisms (SNPs) using as little as 10 ng to 100 ng of genomic DNA without the need to preamplify the target. However, for potential for whole genome association studies involving over 80,000 invader assays and hundreds of thousands of SNPs deployed, the amount of sample DNA is a limiting factor for mass analysis. Thanks to the sensitivity of the invader assay on human genomic DNA (hgDNA) without target amplification, the multiplex PCR method coupled with the invader assay is a large amount of amplification (10 ⁹ to 10 ¹² ) for normal gel detection methods. Only limited target amplification (10 ³ to 10 ⁴ ) is required compared to a typical multiplex PCR reaction that requires The low level of target amplification used for invader assay detection allows larger scale multiplexing by avoiding amplification inhibition that typically occurs as a result of target accumulation.

  In some embodiments, it may be desirable to detect related loci in a multiplex PCR reaction. In some such embodiments, the detection assay technology may not be able to adequately discriminate between closely related sequences, so similarity between loci will allow detection assay analysis of that sequence. Can interfere or complicate. The present invention provides a method for overcoming such problems by producing a unique target sequence using nucleic acid amplification techniques (e.g., PCR), the unique target sequence, rather than its original sample. (Eg genomic DNA) will be tested by the detection assay. This method is compatible with multiplexing in terms of ensuring that the target sequence to be amplified meets several criteria: 1) includes the polymorphism in which the target sequence is analyzed 2) the target sequence exhibits a unique target sequence (ie it is the only sequence in the reaction mixture detected by a detection assay designed to target that target sequence); and 3 ) The target sequence does not contain other polymorphisms detected by any detection assay present in the multiplex reaction. For example, in some embodiments, the software performs a BLAST alignment of the target sequence used in the SNP assay to find similar sequences in the genome that can generate cross-reactive signals. The design of PCR primers in the software program should prevent amplification of any similar locus except the locus containing the SNP. To avoid pre-amplification of sequences except for that particular SNP sequence, the software performs a BLAST alignment of the sequences amplified with a pair of primers against all other detection assay sequences contained in the pool. If there is cross-reactivity or possible cross-reactivity, the set of primers is redesigned or the co-amplified sequences are included in different pools.

  The same type of design analysis can be used for detection assays that are directed to the detection of haplotypes. For example, the primers are made to amplify a set of target sequences each containing only the polymorphism that is detected.

  In some embodiments, multiplex detection assays are provided in multiple arrays. For example, in some embodiments, the first array comprises an assay configured for direct detection from genomic DNA, and the second array is derived from genomic DNA prior to target sequence detection assay analysis. Includes assays configured for pre-amplification of target sequences.

In some preferred embodiments, only limited pre-amplification of the target sequence is performed prior to detection by the detection assay. For example, in some embodiments, a 10 ⁵ fold to 10 ⁶ fold increase or less in target copy number is obtained prior to detection. This is in contrast to typical PCR reactions in which 10 ¹⁰ fold to 10 ¹² fold or more amplification is utilized in the detection reaction. In certain embodiments, using only 10 ng of genomic DNA (eg, less than 0.1 ng of human genomic DNA per SNP), the methods and systems of the present invention allow 100 genotypes from a single PCR amplification .

  In some embodiments, kits are provided for preamplification and detection of target sequences. In some embodiments, the kit includes an amplification primer. For multiplex reactions, amplification primers can be provided in a single container. Amplification primers can also be packaged with a detection assay component. In some embodiments, the amplification primer and detection assay component (eg, invader assay component) are provided in a single container (eg, in one of the wells of a multi-well plate). In some embodiments, the reaction component is provided in a dry form in the reaction chamber. In some such embodiments, the kit is configured to allow the reaction to occur simply by adding a solution containing genomic DNA to the reaction chamber.

  The present invention provides methods and selection criteria that allow primer sets for multiplex PCR to be generated (eg, can be linked to a detection assay such as an invader assay). In some embodiments, the software application of the present invention automates multiplex PCR primer selection, thus allowing for highly multiplexed PCR with primers designed thereby. Using the Invader Medically Associated Panel (MAP) as a corresponding platform for SNP detection, the PCR method as shown in Example 2 (below), the method, software, and selection determination of the present invention The criteria allowed for accurate genotyping of 94 (˜93%) out of 101 possible amplicons from a single PCR reaction. The initial PCR reaction used only 10 ng of hgDNA as template, corresponding to less than 150 pg hgDNA per invader assay.

Multiple primer design systems can be used to design PCR primer sets useful for specific types of assays, such as invader assays. FIG. 2 shows primer pairs (forward primer and reverse) for 101 primer sets from sequences available for analysis in the Invader Medically Associated Panel using one of the software application embodiments of the present invention. One example of the production of both primers) is illustrated. FIG. 2A shows a single entry sample input file (eg, showing target sequence information for a single target sequence containing SNPs processed by the methods and software of the invention). The target sequence information in FIG. 2 includes a sequence having an SNP number indicated by a third wave technologies SNP number, a short identifier, and square brackets. FIG. 2B shows a sample output file of the same entry (eg, showing the target sequence after being processed by the system and method and software of the present invention). The output information includes the footprint region sequence (uppercase adjacent SNP site, indicating the region where the invader assay probe hybridizes to this target sequence to detect SNP in the target sequence), forward primer sequence and reverse primer Contains sequences (bold) and their corresponding T _m .

  In some embodiments, the selection of primers to create a primer set capable of multiplex PCR is performed in an automated format (eg, by a software application). Automated primer selection for multiplex PCR can be achieved using a software program designed as shown by the flowchart of FIG.

  Multiplex PCR generally requires extensive optimization to avoid the amplification of biased amplification of the selected amplicon and the mimic products that result from the construction of primer dimers. To avoid these problems, the present invention provides methods and software that provide selection criteria for generating primer sets that are configured for subsequent use in multiplex PCR and detection assays (e.g., invader detection assays). Provide an application.

  In some embodiments, the methods and software applications of the present invention begin with a user-defined sequence and corresponding SNP position. In certain embodiments, the method and / or software application determines, for each sequence, the footprint area within the target sequence (minimum amplicon required for invader detection) (shown in capital letters in FIG. 2B). . The footprint region is in addition to the region to which the assay probe hybridizes, and any user-defined additional bases extending out therefor (e.g., 5 Additional base). Next, primers are designed out of the footprint region and evaluated against a number of criteria, including the possibility for primer-dimer configuration with previously designed primers in their current multiplexing set. (See bold primer in FIG. 2A and selection step in FIG. 4). As shown in FIG. 4, this step can be continued through multiple interactions of the same set of sequences until primers for all sequences in the current multiplexed set can be designed.

In some embodiments, once a primer set is designed for multiplex PCR, this set can be used as shown in the basic workflow scheme shown in FIG. For example, multiplex PCR can be performed under standard conditions using only 10 ng of hgDNA as a template. After 10 minutes at 95 ° C., Taq (2.5 units) can be added to the 50 μl reaction and PCR can be performed for 50 cycles. PCR reactions can be diluted and loaded directly onto invader MAP plates (3 μl / well) (see FIG. 3). An additional 3 μl of 15 mM MgCl ₂ may be added to each reaction on the Invader MAP plate and covered with 6 μl of mineral oil. The entire plate can then be heated to 95 ° C. for 5 minutes and incubated at 63 ° C. for 40 minutes. The FAM and RED fluorescence can then be measured at the “Fold Over Zero” (FOZ) value calculated for the Cytofluor 4000 fluorescence plate reader and each amplicon. The results obtained from each SNP are color coded in the table as `` pass '' (green), `` mis-call '' (pink), or `` no-call '' (white). (See PCR primer design example 2 below).

  In some embodiments, the PCR reaction is from about 1 to about 10 reactions. In some embodiments, the PCR reaction is about 10 to about 50 reactions. In further embodiments, the number of PCR reactions is from about 50 to about 100 times. In additional embodiments, the number of PCR reactions is greater than 100.

  The present invention also provides a method for optimizing multiplex PCR reactions (eg, when a primer set is made, the concentration of each primer or primer pair can be optimized). For example, once a primer set has been made and used in multiplex PCR at equimolar concentrations, the primers can be evaluated separately in order to determine the optimal primer concentration so that the multiplex primer set works better.

  Multiplex PCR reactions are becoming recognized in the scientific, research, clinical and biotechnology industries as a means of obtaining potentially time-efficient and less expensive nucleic acid information compared to standard single PCR reactions. Instead of performing only a single amplification reaction per reaction vessel (eg, a tube or a well of a multi-well plate), multiple amplification reactions are performed in a single reaction vessel.

  Cost per target is theoretically reduced by eliminating expert time in assay setup and data analysis, and substantial reagent savings (especially enzyme costs). Another advantage of the multiplex method is that a much smaller target sample is required. In genome-wide association studies involving hundreds of thousands of single nucleotide polymorphisms (SNPs), the amount of target or test sample is limited for large-scale analysis, for example, one sample constant to obtain 100 results The concept of performing a single reaction of using 100 sample aliquots to obtain the same data set versus using aliquots is an attractive choice.

  In order to design primers for successful multiplex PCR reactions, attention must be paid to the issue of aberrant interactions between primers. Even if it is only a few bases long, the primer dimer configuration may prevent both primers from hybridizing correctly to the target sequence. Furthermore, if the dimer is constructed at or near the 3 'end of the primer, no amplification occurs or very low levels of amplification occur because the 3' end is required for a priming event Will. Clearly, the more primers used per multiplex reaction, the more aberrant primer interactions can occur. This rescue method, system and application prevent primer dimers in a large number of primer sets and create highly multiplexed sets suitable for PCR.

  When designing primer pairs for a large number of sites (e.g., 100 sites in a multiplex PCR reaction), the order in which the primer pairs are designed affects the total number of compatible primer pairs for a reaction. sell. For example, if a first set of primers is accidentally designed for a first target region that is an A / T rich region, these primers will be A / T rich. If the selected second target region is also accidentally an A / T-rich target region, the primers designed for these two sets will have an aberrant interaction such as primer dimer Because of this, there is a much higher chance of nonconformity. However, if the selected second target region is not A / T rich, it is much more likely that a primer set that does not attempt to interact with the first A / T rich set can be designed. For any given set of input target sequences, the present invention randomizes the order in which primer sets are designed (see FIG. 4). Still further, in some embodiments, the present invention sets a set of input target sequences to a number of different random numbers so as to maximize the number of compatible primer sets for any given multiplex reaction. Redesign to order (see Figure 4). In certain embodiments, primers are designed such that GC-rich and AT-rich regions are avoided.

  The present invention minimizes 3 ′ interactions while maximizing the number of compatible primer pairs for a given set of reaction targets in a multiplex design (e.g., the possibility of primer dimer configuration). Provide a criterion for primer design (avoid primer 3 'complementarity to reduce). For primers described as 5'-N [x] -N [x-1] -....- N [4] -N [3] -N [2] -N [1] -3 ' , N [1] is A or C (in alternative embodiments, N [1] is G or T). The N [2] -N [1] of each designed forward and reverse primer should not be complementary to N [2] -N [1] of any other oligonucleotide. In certain embodiments, N [3] -N [2] -N [1] should not be complementary to N [3] -N [2] -N [1] of any other oligonucleotide . In a preferred embodiment, if a given N [1] does not meet these criteria, the next base in the 5 ′ direction for the forward primer or the next base in the 3 ′ direction for the reverse primer is N [1 It can be evaluated as a site. With target randomization until all criteria are met for all or most of the target sequences (e.g., 95% of target sequences can have primer pairs made as primer sets that meet these criteria) This step is repeated.

Another problem to be overcome in multiplex primer design is the balance between the actually required nucleotide sequence, sequence length, and oligonucleotide melting temperature (T _m ) constraints. Importantly, the primers of one multiplex primer set in one reaction will function under the same reaction conditions of buffer, salt and temperature, therefore they are GC in the region of interest. Regardless of abundance or AT abundance, they should have substantially similar T _m . The present invention allows the minimum requirements in T _m and the maximum in T _m and minimum and maximum length requirements are met primer design. For example, in the formula 5'-N [x] -N [x-1] -....- N [4] -N [3] -N [2] -N [1] -3 'for each primer , X is selected such that the primer has a predetermined melting temperature (eg, include a base in the primer until the primer has a calculated melting temperature of about 50 degrees Celsius). In certain embodiments, each primer in the set has the same melting temperature.

  PCR reaction products are often used as target material for hybridization-type detection assays or other nucleic acid detection means such as invader reaction assays. The position of the primer arrangement to allow the secondary reaction to take place should be taken into account, and again, the atypical interaction between the amplification primer and the secondary reaction oligonucleotide is not accurate results and data Should be kept to a minimum. Selection criteria can be used such that primers designed as a multiplex primer set do not react (eg, do not hybridize or elicit a reaction) with the oligonucleotide components of the detection assay. For example, specific homology criteria are used to prevent primers from reacting with FRET oligonucleotides in a double invader assay. In particular, each primer in the set is 5'-N [x] -N [x-1] -....- N [4] -N [3] -N [2] -N [1] -3 ' If defined, then N [4] -N [3] -N [2] -N [1] -3 'is selected to have less than 90% homology with FRET or invader oligonucleotides. The In other embodiments, N [4] -N [3] -N [2] -N [1] -3 'is selected for each primer such that it has less than 80% homology with FRET or invader oligonucleotides . In certain embodiments, N [4] -N [3] -N [2] -N [1] -3 'is selected for each primer such that there is less than 70% homology with FRET or invader oligonucleotides .

  While using the criteria of the present invention to develop a primer set, some primer pairs may not meet all of the established criteria (these may be rejected as errors). For example, in a set of 100 targets, 30 are designed and meet all the listed criteria, but set 31 fails. In the method of the present invention, set 31 can be flagged as failed, and the method can continue to the end of the list of 100 targets, again flagging the set that does not meet the criteria (Figure 4). reference). If all 100 targets have the opportunity for primer design, the method notes the number of unsuccessful sets, realigns the 100 targets in a new random order, and repeats the design phase (see FIG. 4). After performing a configurable number of times, the set with the most successful primer pair (the minimum number of unsuccessful sets) is selected for the multiplex PCR reaction (see FIG. 4).

  FIG. 4 shows a flowchart for the basic flow of a particular embodiment of the method and software application of the present invention. In a preferred embodiment, the steps detailed in FIG. 4 are incorporated into a software application for ease of use (although the method can also be performed manually, eg, using FIG. 4, as a guide). sell).

  Target sequences and / or primer pairs are input to the system shown in FIG. The first set of squares is the other sequence to the pool of primer sets (eg PDPass, previously processed and without constituting primer dimers or having reactivity to FRET sequences) , Sequences that have been shown to work together), and dimer test entries (e.g., pairs that the user wants to use but has not yet been tested for primer dimer or FRET reactivity) (Or primers) and shows how the target sequence is added to the list of sequences with the determined footprint (see “B” in FIG. 4). In other words, the first set of squares that lead progressively to "end of input" sorts the array so that they can later be processed correctly.

  Beginning with “A” in FIG. 4, the primer pool is basically cleared or “emptied” to initiate a new run. The target sequence is then sent to “B” for processing, and the dimer test pair is sent to “C” for processing. The target sequence is sent to “B” where a user or software application determines the footprint region for that target sequence (eg, where an assay probe to detect mutations (eg, SNPs) in that target sequence Hybridize). This region is generally shown in capital letters in the figure, as in FIG. 2B. It is important to design this region (which may be further expanded by allowing the user to add additional bases beyond the hybridization region) so that the designed primer completely contains this region. is there. (To create any type of probe using the program of any type of system that is interested in the user designing the probe and thus determining the footprint region on the target sequence. In FIG. 4, the software application Invader Creator (INVADER CREATOR) is used to design invader oligonucleotides and downstream probes that will hybridize to the target region. Thus, the footprint region of the core is then limited by the location of these two assay probes on the target.

  The system then starts at the 5 ′ end of the footprint and moves 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 first starting point to limit the sequence of the forward primer (ie, it serves as the first N [1] site). From this first N [1] site, the sequence of the primer as a 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 base selected as N [1] and then adds the next 11 bases found in the target sequence. This 12mer primer is then tested for melting temperature (e.g., using an invader creator) and the sequence is specified by the user at a melting temperature (e.g., about 50 degrees Celsius and no more than 55 degrees Celsius). Additional bases are added from the target sequence until For example, the system uses the formula 5'-N [x] -N [x-1] -....- N [4] -N [3] -N [2] -N [1] -3 ' X is 12 at first. The system then adjusts x to a larger number (eg, a longer sequence) until a preset melting temperature is found.

  The next square in FIG. 4 is used to determine whether previously designed primers cause primer dimer and / or FRET reactivity (eg, the remaining sequences are already in the pool). The criteria used for this determination are described above. If the primer passes this step, the forward primer is added to the primer pool. However, if the forward primer lacks this criterion, the starting point (N [1]) is 1 nucleotide in the 5 ′ direction (or the next A or C, or the next, as shown in FIG. 4. To G or T). The system first checks to see if moving beyond leaves enough room on its target sequence to succeed in creating a primer. If yes, the system loops back and checks this new primer for melting temperature. However, if the sequence cannot be designed, then the target sequence is flagged as an error (eg, indicating that no forward primer can be made for this target).

  This same step is then repeated to design the reverse primer, as shown in FIG. If the reverse primer is successfully created, the pair or primer is put into the primer pool and the system returns to `` B '' (if there are more target sequences to process), or to test the dimer test pair Go to “C”.

  Initiating “C” in FIG. 4 shows how the primer pair entered as a primer (Dimer Test) is processed by the system. If there is no dimer test pair, the system proceeds to “D” as shown in FIG. However, if there are dimer test pairs, these are tested for primer dimer and / or FRET reactivity as described above. If dimer test pairs lack these criteria, they are flagged as errors. If the dimer test pairs pass the criteria, they are added to the primer set pool, and then the system returns to “C” if there are more dimer test pairs to be evaluated, or the dimer to be evaluated. If there are no more test pairs, go to “D”.

  Starting from “D” in FIG. 4, the pool of primers generated is evaluated. The first step in this section is to examine the number of errors (failures) caused by this particular randomized run of sequences. If there are no errors, this set is the best set that can be output to the user. If there are more than zero errors, the system compares this run with any other previous run to see which run produced the least error. If the current run has fewer errors, it is designated as the current best set. At this point, the system may return to “A” and start another run for another randomized set of the same sequence, or to the preset maximum number of runs (eg, 5 runs) It may be reached in a run (for example, this was the fifth run and the maximum number of runs was set as 5). If the maximum number is reached, the best set is output as the best set. This best primer set can then be used to make a set of material oligonucleotides such that a multiplex PCR reaction can be performed.

  Another problem to be overcome for the multiplex PCR reaction is the resulting unequal amplicon concentration in a standard multiplex reaction. Different loci targeted for amplification each behave differently in the amplification reaction and can result in very different concentrations of each different amplicon product. The present invention adjusts the primer concentration relative to the initial detection assay reading (e.g., the invader assay reading) and then substantially equals the different amplicons at a balanced primer concentration. Methods, systems, software applications, computer systems, and computer data storage media that can be used to approximate concentrations are provided. A generalized protocol for such multiple optimization is presented in FIG.

  Concentrations for various primer pairs can be determined based on experiments. In some embodiments, the first run is performed with equimolar concentrations of all primers. Thereafter, a time reading is performed. Based on the time reading, a relative amplification factor for each amplicon is determined. Subsequently, an estimate of what the primer concentration should be was obtained to obtain a closer signal within the same time point based on a unified correction equation. These detection assays are possible in different sized arrays (384 well plates).

  It has been recognized that combining the present invention with detection assays and arrays of detection assays provides substantial processing efficiency. A balanced mixture of primers or primer pairs made using the present invention can be used to perform a single point reading, requiring the average user to be highly sensitive and highly specific across an array of different targets. High efficiency can be obtained in performing the test.

  Optimized primer pair concentrations in a single reaction vessel allow users to amplify multiple or multiple amplification targets in a single reaction vessel and in a single step . The one-stage product is then used, for example, to successfully obtain test result data for hundreds of assays. For example, each well on a 384 well plate can perform a different detection assay thereon. The results of a one-step multiplex PCR reaction amplify 384 different targets of genomic DNA and provide 384 test results for each plate. Even higher efficiencies can be obtained where each well is conducting multiple assays.

  The present invention therefore provides for the use of the concentration of each primer set in highly multiplexed PCR as a parameter to achieve unbiased amplification of each PCR product. Both PCRs include a primer annealing and primer extension step. Under standard PCR conditions, high primer concentrations on the order of 1 μM guarantee a fast reaction rate for primer annealing, while the optimal time for the primer extension step depends on the amount of amplification product and annealing. Can be considerably longer than the stage. By lowering the primer concentration, the primer annealing reaction rate can become the rate-limiting step, and the PCR amplification factor is strongly dependent on the primer concentration, the primer binding reaction rate constant, and the annealing time.

K_aはプライマーアニーリングの結合反応速度定数である。アニーリングはプライマー融解より低い温度で起こり、かつ逆反応を無視することができることを仮定している。 The binding of primer P to target T can be described by the following model.

K _a is the coupling reaction rate constant of primer annealing. It is assumed that annealing occurs at a temperature lower than primer melting and that the reverse reaction can be ignored.

[PT]はプライマーと結合した標的分子の濃度であり、T₀は最初の標的濃度、cは最初のプライマー濃度、およびtはプライマーアニーリング時間である。プライマーと結合した各標的分子は完全なサイズのPCR産物を産生するために複製され、単一PCRサイクルにおける標的増幅因子は以下のとおりである。

The solution for this reaction rate under primer-excess conditions is well known.

[PT] is the concentration of target molecule bound to the primer, T ₀ is the initial target concentration, c is the initial primer concentration, and t is the primer annealing time. Each target molecule bound to the primer is replicated to produce a full size PCR product, and the target amplification factors in a single PCR cycle are:

方程式4から導かれるように、プライマーアニーリング反応速度がPCRの律速段階である条件下において、増幅因子は、プライマー濃度に強く依存することになる。このように、偏りのある遺伝子座の増幅は、それが個々の結合反応速度定数、プライマー伸長段階、またはいくつかの他の因子により引き起こされているにせよ、多重PCRにおける各プライマーセットについてのプライマー濃度を調整することにより補正されうる。調整されたプライマー濃度はまた、PCR前増幅された遺伝子座の分析に用いられるインベーダーアッセイ法の偏りのある実行を補正するためにも使用されうる。この基本的原理を用いて、本発明は、増幅効率とプライマー濃度との間の直線的関係を実証し、この方程式を異なる単位複製配列のプライマー濃度のバランスをとるために用いて、その結果として、PCRプライマー設計実施例1において10個の異なる単位複製配列が均等に増幅された。この技術は、複数のプライマー対の任意の大きさのセットに用いられうる。いくつかの態様において、PCRプライマーは最適化されず、かつインベーダーアッセイ法がその増幅産物を検出するために使用される(参照として本明細書に組み入れられている、Ohnishiら、J. Hum. Genet. 46: 471〜477、2001を参照)。 The total PCR amplification factor after n cycles is given by:

As can be derived from Equation 4, under conditions where the primer annealing reaction rate is the rate limiting step of PCR, the amplification factor will depend strongly on the primer concentration. Thus, amplification of a biased locus is the primer for each primer set in multiplex PCR, whether it is caused by individual binding kinetic constants, primer extension steps, or some other factor. It can be corrected by adjusting the density. Adjusted primer concentrations can also be used to correct the biased performance of invader assays used to analyze pre-PCR amplified loci. Using this basic principle, the present invention demonstrates a linear relationship between amplification efficiency and primer concentration, and uses this equation to balance the primer concentration of different amplicons, and consequently In PCR primer design Example 1, 10 different amplicons were equally amplified. This technique can be used for any size set of primer pairs. In some embodiments, PCR primers are not optimized and invader assays are used to detect the amplification products (Ohnishi et al., J. Hum. Genet, incorporated herein by reference). 46: 471-477, 2001).

i. PCR primer design Example 1
The following experimental example describes the manual design of amplification primers for the detection of amplicons by multiplex amplification reactions and subsequent invader assays.

  Ten target sequences were selected from a previously validated set of SNP-containing sequences available in the TWT in-house oligonucleotide order entry database (see FIG. 4). Each target contains a single nucleotide polymorphism (SNP) for which an invader assay has previously been designed. Invader assay oligonucleotides are designed by Invader Creator software (Third Wave Technologies, Madison, WI), and thus the footprint area in this example is covered by the invader “footprint” or invader and probe oligonucleotides. , Defined as the base of interest, in this case the base optimally located for the detection of single nucleotide polymorphisms (see FIG. 5). Approximately 200 nucleotides of each of the 10 target sequences are analyzed for amplification primer design analysis so that SNP bases are present around the center of the sequence. The sequence is shown in FIG.

The length of the maximum and minimum probe (respectively, default 30 nucleotides and 12 nucleotides) criteria is defined to be the range for the probe melting temperature T _m of a 50 ° C. to 60 ° C.. In this example, the oligonucleotide melting temperature (T _m ) is published for the flanking model and DNA duplex configuration to select the probe sequence that will perform optimally at the preselected reaction temperature. (Allawi and SantaLucia, Biochemistry, 36: 10581 [1997], which is incorporated herein by reference). Determine the optimal temperature at which the reaction will occur because the salt concentration of the assay is different from the solution conditions (1 M NaCl and no divalent metal) from which adjacent parameters are obtained and because the presence and concentration of the enzyme will affect the optimal reaction temperature In order to do so, adjustments should often be made to the calculated T _m . One way to correct for these factors is to change the value given for the salt concentration within the melting temperature calculation. This adjustment is called “salt correction”. The term `` salt correction '' is applied to the value given for the salt concentration for the purpose of reflecting the effect on the _Tm calculation for nucleic acid duplexes of non-salt parameters or conditions that affect the duplex. Refers to fluctuations. Variations in the value given for the chain concentration will also affect the output of these calculations. By using a value of 280 nM NaCl (SantaLucia, Proc Natl Acad Sci USA, 95: 1460 [1998], which is incorporated herein by reference) and a chain concentration of about 10 pM of probe and 1 fM of target The algorithm used to calculate the probe-target melting temperature was adapted to be used to predict the optimal primer design sequence.

  Next, both upstream and downstream sequences adjacent to the footprint region are scanned, and for the primer, N [1] is A or C 5'-N [x] -N [x-1 ] -....- N [4] -N [3] -N [2] -N [1] -3 'was selected as the starting design, as described in the first A or C. N [2] -N [1] of a given oligonucleotide should not be complementary to N [2] -N [1] of any other oligonucleotide and N [3] -N [2 Primer complementarity was avoided by using the rule that] -N [1] should not be complementary to N [3] -N [2] -N [1] of any other oligonucleotide . If these criteria are not met for a given N [1], then the next base in the 5 'direction for the forward primer or the next base in the 3' direction for the reverse primer is N [1 It is evaluated as a part. In the case of manual analysis, the A / C rich region was targeted to minimize 3 'end complementarity.

  In this example, the invader assay was performed after the multiplex amplification reaction. Therefore, the site of the secondary invader reaction oligonucleotide (FRET oligonucleotide sequence) was also incorporated as a criterion for primer design, but the amplification primer sequence was less than 80% relative to the specified region of the FRET oligonucleotide. Should be homologous.

The output primers for the 10-fold multiplex design are shown in FIG. All primers were synthesized according to standard oligonucleotide chemistry, desalted (by standard methods), quantified by absorbance at A260, and diluted to a 50 μM concentrated stock solution. Subsequently, multiplex PCR was performed using 10-fold PCR using equimolar amounts of primers (0.01 uM / primer) under the following conditions: 100 mM KCl, 3 mM MgCl in a 50 μl reaction. ₂ , 10 mM Tris pH 8.0, 200 uM dNTP, 2.5 U taq, and 10 ng of human genomic DNA (hgDNA) template. The reaction was incubated for 30 cycles (94 C / 30 seconds, 50 C / 44 seconds). After incubation, 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, 100 ng CLEAVASE VIII. The invader assay was constructed as a 15 ul reaction, as follows: 1 ul of a 1:10 dilution of PCR reaction, 3 ul of PPI mixture, 22.5 mM MgCl ₂ 5 ul, dH ₂ O 6 ul, Chillout 15 Cover with ul. Samples were denatured in Invader Duplex by incubation at 95 C for 5 minutes, followed by incubation at 63 C, and fluorescence measured at various time points with Cytofluor 4000.

  Using the following criteria to ensure that a genotyping call is made accurately (FOZ_FAM + FOZ_RED-2> 0.6), only 2 of the 10 invader assay calls are incubated at 63C for 10 minutes It was possible to make later, and only 5 out of 10 calls could be made after an additional 50 minute incubation (60 minutes) at 63 C (see FIG. 6A). At 60 minutes, the variation between detectable FOZ values is the strongest signal (Figure 6A, 41646, FAM_FOZ + RED_FOZ-2 = 54.2, which is also far outside the dynamic range of the reader) and the weakest signal (Fig. 6A, 67356, FAM_FOZ + RED_FOZ-2 = 0.2). Using the same invader assay directly on 100 ng of human genomic DNA (equimolar amounts of each target will be available), all readings can be made within the dynamic range of the reader, The variation in FOZ values was approximately 7 times between the strongest (Figure 6, 53530, FAM_FOZ + RED_FOZ-2 = 3.1) and the weakest (Figure 6, 53530, FAM_FOZ + RED_FOZ-2 = 0.43). This suggests that the dramatic discrepancy in FOZ values seen between different amplicons in the same multiplex PCR reaction is a biased effect of amplification and not the variability attributed to invader assays. Under these conditions, FOZ values generated by different invader assays can be directly compared to each other and can be used reliably as an indicator of amplicon efficiency.

について用いられる、経験的に決定されたRED_FOZ値(インベーダーアッセイ41646を用いる)を表す。

Estimating the amplification factor for a given amplicon using the FOZ value To estimate the amplification factor (F) for a given amplicon, the FOZ value for the invader assay is used to estimate the amplicon abundance Can be used. The FOZ of a given amplicon with an unknown concentration at a given time (FOZm) is calculated from the known amount of target at a given point in time (FOZ ₂₄₀ , 240 min) (e.g., 100 ng genomic DNA = It can be directly compared to the FOZ of one gene (30,000 copies) and used to calculate the copy number of an unknown amplicon. In Equation 1, FOZm represents the sum of RED_FOZ and FAM_FOZ at unknown concentrations of the target incubated in the invader assay for a given amount of time (m). FOZ ₂₄₀ is the known number of copies of the target in 240 minutes

Represents the empirically determined RED_FOZ value (using invader assay 41646) used for.

Equation 1a is used to determine the linear relationship between primer concentration and amplification factor F, while equation 1a 'is used in the calculation of amplification factor F for 10-fold PCR (equimolar amount of primer). D values represent the dilution factor of the PCR reaction. For a 1: 3 dilution of a 50 ul multiplex PCR reaction. D = 0.3333.

Equations 1a and 1a ′ would be used to describe a 10-fold multiplex PCR, but a further corrected adaptation of this equation was used to optimize primer concentrations in 107-fold PCR. In this case, FOZ ₂₄₀ = average of FAM_FOZ ₂₄₀ + RED_FOZ ₂₄₀ (FOZ ₂₄₀ = 3.42) and dilution factor D are set to 0.125 across all invader MAP plates using hgDNA as a target.

  It should be noted that for a more accurate estimation of the amplification factor F, the FOZ value should be within the dynamic range of the device from which the reading is obtained. For the Cytofluor 4000 used in this study, the dynamic range was between about 1.5 FOZ and about 12 FOZ.

Item 3.Linear relationship between amplification factor and primer concentration.To determine the relationship between primer concentration and amplification factor (F), four separate single PCR reactions were performed using primers 1117-70-17 and 1117-70-18 was used at concentrations of 0.01 uM, 0.012 uM, 0.014 uM and 0.020 uM, respectively. The four independent PCR reactions were performed under the following conditions: 100 mM KCl, 3 mM MgCl, 10 mM Tris pH 8.0, 200 uM dNTP, and 10 ng hgDNA as template. Incubation was performed for 30 cycles (94 C / 30 seconds, 50 C / 20 seconds). After PCR, the reaction was diluted 1:10 with water and run under standard conditions using an invader assay FRET frets detection plate, 96 well genomic duplex, 100 ng CLEAVASE VIII enzyme. Each 15 ul reaction was set up as follows: 1 ul diluted PCR reaction 1 ul, 3 ul PPI mix SNP # 47932, 5 ul 22.5 mM MgCl ₂ , water 6 ul, Chillout 15 ul. The entire plate was incubated at 95 C for 5 minutes and then at 63 C for 60 minutes, at which point a single reading was obtained on a Cytofluor 4000 fluorescent plate reader. Amplification factor using equation 1a with FOZm = FOZ_FAM plus FOZ_RED in 60 minutes, m = 60, and FOZ ₂₄₀ = 1.7 for each of 4 different primer concentrations (0.01 uM, 0.012 uM, 0.014 uM, 0.020 uM) F was calculated. When the primer concentration of each reaction was plotted against the logarithm log (F) of the amplification factor, a remarkable linear relationship was shown (FIG. 7). Using the data points in FIG. 7, the equation representing the linear relationship between amplification factor and primer concentration is described in Equation 2.
Y '= 1.684X + 2.6837 (Equation 2a)

Using Equation 2, the amplification factor Log (F) = Y ′ for a given amplicon could be manipulated in a predictable manner using a known concentration (X) of primer. In the reverse method, the amplification bias observed under equimolar primer concentration conditions in multiplex PCR could be measured as the “apparent” primer concentration (X) based on its amplification factor F. In multiplex PCR, the value of “apparent” primer concentration between different amplicons is used to estimate the amount of primer for each amplicon required to equalize amplification of different loci. sell.
X = (Y'-2.6837) /1.68 (Equation 2b)

Section 4. Calculation of Apparent Primer Concentration from Balanced Multiple Mixtures As described in the previous section, primer concentration can directly affect the amplification factor for a given amplicon. FOZm readings under equimolar primer conditions can be used to calculate the “apparent” primer concentration for each amplicon using Equation 2. Replacing Y 'in Equation 2 with the log (F) for a given amplification factor and solving for X yields an "apparent" primer concentration based on the relative abundance of a given amplicon in the multiplex reaction. It is done. Using Equation 2 to calculate the “apparent” primer concentration of all primers (supplied as equimolar concentrations) in a multiplex reaction provides a means to normalize the primer set relative to each other. Divide the maximum apparent primer concentration (X _max ) by each of its “apparent” primer concentrations to derive the relative amount of each primer to be added to the “optimized” multiplex primer mixture R. Often, the strongest amplicon is set to a value of 1 and the remaining amplicons are set to a value equal to or greater than 1.
R [n] = Xmax / X [n] (Equation 3)

  Using the R [n] value as an arbitrary value for the relative primer concentration, multiplying the R [n] value by a constant primer concentration provides the working concentration for each primer in a given multiplex reaction. . In the example shown, the amplicon corresponding to SNP assay 41646 has an R [n] value equal to 1. Multiplying all R [n] values by 0.01 uM (first starting primer concentration in an equimolar multiplex PCR reaction) yields a minimum primer concentration of 41646 R [n] set to 1, ie 0.01 uM. is there. The remainder of the primer set also increased proportionally, as shown in FIG. The results of multiplex PCR with “optimized” primer mix are described below.

Section 5 Using optimized primer concentrations in multiplex PCR greatly reduces the variation in FOZ between 10 invader assays.
Multiplex PCR was performed using 10-fold PCR using various amounts of primers based on the volumes shown in FIG. 8 (X [max] is SNP41646, set to 1x = 0.01 uM / primer). Multiplex PCR was performed under the same conditions used with equimolar primer mixtures: 100 mM KCl, 3 mM MgCl, 10 mM Tris pH 8.0, 200 uM dNTP, 2.5 U in a 50 ul reaction. taq and hgDNA template 10 ng. The reaction was incubated for 30 cycles (94 C / 30 seconds, 50 C / 44 seconds). After incubation, the multiplex PCR reaction was diluted 1:10 with water and subjected to invader analysis. Using an invader assay FRET detection plate, (96 well genomic duplex, 100 ng CLEAVASE VIII enzyme), the reaction was constructed as a 15 ul reaction as follows: 1:10 dilution of PCR reaction 1 ul, 3 ul of the appropriate PPI mixture, 22.5 mM MgCl ₂ 5 ul, dH ₂ O 6 ul. An additional 15 ul of CHILL OUT was added to each well, followed by a 5 minute incubation at 95 ° C. Plates were incubated at 63 C and fluorescence was measured on Cytofluor 4000 at 10 minutes.

  Using the following criteria (FOZ_FAM + FOZ_RED-2> 0.6) to accurately generate genotyping calls, all 10 out of 10 (100%) invader calls were incubated at 63 C. Can be created after 10 minutes. In addition, the value of FAM + RED-2 (overall signal production indicator (see Equation 2), directly related to the amplification factor) is the lowest signal (Figure 9, 67325, FAM + RED-2 = 0.7 ) And the highest (Figure 9, 47892, FAM + RED-2 = 4.3) up to less than 7 times.

ii. PCR primer design Example 2
144 sequences less than 200 nucleotides long, using the TWT Oligo Order Entry Database, where SNPs are annotated using square brackets to indicate the SNP position for each sequence (For example, NNNNNNN [N _(wt) / N _(mt) ] NNNNNNNN). To extend the sequence data adjacent to the SNP of interest, the sequence was extended to a length of approximately 1 kB (500 nucleotides adjacent to each side of the SNP) using BLAST analysis. Of the 144 starting sequences, 16 could not be extended by BLAST, resulting in a final set of 128 sequences extended to a length of about 1 kB (see FIG. 10, 29043). And only 34573 are shown). These extended sequences were provided to the user in Excel format with the following information for each sequence: (1) TWT number, (2) short identifier, and (3) sequence (see Figure 10; Only SNP29043 and 34573 are shown). Excel file is converted to comma-separated format, Primer Designer INVADER CREATOR v1.3.3 software (This version of the program does not screen for FRET reactivity, and specifies maximum length of primer to user Used as an input file. INVADER CREATOR Primer Designer v1.3.3 was run using default conditions (eg, minimum primer size 12, maximum primer size 30) except T _mlow set to 60 C. The output file (see Figure 11, only SNP 29043 and 34573 are shown, the lower part of each sheet indicates the footprint area with upper case letters, and SNP with square brackets) is 128 sets of primers Includes set (256 primers, see Figure 12), 4 of which are discarded due to overly long primer sequences (SNP numbers 47854, 47889, 54874, 67396) and 124 primers valid for synthesis The set (248 primers) was left. The remaining primers were synthesized using standard methods on a 200 nmol scale and purified by desalting. After the synthesis failure, 107 primer sets were effective as a collection of equimolar 107-fold primer mixtures (214 primers, see FIG. 12). Of the 107 primer sets effective for amplification, only 101 were present on the Invader MAP plate for evaluating amplification factors.

Multiplex PCR was performed using 101-fold PCR using equimolar amounts (0.025 uM / primer) of primers under the following conditions: 100 mM KCl, 3 mM MgCl, 10 in a 50 ul reaction. mM Tris pH 8.0, 200 uM dNTP, and 10 ng of human genomic DNA (hgDNA) template. After denaturation at 95 C for 10 min, Taq 2.5 units were added and the reaction was incubated for 50 cycles at (94 C / 30 sec, 50 C / 44 sec). After incubation, the multiplex PCR reaction was diluted 1:24 with water and subjected to invader assay analysis using an invader MAP detection platform. Each invader MAP assay was performed as a 6 ul reaction as follows: 3 ul of a 1:24 dilution of PCR reaction (total dilution 1: 8 equal to D = 0.125), 15 mM MgCl ₂ Covered with 3 ul, CHILLOUT 6 ul. Samples were denatured in Invader MAP plates by incubation at 95 C for 5 minutes, followed by incubation at 63 C, and fluorescence measured at various time points during 160 minutes on Cytofluor 4000 (384 well reader) . Analysis of the FOZ values calculated at 10, 20, 40, 80, and 160 minutes shows that the correct call (compared to the genome call of the same DNA sample) is detected by the Invader MAP platform. This shows what was done for 94 of the amplicons (FIGS. 13 and 14). This provides evidence that Invader Creator Primer Designer software can create primer sets that function in highly multiplexed PCR.

In using the FOZ values obtained over 160 minutes, amplification factors F and R [n] were calculated for each of 101 amplicons (FIG. 15). R [nmax] was set to 1.6, but for amplicons that failed to give sufficient FOZm signal at 160 minutes, assign an arbitrary value of 12 to R [n] to correct for low-end correction. went. The high end correction for amplicons that read the FOZm value at 10 minutes was arbitrarily assigned an R [n] value of 1. The 101-fold optimized primer concentration was calculated using the 10-fold example and the basic principle outlined in Equation 1b, with a 0.025 uM primer corresponding to a R [n] of 1. . Multiplex PCR was under the following conditions: 100 mM KCl, 3 mM MgCl, 10 mM Tris pH 8.0, 200 uM dNTP, and 10 ng human genomic DNA (hgDNA) template in a 50 ul reaction. After denaturation at 95 C for 10 min, 2.5 units of Taq DNA polymerase was added and the reaction was incubated for 50 cycles (94 C / 30 sec, 50 C / 44 sec). After incubation, the multiplex PCR reaction was diluted 1:24 with water and subjected to invader assay analysis using an invader MAP detection platform. Each invader MAP assay was performed as a 6 ul reaction as follows: 3 ul of a 1:24 dilution of PCR reaction (total dilution 1: 8 equal to D = 0.125), 15 mM MgCl ₂ 3 ul, this is covered with CHILLOUT 6 ul. Samples were denatured in Invader MAP plates by incubation at 95 C for 5 minutes, followed by incubation at 63 C, and fluorescence measured at various time points for 160 minutes on Cytofluor 4000 (384 well reader). Analysis of FOZ values was performed at 10, 20, and 40 minutes and compared to calls made directly on genomic DNA. Shown in FIG. 13 is a call made in 10 minutes for 101-fold PCR with equimolar primer concentrations versus 10-fold for 101-fold PCR performed under optimized primer concentrations Comparison between calls. Additional data for this example is shown in FIGS. 16a, 16b and 17. Under equimolar primer concentrations, multiplex PCR yields only 50 correct calls at 10 minutes, whereas under optimized primer concentrations, multiplex PCR yields 71 correct calls, resulting in , Resulting in an increase of 21 new calls (42%). All 101 calls could not be made at 10 minutes, but 94 calls could be made at 40 minutes, which is the amplification efficiency of the majority of amplicons. Suggest improvements. Unlike more than 10-fold optimization, which required only one round of optimization, for more complex multiplexed reactions, multiple rounds of optimization were used to balance the amplification of all loci. May be needed.

  Additional primers for CYP2D6 are shown in FIG. FIG. 19 shows one protocol for multiple optimization.

III. Measurement of Allele Frequency in Pooled Samples In certain embodiments, the present invention comprises a plurality of individuals in a population (eg, 10, 50, 100, or 500 individuals) or a nucleic acid sequence Allows detection of polymorphisms in pooled samples combined from one subject that is derived from a large number of cells assayed at once. In this regard, the present invention makes it possible to detect the frequency of rare mutations in a pooled sample and to confirm the allelic frequency for a population. In some embodiments, this allele frequency is then used to statistically analyze the results of applying an invader detection assay to the individual's frequency for that polymorphism (e.g., measured using an invader assay). Can be used. In this regard, mutations that depend on the percentage of mutants found (eg, reduction of heterozygous mutations) can be analyzed to determine disease severity or disease progression (eg, any See U.S. Pat.Nos. 6,146,828 and 6,203,993 by Lapidus, incorporated herein by reference for purposes, genetic testing and statistical analysis to find disease-causing mutations or patients Used to identify that the sample contains a disease-causing mutation).

  In some embodiments of the invention, a broad population screen is performed. In some preferred embodiments, pooled DNA from hundreds or thousands of individuals is optimal. In such a pool, for example, DNA from any one individual is not detectable and provides a measure of the frequency of alleles where any detectable signal is detected in a wider population. For example, the amount of DNA used is not set by the number of individuals in the pool, but rather by the allele frequency detected, as was done in the 15 pool described in Example 3. is there. For example, a 96-well format assay provides sufficient signal from 20 ng to 40 ng of DNA in a 90 minute reaction. At this level of sensitivity, analysis of 1 μg of DNA from a high complexity pool appears to produce a comparable signal from alleles present in only about 3% to 5% of the population. In some embodiments, the reaction is set to run in a smaller volume so that less DNA is required for each analysis. In some preferred embodiments, the reaction is performed in a microwell plate (eg, a 384 well assay plate) and at least two alleles or loci are detected in each reaction well. In particularly preferred embodiments, the signals measured from each of the two or more alleles or loci in each well are compared.

Pooled samples-Example 1
This example describes the detection of polymorphisms in the APOC4 gene. In particular, this example describes the use of an invader assay to detect mutations in the APOC4 gene in pooled samples.

ならびに、T(Leu96)およびC(Pro96)の対立遺伝子それぞれに対する一次シグナルプローブとして

または

のいずれかを使用した。二次標的およびプローブはそれぞれ、

および

であった。 In this example, genomic DNA was isolated from blood samples from several individual donors and characterized by invasive cleavage against the T / C polymorphism at codon 96 of the APOC4 gene (as a reference). (See Allan et al., Genomics, July 20, 1995; 28 (2): 291-300, incorporated herein). The APOC4 assay is an invasive oligonucleotide

And as a primary signal probe for the T (Leu96) and C (Pro96) alleles, respectively.

Or

One of them was used. Each secondary target and probe is

and

Met.

All oligonucleotides were synthesized using standard phosphoramidite chemistry. The primary probe oligonucleotide was not labeled. FRET probes were labeled by incorporation of Cy3 phosphoramidite and fluorescent phosphoramidite (Glen Research, Sterling, VA). While designed for use at the 5 'end, Cy3 phosphoramidites have an additional monomethoxytrityl (MMT) group on the dye that can be removed to allow further synthetic chain extension. The result is an internal labeling with a dye that bridges the gap in the sugar-phosphate backbone of the oligonucleotide. At the 3 ′ end of the primary probe and secondary target oligonucleotides, amine or phosphate modifications as indicated were used so that they were not used as interstitial oligonucleotides. The 2'-O-methyl base in the secondary target oligonucleotide is indicated by underlining, but was also used to minimize enzyme recognition at the 3 'end. The approximate probe melting temperature (T _m ) was calculated using Oligo 5.0 software (National Bioscience, Plymouth, Minn.) And excluded non-homologous regions from the calculation.

  Pooled samples were constructed by diluting heterozygous (het) DNA into DNA that is homozygous T (L96) at this locus. The test reaction contained 0.08 μg to 8 μg of T (L96) genomic DNA per reaction, and het DNA was retained at 0.08 μg, which allowed het DNA to be reduced from 50% to 1% below the total DNA in the sample. A series of indicated mixtures were made (see FIG. 20). The actual representation of the C (P96) allele ranged from 25% of the copy of this gene in the mixed sample to 0.5% below. Controls included reactions with either all T (L96) DNA at each of the various DNA levels, or all het DNA at the 80 ng level. In addition, a sample of DNA that was homozygous for the C (P96) allele was tested (FIG. 20).

For all invader assay reactions, 4 pmol of invasive probe, 40 pmol of FRET probe, and 20 pmol of secondary target oligonucleotide were combined with genomic DNA in 34 μl of 10 mM MOPS (pH 7.5) containing 1.6% PEG. The reaction of the APOC4 gene with the C (Pro96) allele contained 80 ng of heterozygous DNA in this allele and in that indicated ratio homozygous DNA for T (Leu96). The sample was covered with 15 μl of Chill-Out liquid wax and heated to 95 ° C. for 5 minutes to denature the DNA. Upon cooling to 67 ° C, the reaction was initiated with 400 ng of Cleavase VIII enzyme, 15 pmol of either T (Leu96) or C (Pro96) primary signal probe, and MgCl ₂ to a final concentration of 7.5 mM. The plate was incubated at 67 ° C. for 2 hours, cooled to 54 ° C. to initiate a secondary (FRET) reaction, and incubated for an additional 2 hours. The reaction was then stopped by adding 60 μl TE. The fluorescence signal was measured on a Cytofluor fluorescence plate reader at excitation 485/20, emission 530/25, gain 65, temperature 25 ° C. For each reaction and target-free control, calculate the average signal for each target DNA that was run in triplicate, subtract the average background from the target-free control, and use Microsoft Excel, The data was plotted.

  The results of this example are shown in FIG. As shown in this figure, the C (P96) allele was readily detected in all reactions, including reactions in which only 0.5% of the APOC4 allele present in the mixture was present. These data indicate that the invasive cleavage reaction can be used for population analysis using pooled DNA samples. This allows to reduce the number of assays required to demonstrate a new SNP, and the use of a large preparation of one pooled pool for multiple tests, thereby increasing DNA purity Has twice the advantage of reducing the effects of sample-to-sample variation.

  The above examples demonstrate that invader assays can be used to screen populations. The sample of mixed DNA to be analyzed should be large enough to detect low-frequency alleles, for example, to bring the amount of variant genome in these 40 μl reactions to 80 ng to 100 ng It is. As indicated above in this example, a mixed sample of 8 μg to 10 μg of DNA allows detection of alleles present in 0.5% to 1% of the population under these conditions. In addition, DNA from any one individual should ideally not be present in large quantities sufficient to produce a detectable signal when the pool split is tested. Creating a pool of hundreds of individuals should ensure that any detected signal reflects the contributions from multiple individuals in that pool. Finally, the use of a second probe set as an internal standard allows the signal to be normalized from reaction to reaction, and allows any SNP prevalence to be measured more accurately. Seem.

Pooled samples-Example 2
This example describes the detection of polymorphisms in the CFTR gene. In particular, this example describes the use of an invader assay to detect a ΔF508 mutation in the CFTR gene in a pooled sample.

ならびに野生型および変異型の対立遺伝子についてのシグナルプローブとして

または

のいずれかを含んだ。二次反応構成要素は、一次反応温度より少なくとも5度低い温度で最適に機能するように設計された。 For invader assay analysis of the ΔF508 mutation, the primary probe set is an invasive oligonucleotide

As signal probes for wild-type and mutant alleles

Or

Including one of the following. The secondary reaction components were designed to function optimally at temperatures at least 5 degrees below the primary reaction temperature.

  All oligonucleotides were synthesized using standard phosphoramidite chemistry. The primary probe oligonucleotide was not labeled. FRET probes were labeled by incorporation of Cy3 phosphoramidites and fluorescent phosphoramidites (Glen Research, Sterling, VA). While designed for use at the 5 'end, the Cy3 phosphoramidite has an additional monomethoxytrityl (MMT) group on its dye that can be removed to allow further synthetic chain extension. The result is an internal labeling with a dye that bridges the gap in the sugar-phosphate backbone of the oligonucleotide. One nucleotide was deleted at this position to accommodate the dye. Amine modifications were used at the 3 ′ end of the primary probe, secondary target and arrestor oligonucleotide so that they were not used as interstitial oligonucleotides. The 2'-O-methyl base is shown underlined, but is also used to minimize enzyme recognition at the 3 'end. The approximate probe melting temperature was calculated using Oligo 5.0 software (National Bioscience, Plymouth, MN) and excluded non-homologous regions from the calculation.

  DNA samples characterized as CFTR genotypes are heterozygous in the CFTR gene for the Coriell Institute for Medical Research (Camden, NJ), catalog number NA07469 (both ΔF508 and R553X mutations) ) And NA01531 (homozygous ΔF508). DNA that is heterozygous for the ΔF508 mutation in the CFTR gene to determine how much of the variant can be detected in the pooled sample using the FRET time-based invasive cleavage method Was diluted to DNA that was homozygous wild type at that locus. The test reaction contains 0.1 μg to 2.6 μg of total genomic DNA per reaction, and the mutant DNA is retained at 0.1 μg, so that the mutant DNA is below 50% of the total DNA in the sample. Mixture sets displaying up to 4% were made. Because the mutant DNA is heterozygous at the 508 locus, the actual allele designation ranged from 25% to 2% below that DNA in the mixed sample. Controls included reactions with either all at the various DNA levels, or all heterozygous mutant DNA at the 100 ng level. In addition, samples of DNA that were homozygous for the ΔF508 mutation were tested.

DNA concentration was estimated using the PicoGreen method. 4 pmol of invader probe, 40 pmol of FRET probe, and 20 pmol of secondary target oligonucleotide were combined with genomic DNA in 34 μl of 10 mM MOPS (pH 7.5) containing 4% PEG. The sample was covered with 15 μl of Chill-Out liquid wax and heated at 95 ° C. for 5 minutes to denature the DNA. Upon cooling to 62 ° C., the reaction was initiated by the addition of 400 ng of AfuFEN1 enzyme, 15 pmol of either wild type or mutant primary probe, and MgCl ₂ to a final concentration of 7.5 mM. The plate was incubated at 62 ° C. for 2 hours, cooled to 54 ° C. to initiate a secondary (FRET) reaction, and incubated for an additional 2 hours. The reaction was then stopped by adding 60 μl TE. The fluorescence signal was measured on a Cytofluor fluorescence plate reader at excitation 485/20, emission 530/25, increase 65, temperature 25 ° C. Three replicates were performed for each reaction and for target-free controls. The average signal for each target DNA was calculated, the average background from the target-free control was subtracted, and then the data was plotted using Microsoft Excel.

  The results of this example are presented in FIG. Analysis of the signal from the mutant allele shows that it is not significantly inhibited by a substantial increase in the amount of wild-type DNA and is easily detected when ΔF508 mutant DNA is present as only 2% of the mixture (Fig. 21). These data indicate that an invasive cleavage reaction can be used for population analysis using pooled DNA samples. This allows to reduce the number of assays required to demonstrate a new SNP, and the use of a bulk preparation of one pooled DNA used for multiple tests, thereby Has twice the advantage of reducing the effects of intersample variation in DNA purity.

  In view of the results presented in this example, it is possible to apply invader assays to screen populations. In a preferred embodiment, for population screening, the contribution of DNA from each individual should be equal, and DNA from any one individual also produces a detectable signal when pool splits are tested. It is better not to exist in a large enough quantity to do. For example, for this system, any detected signal can be derived from multiple individuals in the pool by creating a sufficiently large pool where any one person contributes less than 1 ng (e.g., 0.5 ng) to each reaction. It will be guaranteed to reflect the contribution of. For other detection systems, limiting the DNA from any one individual to an amount that is less than the detection limit of the system, eg, 1/5 to 1/10 of the detection limit, will produce the desired effect. The use of a secondary probe set as an internal standard would, for example, allow the signal to be normalized from reaction to reaction, and allow any SNP prevalence to be measured more accurately. It is.

Pooled samples-Example 3
This example describes the detection of a Consortium number: TSC 0006429 (SNP 1831) mutation in a pooled sample. DNA from 15 individuals was purchased from the Coriell Cell Repository, and each sample was tested to identify the genotype at the SNP Consortium Number: TSC 0006429 (SNP 1831) locus. Each reaction of 7.5 mM 10 mM MOPS containing MgCl ₂ (pH 7.5) buffer, each individual derived DNA 40 ng, 0.366 [mu] M primary probes, 0.0366 [mu] M INVADER oligonucleotides, 0.183 MyuMFRET probe and 100 ngCLEAVASE VIII enzyme Inclusive.

The probes used are as follows (from 5 ′ to 3 ′):

  The assay was performed as described in Hall et al., PNAS, 97 (15): 8272 (2000). Briefly, the reaction was incubated at a constant temperature of 65 ° C. Data for each sample generated using the ABI7700 instrument for real-time reaction detection is shown in the 15 panels of FIGS. 22 and 23, where the signal from the G allele is a light colored line And the signal from the T allele is shown as a dark line. The signal from each allele present in the mixture is produced as a rising curve that reflects the quadratic nature of signal accumulation, and the signal from the absence of any allele is essentially linear. These DNAs were then pooled in several combinations: Samples 1-5, 6-10, 11-15, 1-10, 6-15, and 1-15. The data panel is shown in FIG. FIG. 25 provides a comparison of the net fluorescence count measured at the end of each reaction. From the results in FIGS. 22 and 23, the allelic representation in each mixture can be calculated. Both FIG. 24 and FIG. 25 demonstrate that the aggregated signal for each pool is proportional with respect to the final allele ratio in the mixture. The net fluorescent signal from the pooled sample is greater than those from the individual because the amount of DNA from each person was kept constant. For example, an assay performed on DNA pooled from 5 individuals had 5 times the amount of DNA as an assay performed on DNA from 1 individual.

  As seen in this example, the real-time detection capability of ABI7700 can prove invaluable in the detection of rare SNPs. Since the reaction is a two-step cascade, the real-time trace of the signal accumulated in the invader assay fits a quadratic equation (i.e. the curves observed in Figure 22, Figure 23 and Figure 24), but the background signal is It remains straight during the course of the reaction. It is therefore obvious to distinguish the signal produced from the genomic target from the background fluorescence. This feature of the assay means that low level signals from rare alleles can be more reliably determined from the background.

Pooled samples-Example 4
Measurement of different alleles within one reaction eliminates concerns about sample-to-sample variations that introduce inaccuracies in the measurements compared in determining allele frequencies. Use of dual (detection of two alleles or loci per reaction) or more complex multiplex (detection of more than two alleles or loci per reaction) increases throughput for allele frequency determination However, comparison of allele frequencies between different populations is facilitated (eg, those that are not affected by those affected by a particular trait).

  The following provides one example of a general protocol for the detection of two alleles in a DNA sample and several examples where the protocol has been applied to the determination of alleles in a sample. In this example, the signal was measured from a fluorescent dye (FAM) and a redmond red (REDMOND RED) dye (Red, Synthetic Genetics, San Diego, Calif.), Each independently combined with a Z28 Eclipse (ECLIPSE) quencher. Used on the FRET probe. This protocol is provided to serve as an example and is not intended to limit the use of the methods or components of the invention to any particular assay protocol or reaction format. Numerous fluorescent dyes and fluorophore / quencher combinations and methods for attaching and detecting such reagents alone and in FRET binding to nucleic acids are known in the art. Such other reagent combinations are contemplated for use in the present invention and their use in these methods is within the scope of the present invention.

および1.17 μM Red FRETプローブ

を含有する溶液3 μlを含む。
6. 次に、384ウェル二重インベーダーアッセイFRET検出プレートの適当なウェルへ、プローブ/ インベーダーオリゴヌクレオチド/MgCl₂混合物3 μlをピペットで移す。
7. 各反応物をミネラルオイル6 μlで表面を覆う。
8. プレートを接着性カバーで覆い、プローブおよび標的をウェルの底に移動させるためにベックマン(Beckman)GS-15R遠心分離機(または等価物)で10秒間、1,000 rpmで振とうする。
9. 反応物をサーマルサイクラーまたはBioOven IIIなどのインキュベーターにおいて63℃で3時間〜4時間、インキュベートする。63℃での3時間〜4時間のインキュベーション後、サーマルサイクラーを使用する場合には4℃まで、またはインキュベーターを使用する場合には室温まで、温度を低下させる。
10. 以下のパラメーターを用いる蛍光プレートリーダー上でマイクロタイタープレートを分析する。

a. Procedure for determination of allele frequency in pooled samples
1. Measure the DNA concentration of each sample used in the invader assay using the PICOGREEN reagent (the procedure follows).
2. Mix the DNA samples in the desired ratio into a simulated pool of genomic samples at the specified allele frequency.
3. Denature the genomic DNA sample by incubating at 95 ° C for 10 minutes. The sample may then be placed on ice (optionally).
4. Prepare a probe / invader oligonucleotide / MgCl ₂ mixture by mixing 1.15 μL probe / invader oligonucleotide mixture (3.5 μM and 0.35 μM invader oligonucleotides for each primary probe) and 1.85 μL 24 mM MgCl ₂ per reaction. . Preparation of a master mixture sufficient for testing a complete set of samples is preferred.
5. Appropriate wells at 80 ng / μl to 100 ng / μl (approximately 240 ng to 300 ng of genomic DNA) into appropriate wells of a 384 well double invader assay FRET detection plate (Third Wave Technologies, Madison, Wis.) Add 3 μl of control or sample DNA target. Each plate well was dried after dispensing, 10 mM MOPS, 8% PEG, 4% glycerol, 0.06% NP 40, 0.06% Tween 20, 12 ug / ml BSA, 50 ng / ul BSA, 33.3 ng / ul CLEAVASE VIII enzyme, 1.17 μM FAM FRET probe

And 1.17 μM Red FRET probe

3 μl of solution containing
6. Next, pipette 3 μl of the probe / invader oligonucleotide / MgCl ₂ mixture into the appropriate wells of a 384 well dual invader assay FRET detection plate.
7. Cover each reaction with 6 μl of mineral oil.
8. Cover the plate with an adhesive cover and shake at 1000 rpm for 10 seconds in a Beckman GS-15R centrifuge (or equivalent) to move the probe and target to the bottom of the well.
9. Incubate the reaction in an incubator such as a thermal cycler or BioOven III at 63 ° C. for 3-4 hours. After a 3 to 4 hour incubation at 63 ° C., the temperature is lowered to 4 ° C. when using a thermal cycler or to room temperature when using an incubator.
10. Analyze the microtiter plate on a fluorescent plate reader using the following parameters.

b. Calculation of fold-over-zero minus 1 (FOZ-1) The signal from each reaction was measured by comparison with the signal from a target-free control ("zero"), Expressed as a multiple of the signal from the “zero” reactant. Factor 1 is subtracted to obtain a factor of the actual signal relative to the background (e.g. for a sample with a 1.5 × signal of zero, i.e. 1.5 foldover zero, the amount of intrinsic signal is 1.5-1, Ie 0.5).

Determine FOZ-1 as follows.
FOZ-1 FAM probe = ((Raw count of FAM probe 1 (raw count), 485/530 ) / (Raw data of target FAM probe without target, 485/530 ))-1
FOZ-1 red probe = ((Raw data for the number of red probes 2, 560/620 ) / (Raw data for the number of control red probes without target, 560/620 ))-1

レッド対立遺伝子頻度計算について：

c. Calculation of Correction Factor (CF) as follows: The correction factor can be calculated to account for any variation between probe sets in cleavage reaction efficiency.
CF _FAM for heterozygous control = (FOZ _FAM -1) / ((FOZ _Red -1); CF _Red = (FOZ _Red -1) / (FOZ _FAM -1)
About FAM allele frequency calculation:

About red allele frequency calculation:

d. Procedure for DNA quantification (Molecular Probes PICOGREEN assay)
PICOGREEN reagent is an asymmetric cyanine dye (Molecular Probes, OR). The free dye does not fluoresce but shows a> 1000-fold increase in fluorescence upon binding to dsDNA. PICOGREEN is 10,000 times more sensitive than the UV absorbance method and is more selective for dsDNA than ssDNA and RNA.

2. ピコグリーン(PICOGREEN)キットに供給されている20×TE保存液から1×TE緩衝液(10 mM トリスHCl、1 mM EDTA、pH 7.5)を調製する(50 mlを作製するためには、DNase非含有滅菌蒸留水47.5 mlへ20×TEを2.5 ml添加する)。50 mlは、250アッセイに対して十分な量である。
3. DNA標準を1×TEで100 μg/mlから2 μg/mlまでに希釈する。2つの標準曲線のために、100 μg/ml保存液8 μlを392 μlの1×TEへ添加することにより2 μg/ml保存液を400 μl調製する。
4. 表1に示されるように、マイクロタイタープレート内の2つの標準曲線を調製する。 1. Spin on fluorescent plate reader for at least 10 minutes before reading results. Use the following settings to read the PICOGREEN results.

2. Prepare 1x TE buffer (10 mM Tris HCl, 1 mM EDTA, pH 7.5) from the 20x TE stock supplied in the picogreen kit (to make 50 ml, Add 2.5 ml of 20 × TE to 47.5 ml of sterile DNase-free distilled water). 50 ml is sufficient for 250 assays.
3. Dilute the DNA standard from 100 μg / ml to 2 μg / ml with 1 × TE. For the two standard curves, prepare 400 μl of 2 μg / ml stock solution by adding 8 μl of 100 μg / ml stock solution to 392 μl of 1 × TE.
4. Prepare two standard curves in the microtiter plate as shown in Table 1.

5. For each unknown sample, add 2 μl of sample to 98 μl of 1 × TE in a microplate well. Mix by pipetting up and down.
6. Prepare a 1: 200 dilution of PICOGREEN reagent in 1 × TE. A volume of 100 μl is required for each standard and each unknown sample. For example, two standard curves with 8 points each would require 1.6 ml. To calculate the total volume of diluted PICOGREEN reagent required, determine the sample to be tested and the total number unknown, and multiply this number by 100 μl (using a multichannel pipette If so, make additional reagents). PICOGREEN reagents are light sensitive and should be covered with foil during thawing and when diluted. Vortex thoroughly.
7. Add 100 μl of diluted PICOGREEN to all standards and samples. Mix by pipetting up and down.
8. Cover the microplate with foil and incubate at room temperature for 2-5 minutes.
9. Read the plate.
10. Create a standard curve using the standard mean and determine the DNA concentration in the unknown sample.

e. Measurement of allele frequencies in genomic DNA samples DNA samples with alleles at various frequencies were made by mixing different homozygous genomic DNA samples in different ratios. Each pool contained a total of 240 ng genomic DNA and the reaction was performed in 384 well plates at 63 ° C. for 3 hours as described above. The measured signal is shown in FIG. 26A. Allele frequencies were calculated based on the relative signals produced by FAM and Red reporter dye and are shown graphically in FIG. 26B. These data are between theoretical or actual allelic frequencies (frequency implied to be made by mixing known amounts of DNA) compared to allelic frequencies calculated from invader assay data. Show correlation.

  Eight pools of genomic DNA from different individuals were also tested. Each of the 8 DNAs has been previously characterized for each of 8 different SNP loci, and the allele frequencies for each of the 8 SNPs in the pool were known. In this test, each pool contained a total of 300 ng genomic DNA and the reaction was performed in 384 well plates at 63 ° C. for 3 hours as described above. The signal measured for the FAM channel is a rarer allele in each case, but is shown in FIG. The graph compares the known frequency for each allele with the frequency calculated from the invader assay data.

  Homozygous DNA for each of the two different SNPs (SNP132505 and SNP131534) were combined in various ratios to simulate a pool of genomes with different allelic frequencies. Each pool contained a total of 240 ng genomic DNA and reactions were performed in 384 well plates at 63 ° C. for 3 hours as described above. Allele frequencies are calculated based on the relative signals produced by the FAM and Red reporter dyes and are shown graphically in FIGS. 28A and 28B.

  Probes used in the above tests and additional probe sets suitable for use in the methods of the invention are shown in FIGS. 30A-30C.

  All publications and patents mentioned in the above specification are herein incorporated by reference as if specifically set forth herein. Various alterations and modifications to the methods and systems described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described with reference to certain preferred embodiments, it is to be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention as would be apparent to those skilled in the art are intended to be within the scope of the following claims.

1 illustrates one embodiment of an invader detection assay. The input target sequence and the results of processing this sequence using the systems and routines of the present invention are shown. An example of the basic work flow for advanced multiplex PCR using the INVADER Medically Associated Panel is shown. 2 shows a flow diagram outlining the steps that can be performed to create a primer set useful for multiplex PCR methods. FIG. 5 shows the sequences used and data generated for PCR primer design Example 1. FIG. 6 shows the sequences used and data generated for PCR primer design Example 1. FIG. 7 shows the sequences used and data generated for PCR primer design Example 1. FIG. 8 shows the sequences used and data generated for PCR primer design Example 1. FIG. 9 shows the sequences used and data generated for PCR primer design Example 1. FIG. 10A shows the sequence used and the data generated for Example 2. FIG. 10B shows the sequence used and the data generated for Example 2. FIG. 11 shows the sequence used and the data generated for Example 2. FIG. 12 shows the sequence used and data generated for Example 2. FIG. 13-1 shows the sequences used for Example 2 and the data generated. FIG. 13-2 is a continuation of FIG. 13-1. FIG. 13-3 is a continuation of FIG. 13-2. FIG. 14 shows the sequence used and the data generated for Example 2. FIG. 15-1 shows the sequences used for Example 2 and the data generated. FIG. 15-2 is a continuation of FIG. 15-1. FIG. 16A shows the sequence used and the data generated for Example 2. FIG. 16B shows the sequence used and the data generated for Example 2. FIG. 17 shows the sequence used and the data generated for Example 2. Specific PCR primers useful for amplifying various regions of CYP2D6 are shown. 18-2 is a diagram showing a continuation of FIG. 18-1. 1 shows one of the protocols for multiplex PCR optimization according to the present invention. 2 shows a graph demonstrating that the invader assay can detect mutations in the APOC4 gene in pooled samples. 2 shows a graph demonstrating that the invader assay can detect mutations in the CFTR gene in pooled samples. FIG. 22 shows a graph of the results of the experiment described in Pooled Sample—Example 3. FIG. 23 shows a graph of the results of the pooled sample—experiment described in Example 3. FIG. 24 shows a graph of the results of the experiment described in Pooled Sample—Example 3. FIG. 25 shows a graph of the results of the pooled sample—experiment described in Example 3. FIG. 26A shows data measuring allelic signals in an invader assay for detection of alleles containing the indicated number of copies of each locus in percent. FIG. 26B shows an Excel graph comparing the theoretical allelic frequency to the allelic frequency calculated from the invader assay data shown in FIG. 5A. Shows Excel graphs and data comparing actual and calculated allele frequencies for each of the 8 SN loci detected in pooled genomic DNA from 8 different individuals. Calculate allele frequencies compared to the fold-over-zero minus 1 (FOZ-1) measurements for SNP locus 132505 in genomic DNA with different mixtures of these alleles. The Excel graph and data shown are shown. Shows calculated allele frequencies compared to fold-over-zero minus 1 (FOZ-1) measurements for SNP locus 131534 in genomic DNA with different mixtures of these alleles Excel (Excel) graph and data are shown. FIG. 30A shows the sequence of the pooled samples—probes configured for use in the assay described in Example 4 and the synthetic target for each allele. “Y” represents an amine blocking group. Shown are the polymorphisms and dyes that would be detected for each probe when used in the exemplary assay configuration described in Example 4. FIG. 30B shows the pooled samples—sequences of probes configured for use in the assay described in Example 4 and the synthetic target for each allele. “Y” represents an amine blocking group. Shown are the polymorphisms and dyes that would be detected for each probe when used in the exemplary assay configuration described in Example 4. FIG. 30C shows the pooled sample—the sequence of the probe configured for use in the assay described in Example 4 and the synthetic target for each allele. “Y” represents an amine blocking group. Shown are the polymorphisms and dyes that would be detected for each probe when used in the exemplary assay configuration described in Example 4.

Claims (37)

A method comprising the following steps:
a) at least Y targets, each target sequence comprising i) a footprint region, ii) a 5 ′ region adjacent upstream of the footprint region, and iii) a 3 ′ region adjacent downstream of the footprint region Providing target sequence information about the sequence; and
b) The primer set includes a forward primer sequence and a reverse primer sequence for each of at least Y target sequences, and each of the forward primer sequence and the reverse primer sequence is 5′-N [x] -N [x-1]-. ...- N [4] -N [3] -N [2] -N [1] -3 ′ comprising a nucleic acid sequence, wherein N represents a nucleotide base, x is at least 6, N [1] is nucleotide A or C, and N [2] -N [1] -3 ′ of each of the forward primer and reverse primer represents which N [2] − of the forward primer and reverse primer in the primer set Processing the target sequence information to produce a primer set that is not complementary to N [1] -3 ′;   2. The method of claim 1, wherein the primer set is configured to perform a multiplex PCR reaction that amplifies at least Y amplicons, each amplicon being defined by the position of a forward primer and a reverse primer.   2. The method of claim 1, wherein the primer set is generated as digital or printed sequence information.   2. The method of claim 1, wherein the primer set is made as a physical primer oligonucleotide.   Each forward primer and reverse primer N [3] -N [2] -N [1] -3 'is the forward primer and reverse primer N [3] -N [2] -N [1] in the primer set 2. The method of claim 1, wherein the method is not complementary to -3 '.   The method of claim 1, wherein the processing step comprises first selecting N [1] for each forward primer as the 3 ′ most A or C in the 5 ′ region.   The method of claim 1, wherein the processing step comprises first selecting N [1] for each reverse primer as the 3 ′ most A or C in the complement of the 3 ′ region.   2. The method of claim 1, wherein the footprint region comprises a single nucleotide polymorphism.   2. The method of claim 1, wherein the footprint region for each target sequence comprises a portion of the target sequence that hybridizes to one or more assay probes configured to detect single nucleotide polymorphisms.   N [5] -N [4] -N [3] -N [2] -N [for each forward and reverse primer such that the processing step is less than 80 percent homologous with the assay component sequence 2. The method of claim 1, further comprising selecting 1] -3 ′.   The method of claim 1, wherein Y is an integer between 2 and 500.   2. The method of claim 1, wherein the processing step comprises selecting x for each forward primer and reverse primer such that each forward primer and reverse primer has a melting temperature for the target sequence of about 50 degrees Celsius.   2. The method of claim 1, wherein an optimized concentration of a forward primer and reverse primer pair is determined for the primer set.   A primer set produced by the method according to claim 1. A method comprising the following steps:
providing a) a) a user interface configured to receive sequence data; and ii) providing a computer system having an internally stored multiplex PCR primer software application; and
b) sequence data includes target sequence information for at least Y target sequences, each of the target sequences i) a footprint region, ii) a 5 ′ region adjacent upstream of the footprint region, and iii) the Transmitting the sequence data from the user interface to the computer system comprising a 3 ′ region adjacent downstream of a footprint region; and
c) a primer set comprising i) a forward primer sequence that is identical to at least a portion of the target sequence adjacent 5 ′ of the footprint region for each of the Y target sequences, and ii) the at least Y A reverse primer sequence that is identical to at least a portion of the complementary sequence of the target sequence adjacent 3 ′ of the footprint region for each target sequence, each of the forward primer sequence and the reverse primer sequence being 5′- N [x] -N [x-1] -....- N [4] -N [3] -N [2] -N [1] -3 ' Represents a nucleotide base, x is at least 6, N [1] is nucleotide A or C, and N [2] -N [1] -3 ′ of each of the forward and reverse primers is in the primer set The forward primer and reverse primer throat Processing the target sequence information with the multiplex PCR primer pair software application to create a primer set that is not complementary to N [2] -N [1] -3 ′.   16. The method of claim 15, wherein the primer set is configured to perform a multiplex PCR reaction that amplifies at least Y amplicons, each amplicon being defined by the position of a forward primer and a reverse primer.   16. The method according to claim 15, wherein the primer set to be produced is digital or printed sequence information.   Each forward primer and reverse primer N [3] -N [2] -N [1] -3 'is the forward primer and reverse primer N [3] -N [2] -N [1] in the primer set 16. The method of claim 15, wherein the method is not complementary to -3 '.   16. The method of claim 15, wherein the processing step comprises first selecting N [1] for each forward primer as the 3 ′ most A or C in the 5 ′ region.   The method of claim 15, wherein the processing step comprises a first selecting step.   16. The method of claim 15, wherein the footprint region comprises a single nucleotide polymorphism.   16. The method of claim 15, wherein the footprint region for each target sequence comprises a portion of the target sequence that hybridizes to one or more assay probes configured to detect single nucleotide polymorphisms.   16. The method of claim 15, wherein Y is a number between 2 and 500.   16. The method of claim 15, wherein the processing step comprises selecting x for each forward primer and reverse primer such that each forward primer and reverse primer has a melting temperature of about 50 degrees Celsius.   A primer set produced by the method according to claim 15. A system that includes:
a) the user interface is configured to receive sequence data, the sequence data including target sequence information for at least Y target sequences, each of the target sequences i) footprint region, ii) the footprint region A computer system configured to receive data from a user interface, comprising a 5 ′ region adjacent upstream of iii) and a 3 ′ region adjacent downstream of the footprint region;
b) A multiplex PCR primer software application is configured to process the target sequence information to create a primer set, and the primer set is i) 5 ′ of the footprint region for each of the Y target sequences. A forward primer sequence that is identical to at least a portion of the adjacent target sequence, and ii) at least one of the complementary sequences of the target sequence adjacent to the 3 ′ of the footprint region for each of the at least Y target sequences The reverse primer sequence is identical, and each of the forward primer sequence and the reverse primer sequence is 5'-N [x] -N [x-1] -....- N [4] -N [3 ] -N [2] -N [1] -3 ', wherein N represents a nucleotide base, x is at least 6, N [1] is nucleotide A or C, and The forward primer N [2] -N [1] -3 ′ of each of the reverse and reverse primers is not complementary to any N [2] -N [1] -3 ′ of the forward and reverse primers in the primer set A multiplex PCR primer pair software application operably linked to an interface; and
c) A computer system that internally stores the multiplex PCR primer pair software application, including computer memory and computer processing equipment.   27. The system of claim 26, wherein the computer system is configured to return the primer set to the user interface.   The multiplex PCR primer software application is further configured to process the target sequence information so that x is selected for each forward and reverse primer so that each forward and reverse primer has a melting temperature of about 50 degrees Celsius. 27. The system of claim 26. A method for detecting polymorphic allele frequencies comprising the following steps:
a) i) a pooled sample comprising target nucleic acid sequences from multiple individuals, and
ii) providing an INVADER detection reagent configured to detect the presence or absence of a polymorphism; and
b) contacting the pooled sample with the invader detection reagent to produce a detectable signal;
c) measuring the detectable signal, thereby determining the number of the target nucleic acid sequence comprising the polymorphism.   30. The method of claim 29, wherein the plurality comprises at least 10 individuals.   32. The method of claim 30, wherein the at least 10 individuals comprises at least 1000 individuals. A method for detecting polymorphic allele frequencies comprising the following steps:
a) i) a pooled sample comprising target nucleic acid sequences from multiple individuals, and
ii) providing an invader assay detection reagent configured to produce a unique signal for each allele of the polymorphic locus in the target nucleic acid sequence;
b) contacting the pooled sample with the invader detection reagent to produce at least one unique signal;
c) measuring the at least one unique signal, thereby determining the proportion of each allele of the polymorphic locus in the pooled sample.   35. The method of claim 32, wherein the measuring step includes detection of fluorescence.   35. The method of claim 32, wherein at least two unique signals are produced in step b) and the measuring step comprises comparing at least two unique signals.   35. The method of claim 34, wherein the comparing step comprises applying a correction factor to the measurement of at least one unique signal. A method for detecting rare mutations, including the following steps:
a) i) a sample from a single subject comprising at least 10,000 target nucleic acid sequences, and
ii) providing a detection assay capable of detecting mutations in a target nucleic acid sequence population present at an allele frequency of 1: 1000 or less compared to a wild-type allele; and
b) assaying the sample using the detection assay under conditions such that the presence or absence of a rare mutation is detected. A method for detecting rare mutations, including the following steps:
a) i) a sample from a single subject comprising at least 10,000 target nucleic acid sequences, and
ii) providing a detection assay capable of detecting mutations in a target nucleic acid sequence population present at an allele frequency of 1: 1000 or less compared to a wild-type allele; and
b) assaying the sample using the detection assay under conditions such that the allele frequency of rare mutations in the sample is determined.

Images