Patent Application
20100112551
The present invention relates to the field of genetic screening and diagnostics. More specifically, the described embodiments concern methods to screen multiple samples, in a single assay, for the presence or absence of mutations or polymorphisms that relate to Hereditary Nonpolyposis Colorectal Cancer (HNPCC).
Hereditary Nonpolyposis Colorectal Cancer (HNPCC) is the most common hereditary form of colon cancer. It is a genetic syndrome caused by mutations in any one of five or more genes that code for proteins involved with repair of damaged or aberrant DNA, two of which are the human mismatch repair genes mutL homolog 1 (MLH1) and mutS homologue 2 (MSH2). Individuals that inherit mutations associated with HNPCC are at a much higher risk for colon cancer than the general population (80% chance of developing color cancer, vs. 4%) and at an earlier age (average age of onset of colon cancer: 44 years old, vs. 65 years of age for the general population). Individuals with HNPCC also have a higher risk of getting certain other forms of cancer (Lynch, H. et al. Cancer 78:1149 (1996)). There is a great need for approaches to identify mutations and polymorphisms that relate to this deadly disease.
Current DNA-based diagnostics allow for the identification of a single mutation or polymorphism or gene per analysis. Although high-throughput methods and gene chip technology have enabled the ability to screen multiple samples or multiple loci within the same sample, these approaches require several independent reactions, which increases the time required to process clinical samples and drastically increases the cost. Further, because of time and expense, conventional diagnostic approaches focus on the identification of the presence of DNA fragments that are associated with a high frequency of mutation, leaving out analysis of other loci that may be critical to diagnose a disease. The need for more approaches for the diagnosis of genetic disease is manifest.
With the advent of multiplex Polymerase Chain Reaction (PCR), the ability to use multiple primer sets to generate multiple extension products from a single gene is at hand. By hybridizing isolated DNA with multiple sets of primers that flank loci of interest on a single gene, it is possible to generate a plurality of extension products in a single PCR reaction corresponding to fragments of the gene. As the number of primers increases, however, the complexity of the reaction increases and the ability to resolve the extension products using conventional techniques fails. Further, since many diseases are caused by changes of a single nucleotide, the rapid detection of the presence or absence of these mutations or polymorphisms is frustrated by the fact that the PCR products that indicate both the diseased and non-diseased state are of the same size.
Developments in gel electrophoresis and high performance liquid chromatography (HPLC), however, have enabled the separation of double-stranded DNAs based upon differences in their melting behaviors, which has allowed investigators to resolve DNA fragments having a single mutation or single polymorphism. Techniques such as temporal temperature gradient gel electrophoresis (TTGE) and denaturing high performance liquid chromatography (DHPLC) have been used to screen for small changes or point mutations in DNA fragments.
The separation principle of TTGE, for example, is based on the melting behavior of DNA molecules. In a denaturing polyacrylamide gel, double-stranded DNA is subject to conditions that will cause it to melt in discrete segments called “melting domains.” The melting temperature Tm of these domains is sequence-specific. When the Tm of the lowest melting domain is reached, the DNA will become partially melted, creating branched molecules. Partial melting of the DNA reduces its mobility in a polyacrylamide gel. Since the Tm of a particular melting domain is sequence-specific, the presence of a mutation or polymorphism will alter the melting profile of that DNA in comparison to the wild-type or non-polymorphic DNA. That is, a heteroduplex DNA consisting of a wild-type or non-polymorphic strand annealed to mutant or poymorphic strand, will melt at a lower temperature than a homoduplex DNA strand consisting of two wild-type or non-polymorphic strands. Accordingly, the DNA containing the mutation or polymorphism will have a different mobility compared to the wild-type or non-polymorphic DNA.
Similarly, the separation principle of DHPLC is based on the melting or denaturing behavior of DNA molecules. As the use and understanding of HPLC developed, it became apparent that when HPLC analyses were carried out at a partially denaturing temperature, i.e., a temperature sufficient to denature a heteroduplex at the site of base pair mismatch, homoduplexes could be separated from heteroduplexes having the same base pair length. (See e.g., Hayward-Lester, et al., Genome Research 5:494 (1995); Underhill, et al., Proc. Natl. Acad. Sci. USA 93:193 (1996); Oefner, et al., DHPLC Workshop, Stanford University, Palo Alto, Calif., (Mar. 17, 1997); Underhill, et al., Genome Research 7:996 (1997); Liu, et al., Nucleic Acid Res., 26:1396 (1998), all of which and the references contained therein are hereby expressly incorporated by reference in their entireties).
Techniques such as Matched Ion Polynucleotide Chromatography (MIPC) and Denaturing Matched Ion Polynucleotide Chromatography (DMIPC) have also been employed to increase the sensitivity of detection. It was soon realized that DHPLC, which for the purposes of this disclosure includes but is not limited to, MIPC, DMIPC, and ion-pair reverse phase high-performance liquid chromatography, could be used to separate heteroduplexes from homoduplexes that differed by as little as one base pair. Various DHPLC techniques have been described in U.S. Pat. Nos. 5,795,976; 5,585,236; 6,024,878; 6,210,885; Huber, et al., Chromatographia 37:653 (1993); Huber, et al., Anal. Biochem. 212:351 (1993); Huber, et al., Anal. Chem. 67:578 (1995); ODonovan et al., Genomics 52:44 (1998), Am J Hum Genet. December; 67(6):1428-36 (2000); Ann Hum Genet. September:63 (Pt 5):383-91 (1999); Biotechniques, April; 28(4):740-5 (2000); Biotechniques. November; 29(5):1084-90, 1092 (2000); Clin Chem. August; 45(8 Pt 1):1133-40 (1999); Clin Chem. April; 47(4):635-44 (2001); Genomics. August 15; 52(1):44-9 (1998); Genomics. March 15; 56(3):247-53 (1999); Genet Test.; 1(4):237-42 (1997-98); Genet Test.:4(2):125-9 (2000); Hum Genet. June; 106(6):663-8 (2000); Hum Genet. November; 107(5):483-7 (2000); Hum Genet. November; 107(5):488-93 (2000); Hum Mutat. December; 16(6):518-26 (2000); Hum Mutat. 15(6):556-64 (2000); Hum Mutat. March; 17(3):210-9 (2001); J Biochem Biophys Methods. November 20; 46(1-2):83-93 (2000); J Biodhem Biophys Methods. January 30; 47(1-2):5-19 (2001); Mutat Res. November 29; 430(1):13-21 (1999); Nucleic Acids Res. March 1; 28(5):E13 (2000); and Nucleic Acids Res. October 15; 28(20):E89 (2000), all of which, including the references contained therein, are hereby expressly incorporated by reference in their entireties. Despite the efforts of many, there remains a need for more approaches to screen and identify mutations and/or polymorphisms in genes, in particular, genes that relate to Hereditary Nonpolyposis Colorectal Cancer.
Aspects of the invention concern rapid and inexpensive but efficient approaches to determine the presence or absence of mutations and/or polymorphisms that relate to Hereditary Nonpolyposis Colorectal Cancer (HNPCC). Several oligonucleotide primers specific for the human mismatch repair genes, mutL homolog 1 (MLH1) and mutS homologue 2 (MSH2), have been developed (e.g., Tables A and 2). These primers and oligonucleotides that are any number between 1-75 nucleotides upstream or downstream of said primers are unique in sequence and in their ability to generate extension products that melt evenly over vast stretches of nucleotides, which greatly improves the sensitivity of detection (e.g., single base mutations). It was then realized that by grouping extension products with similar melting behaviors, one can rapidly and efficiently separate multiple extension products on the basis of melting behavior on the same lane of a TTGE gel or in the same run on a DHPLC. Accordingly, a rapid, inexpensive and efficient approach to diagnose a subject at risk for HNPCC was discovered, whereby extension products are generated from a subject's DNA using the primers described herein, the extension products are grouped or mixed according to their melting behavior, and the grouped or mixed extension products are separated on the basis of melting behavior (e.g., one group per lane of TTGE gel). Not only does the pooling of extension products reduce cost and the time to perform the analysis but, because the extension products are optimized for melting behavior, the sensitivity of detection remains very high.
By one approach, for example, a method of identifying the presence or absence of a genetic marker in the human mismatch repair genes MLH1 and MSH2 of a subject is conducted by providing a DNA sample from said subject; providing at least one primer set from Table A; contacting said DNA and said at least one primer set; generating an extension product from said at least one primer set that comprises a region of DNA that includes the location of said genetic marker; separating said extension product on the basis of melting behavior; and identifying the presence or absence of said genetic marker in said subject by analyzing the melting behavior of said extension product. In related embodiments, at least 2, 3, 4, 5, 6, 7, or 8 primer sets from Table A are contacted with said DNA. In more related embodiments, the extension products generated from said 2, 3, 4, 5, 6, 7, or 8 primer sets are grouped according to Table D and separated on the basis of melting behavior. Optionally, the extension products and/or the sample nucleic acid used in the approaches above can be sequenced so as to verify and/or identify the mutation or polymorphism.
In another set of embodiments, a method of identifying the presence or absence of a genetic marker in the human mismatch repair genes mutL homolog 1 (MLH1) and mutS homologue 2 (MSH2) of a subject is conducted by providing a DNA sample from said subject; providing at least one primer set that is any number between 1-75 nucleotides upstream or downstream of a primer set from Table A; contacting said DNA and said at least one primer set; generating an extension product from said at least one primer set that comprises a region of DNA that includes the location of said genetic marker, separating said extension product on the basis of melting behavior; and identifying the presence or absence of said genetic marker in said subject by analyzing the melting behavior of said extension product. In related embodiments, at least 2, 3, 4, 5, 6, 7, or 8 primer sets from Table A are contacted with said DNA. In more related embodiments, the extension products generated from said 2, 3, 4, 5, 6, 7, or 8 primer sets are grouped according to Table D and separated on the basis of melting behavior. As above, optionally, the extension products and/or the sample nucleic acid used in these approaches can be sequenced so as to verify and/or identify the mutation or polymorphism.
Embodiments described herein concern a novel approach to screen for the presence or absence of multiple mutations or polymorphisms in a plurality of genes, in particular, genes associated with Hereditary Nonpolyposis Colorectal Cancer (HNPCC). Particularly preferred embodiments concern approaches to screen multiple loci in the human mismatch repair genes mutL homolog 1 (MLH1) and mutS homologue 2 (MSH2) so as to determine the presence or absence of a mutation or polymorphism that may indicate a suseptibility to Hereditary Nonpolyposis Colorectal Cancer (HNPCC) and/or other cancers. Similar approaches have been used to identify the presence or absence or polymorphisms or mutations related to cystic fibrosis, which are described in U.S. patent application Ser. Nos. 10/300,683; 60/333,351; and 60/486,864, all of which are hereby expressly incorporated by reference in their entireties.
Several embodiments permit very sensitive detection of single base mutations, single base mismatches, and small nuclear polymorphisms (SNPs), as well as, larger alterations in DNA at multiple loci, in a plurality of genes, in multiple samples. Additionally, by employing a DNA standard or by screening a plurality of DNA samples in the same assay, improved sensitivity of detection can be obtained. A novel approach to designing primers and extension products generated therefrom is described in the context of an assay that was performed to detect the presence or absence of genetic markers, polymorphisms, or mutations on the human mismatch repair genes mutL homolog 1 (MLH1) and mutS homologue 2 (MSH2). By identifying the presence or absence of these polymorphisms or mutations, an understanding of susceptibility to Hereditary Nonpolyposis Colorectal Cancer (HNPCC) can be obtained.
Embodiments include methods of identifying the presence or absence of a plurality of genetic markers in a subject in the same gene or separate genes. One method is practiced, for example, by providing a DNA sample from said subject, providing a plurality of nucleic acid primer sets that hybridize to said DNA at regions that flank said plurality of genetic markers, wherein each primer set has a first and a second primer and, wherein said plurality of genetic markers exist on the same gene or a plurality of genes, contacting said DNA and said plurality of nucleic acid primer sets in a single reaction vessel or multiple reaction vessels, generating, in said reaction vessel(s), a plurality of extension products that comprise regions of DNA that include the location of said plurality of genetic markers, separating said plurality of extension products on the basis of melting behavior in a single lane or multiple lanes of a gel or a single run or multiple runs on a column, and identifying the presence or absence of said plurality of genetic markers in said subject by analyzing the melting behavior of said plurality of extension products. In some aspects of this method the separation on the basis of melting behavior is accomplished by TTGE and in other embodiments the separation on the basis of melting behavior is accomplished by DHPLC. In some embodiments, said extension products are first separated by size for a period sufficient to separate populations of extension products and then separated by melting behavior. The size separation can be accomplished on the TTGE gel or DHPLC column prior to separating on the basis of melting behavior.
Preferably, after generating the extension products by an amplification technique (e.g., Polymerase Chain Reaction or PCR), the extension products are grouped and pooled according to their predicted and/or actual melting behavior. In this way, multiple extension products, which correspond to different regions on the same gene or different' regions on a plurality of genes can be separated on the same lane of a TTGE gel or in the same run on a DHPLC column. By carefully designing the primers, such that the extension products generated therefrom melt over large stretches of DNA (approximately 25, 50, 75, 100, 125, or 150 nucleotides) at roughly the same temperature (within up to 1.5° C. of one another), it was unexpectedly discovered that multiple extension products (2, 3, 4, 5, 6 or more) can be separated on the same lane of a TTGE gel or in the same run on an DHPLC column, thereby substantially reducing the cost of conducting the analysis and increasing the speed of analysis.
In some embodiments, either the first or the second primer comprise a GC clamp. In other aspects of this embodiment, either the first or the second primer hybridize to a sequence within an intron. Preferably, at least one of the plurality of genetic markers is indicative of Hereditary Nonpolyposis Colorectal Cancer (HNPCC). In other embodiments, the plurality of primer sets consist of at least 3, 4, 5, 6, or 7 primer sets. Additionally, in some embodiments, the plurality of genes consist of at least 2, 3, 4, 5, 6, or 7 genes that are related to Hereditary Nonpolyposis Colorectal Cancer (HNPCC). The method above preferably generates the extension products using the Polymerase Chain Reaction (PCR) and the method can be supplemented by a step in which a control DNA is added.
Another embodiment concerns a method of identifying the presence or absence of a plurality of genetic markers in a plurality of subjects. This method is practiced by: providing a DNA sample from said plurality of subjects, providing a plurality of nucleic acid primer sets that hybridize to said DNA at regions that flank said plurality of genetic markers, wherein each primer set has a first and a second primer and, wherein said plurality of genetic markers exist on the same gene or on a plurality of genes, contacting said DNA and said plurality of nucleic acid primer sets in a single reaction vessel or multiple vessels, generating, in said reaction vessel(s), a plurality of extension products that comprise regions of DNA that include the location of said plurality of genetic markers, separating said plurality of extension products on the basis of melting behavior in a single lane or multiple lanes of a gel or a single run or multiple runs on a column, and identifying the presence or absence of said plurality of genetic markers in said plurality of subjects by analyzing the melting behavior of said plurality of extension products. In some aspects of this embodiment, the separation on the basis of melting behavior is accomplished by TTGE and in other embodiments the separation on the basis of melting behavior is accomplished by DHPLC. Again, preferred genetic markers for identification using the approaches above, concern genes that are associated with Hereditary Nonpolyposis Colorectal Cancer (HNPCC).
As above, preferably, after generating the extension products by the amplification technique (e.g., PCR) from the plurality of subjects, the extension products are grouped and pooled according to their predicted and/or actual melting behavior. By separating multiple extension products generated from a plurality of subjects in the same lane of a TTGE gel or in the same run on a DHPLC column, the cost of analysis is substantially reduced. Because the incidence of polymorphism or mutation in the population as a whole is small, the large scale screening, described above, can be performed. When a polymorphism and/or mutation is detected in this type of assay, single subject assays can be performed, as described above, to identify the subject(s) that have the polymorphism and/or mutation. Optionally, the extension products and/or the nucleic acid samples themselves can be sequenced so as to verify and/or identify the mutation or polymorphism.
In more embodiments, the plurality of subjects consist of at least 2, 3, 4, 5, 6, or 7 subjects. In more aspects of this embodiment, the plurality of primer sets consist of at least 3, 4, 5, 6, or 7 primer sets. Additionally, in some embodiments, the plurality of genes consist of at least 2, 3, 4, 5, 6, or 7 genes. The method above preferably generates the extension products using PCR and the method can be supplemented by a step in which a control DNA is added.
Still another embodiment involves a method of identifying the presence or absence of a mutation or polymorphism in a subject related to Hereditary Nonpolyposis Colorectal Cancer (HNPCC). This method is practiced by: providing a DNA sample from said subject, generating a population of extension products from said sample, wherein said extension products comprise a region of said DNA that corresponds to the location of said mutation or polymorphism, providing at least one control DNA, wherein said control DNA corresponds to the extension product but lacks said mutation or polymorphism, contacting said control DNA and said population of extension products in a single reaction vessel, thereby forming a mixed DNA sample, heating said mixed DNA sample to a temperature sufficient to denature said control DNA and said DNA sample, cooling said mixed DNA sample to a temperature sufficient to anneal said control DNA and said DNA sample, separating said mixed sample on the basis of melting behavior in a single lane or multiple lanes of a gel or a single run or multiple runs on a column, and identifying the presence or absence of said mutation or polymorphism by analyzing the melting behavior of said mixed DNA sample.
By this approach, the addition of the control DNA followed by the heating and cooling steps, forces heteroduplex formation, if a polymorphism or mutation is present, which facilitates identification. In some aspects of this embodiment, the control DNA is DNA obtained or amplified from a second subject and the presence or absence of a mutation or polymorphism is known. In other aspects of the invention, heteroduplex formation can be forced by pooling the extension products generated from a plurality of subjects and denaturing and annealing, as above. Because the predominant genotype in a plurality of subjects lacks polymorphisms or mutations in the gene(s) analyzed, the majority of the DNA will force heteroduplex formation with any polymorphic or mutant DNA in the pool. Accordingly, the identification of mutant and/or polymorphic DNA is facilitated and the cost of the analysis is reduced. In some aspects of this embodiment, the separation on the basis of melting behavior is accomplished by TTGE and in other embodiments the separation on the basis of melting behavior is accomplished by DHPLC.
Still more embodiments concern the primers or groups of primers disclosed herein (preferably MLH1 and MSH2 specific primers), extension products generated from said primers, kits containing said nucleic acids, and methods of using these primers, groups of primers, or extension products to diagnose a risk for a disease (e.g., HNPCC). These nucleic acid primers can be used to efficiently determine the presence or absence of a polymorphism or mutation in a multiplex PCR reaction that screens a plurality of genes and a plurality of subjects in a single reaction vessel or multiple reaction vessels. Additionally, reaction vessels comprising a DNA sample, and a plurality of nucleic acid primer sets that hybridize to said DNA sample at regions that flank a plurality of genetic markers, wherein said plurality of genetic markers exist on a single gene or a plurality of genes are embodiments. Further, a reaction vessel comprising a plurality of DNA samples obtained from a plurality of subjects and a plurality of nucleic acid primer sets that hybridize to said plurality of DNA samples at regions that flank a plurality of genetic markers, wherein said plurality of genetic markers exist on a plurality of genes or on a single gene are embodiments.
Still more aspects of the invention include a reaction vessel containing a plurality of extension products (2, 3, 4, 5, 6, 7, 8, 9, or 10 or more), which melt at approximately the same temperature (e.g., 0° C.-1.5° C. from one another). That is, in some approaches, the extension products are generated in separate vessels using individual primers sets but the extension products with similar melting behaviors are pooled prior to loading onto a TTGE gel or DHPLC. The pooled extension products are loaded onto a single lane of a gel and resolved by melting behavior. In some embodiments, differing fluorescent labels are employed in the individual PCR reactions so that the extension products generated therefrom fluoresce at different wavelengths (e.g., produce a different color under a detector) so as to facilitate identification after the pooled extension products are resolved on the gel or column.
Other embodiments concern a gel having lanes and adapted to separate different DNAs comprising a plurality of extension products, in a single lane of said gel, wherein said plurality of extension products melt at approximately the same temperature but are resolvable on said gel and, which correspond to regions of DNA located on a plurality of genes or on a single gene and, wherein said regions of DNA comprise loci that indicate a genetic trait and a gel having lanes and adapted to separate different DNAs comprising a plurality of extension products, in a single lane of said gel, wherein said plurality of extension products correspond to regions of DNA located on a plurality of genes or on a single gene in a single individual or a plurality of subjects and, wherein said regions of DNA comprise loci that indicate a genetic trait.
Additional embodiments include a DHPLC column adapted to separate different DNAs comprising a plurality of extension products, wherein said plurality of extension products melt at approximately the same temperature but are resolvable on said column and, which correspond to regions of DNA located on a plurality of genes or a single gene or and, wherein said regions of DNA comprise loci that indicate a genetic trait and a DHPLC column adapted to separate different DNAs comprising a plurality of extension products, wherein said plurality of extension products correspond to regions of DNA located on a plurality of genes or on a single gene in a single individual or a plurality of subjects and, wherein said regions of DNA comprise loci that indicate a genetic trait. More description of the compositions and methods described above is provided in the in the following sections.
Approaches to Facilitate and Reduce the Cost of Genetic Analysis
Aspects of the invention described herein concern approaches to analyze DNA, samples for the presence or absence of a plurality of genetic markers that reside on a plurality of genes in a single assay. Some embodiments allow one to rapidly distinguish a plurality of DNA fragments in a single sample that differ only slightly in size and/or composition (e.g., a single base change, mutation, or polymorphism). Other embodiments concern methods to screen multiple genes from a subject, in a single assay, for the presence or absence of a mutation or polymorphism. An approach to achieve greater sensitivity of detection of mutations or polymorphisms present in a DNA sample is also provided. Preferred embodiments, however, include methods to screen multiple genes, in a plurality of DNA samples, in a single assay, for the presence or absence of mutations or polymorphisms.
It was discovered that multiple extension products that have slight differences in length and/or composition can be resolved by separating the DNA on the basis of melting temperature. By one approach, a plurality of varying lengths of double-stranded DNA are applied to a denaturing gel and the double-stranded DNAs are separated by applying an electrical current while the temperature of the gel is raised gradually. By slowly increasing the temperature while the DNA is electrically separated on a polyacrylamide gel containing a denaturant (e.g., urea), the dsDNA eventually denatures to partially single stranded (branched molecules) DNA. Because branched or heteroduplex DNA migrates more rapidly or more slowly than dsDNA or homoduplex DNA, one can quickly determine the differences in melting behavior between DNA fragments, compare this melting temperature to a standard DNA (e.g., a wild-type DNA or non-polymorphic DNA), and identify the presence or absence of a mutation or polymorphism in the screened DNA. This technique efficiently separates multiple DNA fragments, generated by a single multiplex PCR reaction on a plurality of loci from different genes (e.g., in one experiment, 10 different loci were analyzed in the same reaction and each of the extension products, some that differed by only a single mutation, were efficiently resolved).
It was also discovered that multiple extension products that have slight differences in length and/or composition can be resolved by separating the DNA by DHPLC. By one approach, a plurality of varying lengths of double-stranded DNA are applied to a ion-pair reverse phase HPLC column (e.g., alkylated non-porous poly(styrene-divinylbenzene)) that has been equilibrated to an appropriate denaturing temperature, depending on the size and composition of the DNA to be separated (e.g., 53° C. to 63° C.) in an appropriate buffer (e.g., 0.1 mM triethylamine acetate (TEAA) pH 7.0). Once applied to the column, the double stranded DNA binds to the matrix. By slowly increasing the presence of a denaturant (e.g., acetonitrile in TEAA), the dsDNA eventually denatures to partially single stranded (branched molecules) DNA and elutes from the column. Preferably a linear gradient is used to slowly elute the bound DNA. Detection can be accomplished using a U.V. detector, radioactivity, dyes, or fluorescence. In some embodiments, the extension products are first separated on the basis of size using a shallow gradient of denaturant for a time sufficient to separate individual populations of extension products and then on the basis of melting behavior using a deeper gradient of denaturant. The techniques described in the following references can also be modified for use with aspects of the invention: U.S. Pat. Nos. 5,795,976; 5,585,236; 6,024,878; 6,210,885; Huber, et al., Chromatographia 37:653 (1993); Huber, et al., Anal. Biochem. 212:351 (1993); Huber, et al., Anal. Chem. 67:578 (1995); ODonovan et al., Genomics 52:44 (1998), Am J Hum Genet. December; 67(6):1428-36 (2000); Ann Hum Genet. September:63 (Pt 5):383-91 (1999); Biotechniques, April; 28(4):740-5 (2000); Biotechniques. November; 29(5):1084-90, 1092 (2000); Clin Chem. August; 45(8 Pt 1):1133-40 (1999); Clin Chem. April; 47(4):635-44 (2001); Genomics. August 15; 52(1):44-9 (1998); Genomics. March 15; 56(3):247-53 (1999); Genet Test.; 1(4):237-42 (1997-98); Genet Test:4(2):125-9 (2000); Hum Genet. June; 106(6):663-8 (2000); Hum Genet. November; 107(5):483-7 (2000); Hum Genet. November; 107(5):488-93 (2000); Hum Mutat. December; 16(6):518-26 (2000); Hum Mutat. 15(6):556-64 (2000); Hum Mutat. March; 17(3):210-9 (2001); J Biochem Biophys Methods. November 20; 46(1-2):83-93 (2000); J Biochem Biophys Methods. January 30; 47(1-2):5-19 (2001); Mutat Res. November 29; 430(1):13-21 (1999); Nucleic Acids Res. March 1; 28(5):E13 (2000); and Nucleic Acids Res. October 15; 28(20):E89 (2000), all of which are hereby expressly incorpo'rated by reference in their entireties including the references cited therein.
Because branched or heteroduplex DNA elutes either more rapidly or more slowly than homoduplex DNA, one can quickly determine the differences in melting behavior between DNA fragments, compare this melting temperature to a standard DNA (e.g., a wild-type or non-polymorphic homoduplex DNA), and identify the presence or absence of a mutation or polymorphism in the screened DNA. This technique efficiently separates multiple DNA fragments, generated by a single multiplex PCR reaction on a plurality of loci from different genes.
Some of the embodiments described herein have adapted the DNA separation techniques described above to allow for high-throughput genetic screening of organisms (e.g., plant, virus, bacteria, mold, yeast, and animals including humans). Typically, multiple primers that flank genetic markers (e.g., mutations or polymorphisms that indicate a congenital disease or a trait) on different genes are employed in a single amplification reaction or multiple amplification reactions and the multiple extension products are separated on a denaturing gel or by DHPLC according to their melting behavior. The presence or absence of mutations or polymorphisms, also referred to as “genetic markers”, in the subject's DNA are then detected by identifying an aberrant melting behavior in the extension products (e.g., migration on a gel that is too fast or too slow or elution from a DHPLC column that is too fast or too slow). Advantageously, some embodiments provide a greater understanding of a subject's health because more loci that are indicative of disease, for example, are analyzed in a single assay. Further, some embodiments drastically reduce the cost of performing such diagnostic assays because many different genes and markers for disease can be screened simultaneously in a single assay.
By one approach, for example, a biological sample from the subject (e.g., blood) is obtained by conventional means and the DNA is isolated. Next, the DNA is hybridized with a plurality of nucleic acid primers that flank regions of a plurality of genetic loci or markers that are associated with or linked to the plurality of traits to be analyzed. Although 10 different loci have been detected in a single assay (requiring 20 primers), more or less loci can be screened in a single assay depending on the needs of the user. Preferably, each assay has sufficient primers to screen at least three different loci, which may be located on three different genes. That is, the embodied assays can employ sufficient primers to screen at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24 or more, independent loci or markers that are indicative of a disease in a single assay (e.g., in the same tube or multiple tubes) and these loci can be on different genes. Because more than one loci or marker can be detected by a single set of primers, the detection of 20 different markers, for example, can be accomplished with less than 40 primers. However, in many assays, a different set of primers is needed to detect each different loci. Thus, in several embodiments, at least 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, or more primers are used.
Desirably, the primers hybridize to regions of human DNA that flank markers or loci associated with or linked to human diseases such as: familial hypercholesterolemia (FH), cystic fibrosis, Tay-sachs, thalassemia, sickle cell disease, phenylketonuria, galactosemia, fragile X syndrome, hemophilia A, myotonic dystrophy, medium-chain acyl CoA dehydrogenase, maturity onset diabetes, cystinuria, methylmolonic acidemia, urea cycle disorders, hereditary fructose intolerance, hereditary hemachromatosis, neonatal thrombocytopenia, Gaucher's disease, tyrosinemia, Wilson's disease, alcaptonuria, hypolactasia, Baker's disease, argininemia Adenomatous polyposis coli (APC), Adult Polycystic Kidney disease, a-1-antitrypsin deficiency, Duchenne Muscular Dystrophy, Hemophilia A, Hereditary Nonpolyposis colorectal cancer, Huntingtons disease, Marfan syndrome, Myotonic dystrophy, Neurofibromatosis, Osteogenesis imperfecta, Retinoblastoma, Sickle cell disease, Freidrichs ataxia, Hemoglobinopathies, Leber's hereditary optic neuropathy, MCAD, Canavan's disease, Retintitus Pigmentosa, Bloom Syndrome, Fanconi anemia, and Neimann Pick disease. It is particularly preferred that the primers hybridize to regions of DNA that flank markers associated with Hereditary Nonpolyposis Colorectal Cancer (HNPCC). It should be understood, however, that the list above is not intended to limit the invention in any way and the techniques described herein can be used to detect and identify any gene or mutation or polymorphism desired (e.g., polymorphisms or mutations associated with alcohol dependence, obesity, and cancer).
Once the primers are hybridized to the subject's DNA, a plurality of extension products having the marker or loci indicative of the trait are generated. Preferably, the extension products are generated through a polymerase-driven amplification reaction, such as multiplex PCR or multiplex Ligase Chain Reaction (LCR). In some embodiments, one or more fluorescent labels are employed. That is, by some methods, individual extension products are generated by PCR in the presence of different fluorescent labels so that the resulting extension products are fluoresce at different wavelengths (e.g., different colors are seen for each individual extension product on a detector). These embodiments facilitate the analysis of multiple patient samples in the same assay or multiple markers on the same or different genes. The extension products are then pooled according to similar melting behaviors and then the pooled samples are separated on the basis of melting behavior (e.g., TTGE or DHPLC).
In some approaches, for example, the extension products are isolated from the reactants in the amplification reaction, suspended in a non-denaturing loading buffer, and are loaded on a TTGE denaturing gel (e.g., an 8%, 7M urea polyacrylamide gel). The sample can be heated to a temperature sufficient to denature a DNA duplex and then cooled to a temperature that allows reannealing, prior to suspending the DNA in the non-denaturing loading buffer. The extension products are then loaded into a single lane or multiple lanes, as desired. Next, an electrical current is applied to the gel and extension products.
Subsequently, the temperature of the denaturing gel is gradually raised, while maintaining the electrical current, so as to separate the extension products on the basis of their melting behaviors. Once the fragments have been separated by size and melting behavior, one can identify the presence or absence of mutations or polymorphisms at the screened loci by analyzing the migration behavior of the extension products. By employing the fluorescent labels above, one can rapidly identify the differing extension products or patient samples, as well.
In other approaches, the extension products are isolated from the reactants and suspended in a DHPLC buffer (e.g., 0.1M TEAA pH 7.0). The extension products are then injected onto a DHPLC column (e.g., an ion-pair reverse phase HPLC column composed of alkylated non-porous poly(styrene-divinylbenzene)) that has been equilibrated to an appropriate denaturing temperature, depending on the size and composition of the DNA to be separated (e.g., 53° C. to 63° C.) in an appropriate buffer (e.g., 0.1 mM triethylamine acetate (TEAA) pH 7.0) and the extension products are allowed to bind. The presence of a denaturant (e.g., acetonitrile in TEAA) on the column is gradually raised over time so as to slowly elute the extension products from the column. Preferably a linear gradient is used. Presence of the extension products in the eluant is preferably accomplished using a UV detector (e.g., at 260 and/or 280 nm), however, greater sensitivity may be obtained using radioactivity, binding dyes, fluorescence or the techniques described in U.S. Pat. Nos. 5,795,976; 5,585,236; 6,024,878; 6,210,885; Huber, et al., Chromatographia 37:653 (1993); Huber, et al., Anal. Biochem. 212:351 (1993); Huber, et al., Anal. Chem. 67:578 (1995); and O'Donovan et al., Genomics 52:44 (1998), which are all hereby incorporated by reference in their entireties including the references cited therein.
The appearance of a slower or faster migrating band at a temperature below or above the predicted melting point for the particular extension product in the TTGE approach, for example, indicates the presence of a mutation or polymorphism in the subject's DNA. Similarly, the appearance of a slower or faster eluting peak at a concentration of denaturant predicted to elute a wild-type or non-polymorphic homoduplex extension product in the DHPLC approach indicates the presence of a mutation or polymorphism in the subject's DNA. A heterozygous sample will display both homoduplex bands (wild-type homoduplexes and mutant homoduplexes), as well as, two heteroduplex bands that are the product of mutant/wild-type annealing. Because of base pair mismatches in these fragments, they melt significantly sooner than the two homoduplex bands. Accordingly, a user can rapidly identify the presence or absence of a mutation or polymorphism at the screened loci by either the TTGE or DHPLC approach and determine whether the tested subject has a predilection for a disease.
In a related embodiment, greater sensitivity is obtained by adding a “standard” DNA or “control” DNA to the DNA to be screened prior to amplification or after amplification, prior to separation of the DNA on the TTGE gel or DHPLC column. This insures the presence of heteroduplexes in the case of either a homozygous mutant, which normally would not display heteroduplexes, or a heterozygous mutant. Desired DNA standards include, but are not limited to, DNA that is wild-type for at least one of the traits that are being screened. Preferred standards include, but are not limited to, DNA that is wild-type for all of the traits that are being screened. A DNA standard can also be a mutant or polymorphic DNA. In some embodiments, particularly when the control DNA is added after amplification, the DNA standard is an extension product generated from a wild-type genomic DNA or a mutant genomic DNA. By this approach, the amplification phase of the method is performed as described above. That is, DNA from the subject to be screened and the DNA standard are hybridized with nucleic acid primers that flank regions of the genetic loci or markers that are associated with or linked to the traits being tested. In some embodiments, the DNA standard extension products are fluorescently labeled differently than the extension products generated from the screened samples so as to facilitate identification.
Extension products are then generated. If the subject being tested has at least one trait that is detected by the assay (e.g., a congenital disorder), then two populations of extension products are generated, a first population that corresponds to the standard DNA and a second population that corresponds to the subject's DNA having at least one mutation or polymorphism. Next, preferably, the two populations of extension products are isolated from the amplification reactants and are denatured by heat (e.g., 95° C. for 5 minutes), then are allowed to anneal by cooling (e.g., ice for 5 minutes). This ensures the formation of the heteroduplex bands in the presence of any relatively small mutation (e.g., point mutation, small insertion, or small deletion). The isolation and denaturing/annealing steps are not practiced with some embodiments, however.
Subsequently, by the TTGE approach, the two populations of extension products are suspended in a non-denaturing loading buffer and loaded on a denaturing polyacrylamide gel and separated on the basis of melting behavior, as described above. By the DHPLC approach, the two populations of extension products are suspended in a suitable buffer (e.g., 0.1M TEAA pH 7.0), loaded onto a buffer and temperature equilibrated DHPLC column and a linear gradient of denaturant is applied, as described above. Because the two populations of extension products are not perfectly complementary, they form heteroduplexes. Heteroduplexes are less stable than homoduplexes, have a lower melting temperature, and are easily differentiated from homoduplexes using the DNA separation techniques described above. One can identify the presence or absence of mutations or polymorphisms at the screened loci, for example, by comparing the migration behavior or elution behavior of the extension products generated from the screened DNA with the migration behavior or elution behavior of the DNA standard. If heteroduplexes are present, generally, two additional bands that correspond to the single extension product will appear on the gel or the extension products will elute from the column more rapidly than the control or standard DNA alerting the user to the presence of a mutation or polymorphism. Accordingly, a significant increase in sensitivity is obtained and a user can rapidly identify the presence or absence of a mutation or polymorphism in the tested DNA sample and, thereby, determine whether the screened subject has a predilection for a particular trait (e.g., a congenital disease). As stated above, by employing different fluorescent labels during individual amplification reactions, different fluorescently labeled extension products can be generated and the identification of particular markers can be facilitated.
Similarly, an increase in sensitivity can be obtained by mixing DNA from a plurality of subjects prior to amplification. Because the frequency of mutations or polymorphisms for most disorders are very low in the population, most of the extension products generated are wild-type DNA. Thus, most of the pool of DNA behaves as a DNA standard. That is, the predominant structure formed upon annealing after denaturation is a homoduplex, which can be rapidly distinguished from any heteroduplex that would appear if a subject were to have a polymorphism or mutation. Of course, extension products previously generated from multiple subjects can be used as control DNA by mixing the previously generated extension products with the extension products generated from the DNA that is being screened prior to electrophoresis. In several embodiments, the DNA from at least 2 subjects is mixed. Desirably, the DNA from at least 3 subjects is mixed. Preferably, the DNA from at least 4 subjects is mixed. It should be understood, however, that the DNA from at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more subjects can be mixed prior to amplification or prior to separation on the basis of melting behavior, in accordance with some of the described embodiments. Again, by employing different fluorescent labels during individual amplification reactions, different fluorescently labeled extension products can be generated and the identification of genetic markers, in particular the same markers on different subjects (e.g., the amplification reactions for different subjects employ different fluorescent markers) can be facilitated.
In one embodiment, for example, DNA from a plurality of subjects to be tested is obtained by conventional methods, pooled, and hybridized with the desired nucleic acid primers. Extension products are then generated, as before. If at least one of the subjects being tested has at least one congenital disorder that is detected by the screen then two populations of extension products will be generated, a first population that corresponds to DNA from subjects that have the wild-type gene and a second population that corresponds to DNA from subjects having at least one mutant or polymorphic gene.
By one approach, the two populations of extension products are then isolated from the amplification reactants, suspended in a non-denaturing loading buffer, denatured by heat, annealed by cooling, and are separated by TTGE, as described above. By another approach, the two populations of extension products are isolated from the amplification reactants, suspended in a DHPLC loading buffer (0.1M TEAA pH 7.0), denatured by heat, annealed by cooling, and are separated on a DHPLC column, as described above. The presence of a subject in the DNA pool having at least one mutation or polymorphism is identified by analyzing the migration behavior of the DNA on the gel or the elution behavior from the column. The appearance of a slower or faster migrating band at a temperature below or above the predicted melting point for a particular extension product on the gel indicates the presence of a mutation or polymorphism in the DNA from one of the subjects. Similarly, the appearance of a slower or faster eluting extension product from the DHPLC column indicates the presence of a mutation or polymorphism in the DNA from one of the subjects. By repeating the analysis with smaller and smaller pools of samples, one can identify the individual(s) in the pool that has the mutation or polymorphism. Additionally, DNA standards can be used, as described above, to facilitate identification of the individual(s) having the mutation or polymorphism. Advantageously, some embodiments can be used to screen multiple samples at multiple loci that are on found on a plurality of genes in a single assay, thus, increasing sample throughput. The analysis of a plurality of DNA samples in the same assay also unexpectedly provides greater sensitivity. The section below describes a DNA separation technique that can be used with the embodiments described herein.
Multiple Extension Products of Similar Composition can be Separated on the Same Lane of a Denaturing Gel or in the Same Run on a DHPLC Column
It was discovered that multiple fragments of DNA, which vary slightly in length and/or composition, can be rapidly and efficiently resolved on the basis of melting behavior. Although the preferred methods for differentiating multiple fragments of DNA on the basis of melting behavior involve TTGE gel electrophoresis and DHPLC, it is contemplated that other conventional techniques that are amenable to DNA separation on the basis of melting behavior can be equivalently employed (e.g., size exclusion chromatography, ion exchange chromatography, and reverse phase chromatography on high pressure (e.g., HPLC), low pressure (e.g., FPLC), gravity-flow, or spin-columns, as well as, thin layer chromatography).
By one approach, a polyacrylamide gel having a porosity sufficient to resolve the DNA fragments on the basis of size (e.g., 4-20% acrylamide/bis acrylamide gel having a set concentration of denaturant) is used. The amount of denaturant in the gel (e.g., urea or formamide) can vary according to the length and composition of the DNA to be resolved. The concentration of urea in a polyacrylamide gel, for example, can be 3M, 3.5M, 4M, 4.5M, 5M, 5.5M, 6M, 6.5M, 7M, 7.5M, or 8M. In preferred embodiments, an 8% polyacrylamide gel with 7M urea is used. It should be emphasized, however, that other types of polyacrylamide gels, equivalents thereof, and agarose gels can be used.
The DNA samples to be resolved are placed in a non-denaturing buffer and can be loaded directly to the gel. In some embodiments, for example, when heteroduplex formation is desired to increase the sensitivity of the assay, it is desirable to heat the double stranded DNA to a temperature that permits denaturation (e.g., 95° C. for 5-10 minutes) and then slowly cool the DNA to a temperature that allows annealing (e.g., ice for 5-10 minutes) prior to mixing with the loading buffer. Preferably, the DNA is loaded onto the gel in a total volume of 10-20 μl. Preferably, a Temporal Temperature Gradient Gel Electrophoresis (TTGE) apparatus is used. A commercially available system that is suitable for this technique can be obtained from BioRad. The gel can be run at 120, 130, 140, 150, 175, 200, 220, 250, 275, or 300 V for 1.5-10 hours, for example.
Once the DNA has been loaded, an electrical current is applied to begin separating the fragments on the bass of size and the temperature of the gel is raised gradually. In one embodiment, for example, the melting behavior separation is performed by raising the temperature beyond 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., or 75° C. at approximately 5.0 C.°/hour-0.5° C./hour in 0.1° C. increments.
Once the extension products have been separated by melting behavior, the gel can be stained to reveal the separated DNA. Many conventional stains are suitable for this purpose including, but not limited to, ethidium bromide stain (e.g., 1% ethidium bromide in a 1.25×Tris Acetate EDTA pH 8.0 (TAE) solution), fluorescent stains, silver stains, and colloidal gold stains. In some embodiments, it is desirable to destain the gel (e.g., 20 minutes in a 1.25×TAE solution). After staining, the gel can be analyzed visually (e.g., under a U.V. lamp) and/or with a digital camera and computer software such as, the Eagle Eye System by Stratagene or the Gel Documentation System (BioRad). Additionally, when fluorescent markers are employed, conventional detectors that emit various wavelengths of light can be used so as to identify the presence and position of separated fluorescently labeled extension products.
Mutations or polymorphisms are easily identified by comparing the migration behavior of the DNA to be screened with the migration behavior of a control DNA and/or by monitoring the melting temperature of the extension products generated from the screened DNA. Desirable “control” DNA or “standard” DNA includes a DNA that is wild-type or non-polymorphic for at least one loci that is screened and preferred standard DNA is wild-type or non-polymorphic for all of the loci that are being screened. Because this DNA separation technique is sufficiently sensitive to identify a single base pair substitution in a DNA fragment up to 600 base pairs in length, small changes in the melting behaviors and migration of the extension products can be rapidly identified. The standard or control DNA can also be fluorescently labeled (preferably with a fluorescent label that is different than the one employed for the screened samples) to facilitate the analysis.
By another approach, DHPLC is used to resolve heteroduplex and homoduplex molecules of several PCR extension products in a single assay. Preferably, the heteroduplex and homoduplex extension products are separated from each other by ion-pair reverse phase high performance liquid chromatography. In one embodiment, a DHPLC column that contains alkylated non-porous poly(styrene-divinylbenzene) is used. Preferably, the DHPLC column is equilibrated in an appropriate degassed buffer, referred to as Buffer “A” (e.g., 0.1M TEAA pH 7.0) and is kept at a constant temperature somewhat below the predicted melting temperature of the extension products (e.g., 53° C.-60° C., preferably 50° C.). A plurality of extension products that may be generated from a plurality of different loci, as described herein, are suspended in Buffer A and are injected onto the DHPLC column. The Buffer A is then allowed to run through the column for a time sufficient to insure that the extension products have adequately bound to the column. Preferably, flow rate and the amount of gas (e.g., argon or helium) are adjusted and kept constant so that the pressure on the column does not exceed the recommended level. Gradually, degassed denaturing buffer, referred to as Buffer “B”, (e.g., 0.1M TEAA pH 7.0 and 25% acetonitrile) is applied to the column. Although an isocratic gradient can be used, a gradual linear gradient is preferred. By one approach, to separate fragments that range in size from 200-450 bp, for example, a gradient of 50%-65% Buffer B (0.1M TEAA pH 7.0 and 25% acetonitrile) is used. Of course, as the size of extension products to be separated on the DHPLC column decreases, the gradient and/or the amount of denaturant in Buffer B can be reduced, whereas, as the size of extension products to be separated on the DHPLC column increases, the gradient and/or the amount of denaturant in Buffer B can be increased.
The DHPLC column is designed such that double stranded DNA binds well but as the extension products become partially denatured the affinity to the column is reduced until a point is reached at which the particular extension product can no longer adhere to the column matrix. Typically, heteroduplexes denature before homoduplexes, thus, they would be expected to elute more rapidly from the column than homoduplexes.
In some embodiments, particularly embodiments concerning the separation of a plurality of different extension products (e.g., extension products generated from a plurality of loci), the choice of primers and, thus, the extension products generated therefrom, requires careful design. For example, a GC-clamp or other artificial sequence can be used to adjust the melting characteristics and increase the length of a particular DNA fragment, if needed, to facilitate separation on the DHPLC or improve resolution of the extension products. By one approach, each set of primers in a multiplex reaction are designed and selected to generate an extension product that has a unique homoduplex and heteroduplex elution behavior. In this manner, each species can be easily identified.
By another approach, each set of primers are designed to generate extension products that have homoduplexes with very similar melting characteristics. By this strategy, all of the homoduplexes will elute at the same or very similar concentration of denaturant, which is different than the concentration of denaturant required to elute the heteroduplexes. Accordingly, the elution of a species of extension product outside of the expected range for the homoduplexes indicates the presence of a mutation or polymorphism.
In the case that the extension products happen to have overlapping retention times/elution behaviors, the DHPLC conditions can be adjusted to include a primary separation on the basis of size prior to increasing the concentration of the denaturant on the column to improve resolution. The techniques described in Huber, et al., Anal. Chem. 67:578 (1995), hereby expressly incorporated by reference in its entirety, can be adapted for use with the novel DHPLC separation approach described herein. In one embodiment, for example, the alkylated non-porous poly(styrene-divinylbenzene) DHPLC column can be used to separate the extension products on the basis of size for a time sufficient to group the various populations of extension products (i.e., the homoduplexes and heteroduplexes generated from a single independent set of primers constitute a single population of extension products) prior to separating on the basis of melting behavior.
By one approach, the extension products are applied to the column, as above, in Buffer A and a shallow linear gradient of Buffer B (e.g., 30%-50% of a solution of 0.1M TEAA pH 7.0 and 25% acetonitrile for 200-450 by extension products) is applied so as to resolve the various populations of extension products. Then, a deeper linear gradient of Buffer B (e.g., 50%-65% of a solution of 0.1M TEAA pH 7.0 and 25% acetonitrile for 200-450 by extension products) is applied to resolve the homoduplexes from the heteroduplexes within each individual population of extension product. In this manner, the homoduplexes and heteroduplexes from each population of extension product can be resolved despite having overlapping elution behaviors.
It should be understood that the separation based on size can be performed at virtually any temperature as long as the extension products do not denature on the column, however, the amount of denaturant in Buffer B and the type of gradient may have to be adjusted. For example, the size separation can be accomplished at 4° C.-23° C., or 23° C.-40° C., or 40°-50° C., or 50° C.-60° C. Additionally, the size separation can be accomplished while the column is being gradually equilibrated to the temperature that is going to be used for the DHPLC. It should also be understood that the size separation can be performed on the same column with the appropriate gradient (shallow for a time sufficient to separate on the basis of size followed by a deeper gradient to separate on the basis of melting behavior). Additionally, columns in series can be used to separate extension products that have overlapping retention times/elution behaviors. For example, a first DHPLC column can be used to separate on the basis of size and a second DHPLC column can be used to separate on the basis melting behavior.
Mutations or polymorphisms are easily identified using the DHPLC techniques above by comparing the elution behavior of the DNA to be screened with the elution behavior of a control DNA. As above, desirable “control” DNA or “standard” DNA includes a DNA that is wild-type or non-polymorphic for at least one loci that is screened and preferred standard DNA is wild-type or non-polymorphic for all of the loci that are being screened. Control or standard DNA can also include extension products that are homoduplexes by virtue of a mutation or polymorphism or plurality of mutations or polymorphisms. Since the elution behavior of the wild type or non-polymorphic DNA or a homozygous mutant or polymorphism, represents the elution behavior of a homoduplex, one can use DHPLC values obtained from separating these controls, such as the retention time, elution time, or amount of denaturant required to elute the homoduplex as a basis for comparison to a screened sample to identify the presence of homoduplexes. Similarly, a control DNA can be a known heteroduplex and the elution behavior values described above can be used to identify the presence of a heteroduplex in a screened sample.
Additionally, the separated extension products can be collected after passing through the DHPLC column or TTGE gel or reamplified and sequenced to verify the existence of the mutation or polymorphism. Further, the identified products can be isolated from the gel and sequenced. Sequencing can be performed using the conventional dideoxy approach (e.g., Sequenase kit) or an automated sequencer. Preferably, all possible mutant fragments are sequenced using the CEQ 2000 automated sequencer from Beckman/Coulter and the accompanying analysis software. The mutations or polymorphisms identified by sequencing can be compiled along with the respective melting behaviors and the sizes of extension products. This data can be recorded in a database so as to generate a profile for each loci.
Additionally, this profile information can be recorded with other subject-specific information, for example family or medical history, so as to generate a subject profile. By creating such databases, individual mutations can be better characterized. Mutation analysis hardware and software can also be employed to aid in the identification of mutations or polymorphisms. For example, the “ALFexpress II DNA Analysis System”, available from Amersham Pharmacia Biotech and the “Mutation Analyser 1.01”, also available from Amersham Pharmacia Biotech, can be used. Mutation Analyser automatically detects mutations in sample sequence data, generated by the ALFexpress II DNA analysis instrument. The section below describes embodiments that allow for the identification of a mutation or polymorphism at multiple loci in a plurality of genes in a single assay.
Identification of the Presence or Absence of a Mutation or Polymorphism at Multiple Loci in a Plurality of Genes in a Single Assay
The DNA separation techniques described herein can be used to rapidly identify the presence or absence of a mutation or polymorphism at multiple loci in a plurality of genes in a single assay (e.g., in a single reaction vessel or multiple reaction vessels). Accordingly, a biological sample containing DNA is obtained from a subject and the DNA is isolated by conventional means. For some applications, it may be desired to screen the RNA of a subject for the presence of a genetic disorder (e.g., a congenital disease that arises through a splicing defect). In this case, a biological sample containing RNA is obtained, the RNA is isolated, and then is converted to cDNA by methods well known to those of skill in the art. DNA from a subject or cDNA synthesized from the mRNA obtained from a subject can be easily and efficiently isolated by various techniques known in the art. Also known in the art is the ability to amplify DNA fragments from whole cells, which can also be used with the embodiments described herein. Thus, the DNA sample for use with the embodiments described herein need only be isolated in the sense that the DNA is in a form that allows for PCR amplification.
In some embodiments, genomic DNA is isolated from a biological sample by using the Amersham Pharmacia Biotech “GenomicPrep Blood DNA Isolation Kit”. The isolation procedure involves four steps: (1) cell lysis (cells are lysed using an anionic detergent in the presence of a DNA preservative, which limits the activity of endogenous and exogenous Dnases); (2) RNAse treatment (contaminating RNA is removed by treatment with RNase A); (3) protein removal (cytoplasmic and nuclear proteins are removed by salt precipitation); and (4) DNA precipitation (genomic DNA is isolated by alcohol precipitation). EXAMPLE 1 also describes an approach that was used to isolate DNA from human blood.
Once the sample DNA has been obtained, primers that flank the desired loci to be screened are designed and manufactured. Preferably, optimal primers and optimal primer concentrations are used. Desirably, the concentrations of reagents, as well as, the parameters of the thermal cycling are optimized by performing routine amplifications using control templates. Primers can be made by any conventional DNA synthesizer or are commercially available. Optimal primers desirably reduce non-specific annealing during amplification and also generate extension products that resolve reproducibly on the basis of size or melting behavior and, preferably, both. Preferably, the primers are designed to hybridize to sample DNA at regions that flank loci that can be used to diagnose a trait, such as a congenital disease (e.g., loci that have mutations or polymorphisms that indicate a human disease).
Desirably, the primers are designed to detect loci that diagnose conditions selected from the group consisting of familial hypercholesterolemia (FH), cystic fibrosis, Tay-sachs, thalassemia, sickle cell disease, phenylketonuria, galactosemia, fragile X syndrome, hemophilia A, myotonic dystrophy, medium-chain acyl CoA dehydrogenase, maturity onset diabetes, cystinuria, methylmolonic acidemia, urea cycle disorders, hereditary fructose intolerance, hereditary hemachromatosis, neonatal thrombocytopenia, Gaucher's disease, tyrosinemia, Wilson's disease, alcaptonuria, hypolactasia, Baker's disease, argininemia Adenomatous polyposis coli (APC), Adult Polycystic Kidney disease, a-1-antitrypsin deficiency, Duchenne Muscular Dystrophy, Hemophilia A, Hereditary Nonpolyposis colorectal cancer, Huntingtons disease, Marfans syndrome, Myotonic dystrophy, Neurofibromatosis, Osteogenesis imperfecta, Retinoblastoma, Sickle cell disease, Freidrichs ataxia, Hemoglobinopathies, Leber's hereditary optic neuropathy, MCAD, Canavan's disease, Retintitus Pigmentosa, Bloom Syndrome, Fanconi anemia, and Neimann Pick disease. Preferably, the primers are designed to detect the presence or absence of polymorphisms or mutation associated with Hereditary Nonpolyposis Colorectal Cancer (HNPCC). Primers can be designed to amplify any region of DNA, however, including those regions known to be associated with diseases such as alcohol dependence, obesity, and cancer. It should be understood that the embodiments described herein can be used to detect any gene, mutation, or polymorphism found in plants, virus, molds, yeast, bacteria, and animals.
Preferred primers are designed and manufactured to have a GC rich “clamp” at one end of a primer, which allows the dsDNA to denature in a “zipper-like” fashion. As one of skill will appreciate, PCR requires a “primer set”, which includes a first and a second primer, only one of which has the GC clamp so as to allow for separation of the double stranded molecule from one end only. Since the GC clamp is significantly stable, the rest of the fragment melts but does not completely separate until a point after the inflection point of the DNA, which contains the mutation or polymorphism of interest. The denaturant in the gel or on the column allows the temperature of melting to be lower and allows the inflection point of the melt to be longer in terms of temperature and, thus, the sensitivity to temperature at the inflection point is less (i.e., increment temperature=less increment melting), which increases the resolution.
Additionally, desirable primers are designed with a properly placed GC-clamp so that extension products that contain a single melting domain are produced. Preferably, the primers are selected to complement regions of introns that flank exons containing the genetic markers of interest so that polymorphisms or mutations that reside within the early portions of exons are not masked by the GC clamp. For example, it was discovered that GC clamps significantly perturb melting behavior and can prevent the detection of a polymorphism or mutation by melting behavior if the mutation or polymorphism resides too close to the GC clamp (e.g., within 40 nucleotides). By performing amplification reactions with control templates, optimal primer design and optimal concentration can be determined. The use of computer software, including, but not limited to, WinMelt or MacMelt (Bio-Rad) and Primer Premire 5.0 can aid in the creation and optimization of primers and proper positioning of the GC-clamp. Accordingly, many of the primers and groupings of primers described herein, as used in a particular assay (e.g., to screen for HNPCC) are embodiments of the invention. EXAMPLE 2 further describes the design and optimization of primers that allowed for the high-throughput multiplex PCR technique described herein.
Once optimal primers are designed and selected, the DNA sample is screened using the inventive multiplex PCR technique. In some embodiments, for example, approximately 25 ng-500 ng of template DNA (preferably, 200 ng for human genomic DNA) is suspended in a buffer comprising: 10 mM Tris (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 200 μM dNTPs, 50 pmol of each primer, and 1 unit Taq polymerase per primer set in a total volume of 50 μl. Preferably, amplification is performed under the same conditions that were used to design the primers. In some embodiments, for example, amplification is performed on a conventional thermal cycler for 30 cycles, wherein each cycle is: 1 minute @ 95° C., 58° C. for 1 minute, 72° C. for 1 minute. Final extension is performed at 72° C. for 5 minutes. When the primers have a GC clamp, it was found that conditions often favor an amplification reaction having over 40 cycles, wherein each cycle is: 35 seconds @ 95° C., 120 seconds @ 50-57° C., and 60 seconds+3 seconds/cycle @ 72° C. Thermal cyclers are available from a number of scientific suppliers and most are suitable for the embodiments described herein.
Once the PCR reaction is complete, the extension products are desirably isolated by centrifugal microfiltration using a standard PCR cleanup cartridge, for example, Qiagen's QIAquick 96 PCR Purification Kit, according to manufacture's instructions. Isolation or purification of the extension products is not necessary to practice the invention, however. The isolated extension products can then be suspended in a non-denaturing loading buffer and either loaded directly on a DHPLC column or TTGE denaturing gel. The sample can also be denatured by heating (e.g., 95° C. for 5-10 minutes) and annealed by cooling (e.g., ice for 5-10 minutes) prior to loading onto the DHPLC column or TTGE denaturing gel. The various extension products are then separated on a TTGE denaturing gel or DHPLC column on the basis of melting behavior, as described above and, after separation, the extension products can be analyzed for the presence or absence of polymorphisms or mutations. EXAMPLES 3 and 4 describe experiments that verified that multiple loci on a plurality of genes can be screened in a single assay. The section below describes a method of genetic analysis, wherein improved sensitivity of detection was obtained by adding a DNA standard to the screened DNA.
Improved Sensitivity was Obtained Wizen a DNA Standard was Mixed with the Screened DNA
It was also discovered that greater sensitivity in the inventive multiplex PCR reactions described herein can be obtained by mixing a DNA standard with the DNA to be tested prior to conducting amplification or after amplification but prior to separation on the basis of melting behavior. Desired DNA standards include, but are not limited to, DNA that is wild-type for at least one of the traits that are being screened and preferred DNA standards include, but are not limited to, DNA that is wild-type for all of the traits that are being screened. DNA standards can also be mutant or polymorphic DNA. In some embodiments, particularly when the control DNA is added after amplification, the DNA standard is an extension product generated from a wild-type genomic DNA or a mutant genomic DNA. Optionally, the control DNA can be labeled with a fluorescent label, which can be a label that is different than the fluorescent label used to label the extension products generated from the screened sample DNA. In this manner, the standard or control DNA is easily differentiated from the DNA that is being screened.
By one approach, the DNA from the subject to be screened and the DNA standard are pooled and then the amplification reaction, as described above, is performed. Accordingly, optimal primers are designed and selected and approximately 25 ng-500 ng of template DNA (preferably, 200 ng for human genomic DNA) is suspended in a buffer comprising: 10 mM Tris (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 200 μM dNTPs, 50 pmol of each primer, and 1 unit Taq polymerase per primer set in a total volume of 50 μl. Preferably, amplification is performed under the same conditions that were used to design the primers. In some embodiments, amplification is performed on a conventional thermal cycler for 30 cycles, wherein each cycle is: 1 minute @ 95° C., 58° C. for 1 minute, 72° C. for 1 minute. Final extension is performed at 72° C. for 5 minutes. When the primers have a GC clamp, however, conditions often favor an amplification reaction having over 40 cycles, wherein each cycle is: 35 seconds @ 95° C., 120 seconds @ 50-57° C., and 60 seconds+3 seconds/cycle @ 72° C.
If the subject being tested has at least one disorder that is detected by the assay then two populations of extension products are generated, a first population that corresponds to the standard DNA and a second population that corresponds to the subject's DNA having at least one mutation or polymorphism. The pool of extension products are desirably isolated from the amplification reactants, as above, and are suspended in a non-denaturing loading buffer. Preferably, the extension products are then denatured by heat (e.g., 95° C. for 5 minutes), and are allowed to anneal by cooling (e.g., ice for 5 minutes) prior to loading on the TTGE denaturing gel or DHPLC column. In this manner, the formation of heteroduplexes will be favored if the subject has a mutation or polymorphism because the two populations of extension products are not perfectly complementary. However, the isolation and denaturing/annealing steps are not necessary for some embodiments.
By another approach, the DNA standard is added to the extension products generated from the tested subject's DNA after the amplification reaction. As above, the pooled DNA sample is preferably denatured by heat (e.g., 95° C. for 5 minutes), and allowed to anneal by cooling (e.g., ice for 5 minutes). This second approach also produces heteroduplexes if the extension product and the DNA standard are not perfectly complementary.
Next, the TTGE denaturing gel or DHPLC column is loaded and the extension products are separated on the basis of melting behavior, as described above. Since heteroduplexes are less stable than homoduplexes and have a lower melting temperature, the presence or absence of a mutation or polymorphism in the tested DNA sample is easily determined. By comparing the migration behavior or elution behavior of the extension products generated from the screened DNA with the migration behavior of the DNA standard, a user can rapidly determine the presence or absence of a mutation or polymorphism (e.g., two additional bands that correspond to the single extension product will appear on the gel when a mutation or polymorphism is present in the tested DNA or a population of extension products will elute from the DHPLC column earlier than homoduplex controls or the majority of homoduplexes present in the sample). The section below describes a method of genetic analysis, wherein improved efficiency and sensitivity of detection was obtained by screening multiple DNA samples in the same assay.
Improved Sensitivity was Obtained when Multiple DNA Samples were Screened in the Same Assay
It was also discovered that an improved sensitivity of detection and increased throughput could be obtained by mixing DNA from a plurality of subjects prior to amplification. Because the frequency of mutations or polymorphisms for most disorders are very low in the population, most of the extension products generated correspond to wild-type or non-polymorphic DNA. Accordingly, most of the DNA in a reaction comprising DNA from a plurality of subjects behave similar to a DNA standard. That is, the predominant structure formed upon annealing after denaturation is a homoduplex, which can be rapidly distinguished from any heteroduplex that would appear if a subject were to have a mutation or polymorphism. Although the reaction is “dirty” from the perspective that the identity of each subject's DNA is not known initially, the identity of any polymorphic or mutant DNA can be determined through a process of elimination. For example, by repeating the analysis with smaller and smaller pools of samples, one can identify the individual(s) in the pool that have the mutation or polymorphism. Additionally, DNA standards can be used, as described above, to facilitate identification of the individual(s) having the mutation or polymorphism. Optionally, the each DNA can be labeled with a different fluorescent label so that identification of the variant is easily determined.
By one approach, DNA from a plurality of subjects to be tested is obtained by conventional methods, pooled, and hybridized with the desired nucleic acid primers. Accordingly, optimal primers are designed and selected and approximately 25 ng-500 ng of template DNA (preferably, 200 ng for human genomic DNA) is suspended in a buffer comprising: 10 mM Tris (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 200 μM dNTPs, 50 pmol of each primer, and 1 unit Taq polymerase per primer set in a total volume of 50 μl. Preferably, amplification is performed under the same conditions that were used to design the primers. In some embodiments, amplification is performed on a conventional thermal cycler for 30 cycles, wherein each cycle is: 1 minute @ 95° C., 58° C. for 1 minute, 72° C. for 1 minute. Final extension is performed at 72° C. for 5 minutes. When the primers have a GC clamp, however, conditions often favor an amplification reaction having over 40 cycles, wherein each cycle is: 35 seconds @ 95° C., 120 seconds @ 50-57° C., and 60 seconds+3 seconds/cycle @ 72° C.
The pool of extension products are preferably isolated from the amplification reactants, as above, and are suspended in a non-denaturing loading buffer. Preferably, the extension products are then denatured by heat (e.g., 95° C. for 5 minutes), and are allowed to anneal by cooling (e.g., ice for 5 minutes). In this manner, the formation of heteroduplexes will be favored if the subject has a mutation or polymorphism because the two types of extension products are not perfectly complementary. Again, the isolation and denaturing/annealing steps are not performed in some embodiments and fluorescent labels can be employed.
Next, the TTGE denaturing gel or DHPLC column is loaded and the extension products are separated on the basis of melting behavior, as described above. When one of the subjects being tested has at least one trait that is detected by the screen, heteroduplexes are detected on the gel or eluting from the DHPLC column. The assay can be then repeated with smaller pools of samples and assays with a DNA standard can be conducted with individual samples to confirm the identity of the subject having the mutation or polymorphism. EXAMPLE 5 describes an experiment that verified that an improved sensitivity can be obtained by mixing a plurality of DNA samples. EXAMPLE 6 describes an experiment that verified that multiple genes and multiple loci therein can be screened in a plurality of subjects, in a single assay. EXAMPLE 7 describes the screening of multiple genes and multiple loci therein, in a plurality of subjects, in a single assay using a DHPLC approach. The section below describes the optimization of primer design in the context of an approach that was used to detect mutations and/or polymorphisms in the CFTR gene.
Optimization of Primer Design and Extension Product Design Facilitates Identification of Genetic Markers Associated with HNPCC
Using the approaches detailed in the previous sections, a preferred embodiment concerns the identification of the presence or absence of genetic markers, mutations, or polymorphisms that are associated with HNPCC. The sequences of genes associated with HNPCC can be found in U.S. Pat. Nos. 5,922,855; 6,165,713; 6,191,268; 6,538,108 and U.S. patent application Ser. Nos. 08/209,521 and 08/154,792, all of which are hereby expressly incorporated by reference in their entireties.
By one approach, almost the entire coding sequences for the mismatch repair genes mutL homolog 1 (MLH1) and mutS homologue 2 (MSH2) are scanned for the presence or absence of genetic markers, mutations, or polymorphisms that contribute to HNPCC. (See EXAMPLE 8). TABLE A provides the sequences of exons of the MLH1 and MSH2 genes and several oligonucleotide primers that have been used to screen regions of these genes for the presence or absence of genetic markers, polymorphisms, and mutations that are associated with HNPCC. Where indicated, the notation (*) refers to a GC clamp, an additional non-genetic GC rich sequence that is added to one of the two primers in a pair to add stability to the PCR product, as explained above and in Example 2 below. TABLE B also lists many oligonucleotide primers that have been used to screen regions of the MLH1 and MSH2 genes for the presence or absence of genetic markers, polymorphisms, and mutations that are associated with HNPCC. TABLE B also shows the starting and ending point for each primer as it relates to the publicly available gene sequence for the MLH1 and MSH2 genes (GenBank Accession NoS. AY217549 and NM000251, the contents of which are expressly incorporated by reference in its entirety). It is contemplated that primers that are any number between 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides upstream or downstream of the primers identified in TABLE A or B can be used with embodiments of the invention so long as these primers produce extension products that melt over long stretches of DNA (approximately 25, 50, 75, 100, 125, or 150 nucleotides) at approximately the same temperature (within 0° C.-1.5° C.) and are resolvable on a TTGE gel or DHPLC column.
As detailed above, the sequences of the MLH1 and MSH2 genes are readily available. Accordingly, embodiments include methods of diagnosing HNPCC with primers that are any number from 1-75 nucleotides upstream or down stream from the beginning or ending of the primers listed in TABLE A or B, preferably using the approaches described herein. It is also preferred that said methods use primers that produce extension products that melt over long stretches of DNA (approximately 25, 50, 75, 100, 125, or 150 nucleotides) at approximately the same temperature (within 0° C.-1.5° C.) and are resolvable on a TTGE gel or DHPLC column. Preferably, these extension products are obtained, grouped, and separated as described below.
By one approach, samples of DNA were obtained from several subjects to be screened using the approaches described herein and were disposed in a plurality of 96-well micro-titer plates such that a single row of each plate corresponded to a single tested subject. In some cases, 7 total plates were used per assay, wherein each plate has 7 sample lanes (i.e., 7 subjects analyzed) and an eighth lane was used for positive control sample DNA. Amplification buffer, amplification enzyme (e.g., Taq polymerase), and DNTPs were added to the sample DNA in each well, as described above, and a plurality of primer sets that encompass most of the gene (e.g., 84 primer sets) were to yield a final volume of 10 μl. The primer sets that were employed in a first set of tests are identified in TABLE A. TABLE C describes the plate setup for these amplification reactions as well as a protocol for PCR reactions, whereas TABLE D describes the conditions for the TTGE separation for these tests and describes the groupings for the various fragments for TTGE separation. Preferred methods of diagnosing HNPCC employ the primers of TABLE A to generate extension products that are grouped according to TABLE D and separated by melting behavior (e.g., TTGE). By using this approach, a rapid, inexpensive, and efficient diagnosis of the presence or absence of a marker associated with HNPCC can be ascertained. The names of the extension products, “fragments” in TABLE C and TABLE D correspond to the names of the primer sets used throughout. The top line numbering on the master plate chart of TABLE C refers to the location of the well on the 96 well plate, the “MLH stack” or “MSH stack” of TABLE D refers to the grouping pool of the extension products prior to TTGE and the alternating shaded and unshaded sections of TABLE D show grouping pools of extension products that can be run under the same TTGE conditions (which are shown under “Run group”).
Although multiplex PCR reactions can be employed, preferably, each primer set is run in an individual reaction. Conditions for PCR were, in one case for example: 5 minutes at 96° C. for initial denaturing followed by 35 total cycles of: 30 seconds at 94° C. and 30 seconds at the annealing temperature or at a gradient of 49° C. to 63° C. and a final 10 minutes at 72° C. to complete synthesis of any partial products. Most preferred are primers that have an annealing temperature between 49° C. and 63° C., though many of the primer sets have annealing temperatures that are at 49° C., 52° C., 59° C., and 62.4° C. An approximately 3° C. window is allowed for each plate (e.g., primers having annealing temperatures that are within 3° C. of one another are grouped on a single plate). Programs such as WINMELT were used to determine whether the primers could be grouped into various primer sets that have similar annealing temperatures so that individual groups of primers can be amplified by Polymerase Chain Reaction (PCR) on the same plate.
Once the extension products had been generated they were grouped, pooled, and mixed with loading dye. Eight Multi G groups (Multi-Grouping pools of extension products) were used for the extension products “fragments” generated by the various primer sets, which belong to one of the eight groups are identified in TABLE C and TABLE D (some of the run groups on the separate MLH1 and MSH2 Table have identical conditions). After grouping and pooling, the samples were loaded onto a TTGE gel. TABLE D also lists the start and stop temperatures for the TTGE, for each Multi G group, under ‘run conditions’. Preferably, the TTGE is run with a very shallow temperature gradient, e.g., about 1.0° C./hour for a total of three hours, at high voltage, e.g., 150 volts. Once the separation was complete, the gels were grouped, stained with ethidum bromide, and analyzed by the Decode system. The analysis above was rapid, inexpensive, and very effective at detecting mutations and/or polymorphisms, many of which go undetected or are not analyzed by others in the field.
Whereas many in the field seek to design primers that optimally anneal with a template DNA, it has been discovered that primers can also be designed to produce an optimal extension product (e.g., a fragment of short length with a reliable and rapid melting point). Preferably, primers are designed to generate extension products that are approximately 100-300 nucleotides in length and that have long stretches of DNA that melt at approximately the same temperature (e.g., DNA stretches that are 25, 35, 45, 55, 65, 75, 85, 95, 100, 125, 15, 175, or 200 nucleotides that melt at the same temperature or within about a 0° C. to about a 1.5° C. temperature difference).
Programs such as WINMELT were used to evaluate the melting behavior of extension products generated from the various primer sets and test TTGE separation of the extension products generated by the various primer sets were also performed to ensure that the predicted melting behavior was represented on the gel. For example,
By analogy, TABLE D shows several of the characteristics of the extension products generated by the primers described herein. In particular, the PCR annealing temperature for the primer set used to generate the extension product (“PCR temp.”) is provided. Further, the Multi
G/stack group is also listed. The following examples describe the foregoing methodologies in greater detail. The first example describes an approach that was used to isolate DNA from human blood.
A sample of blood was obtained from a subject to be tested by phlebotomy. A portion of the sample (e.g., approximately 1.0 ml) was added to approximately three times the sample volume or 3.0 ml of a lysis solution (10 mM KHCO3, 155 mM NH4Cl, 0.1 mM EDTA) and was mixed gently. The lysis solution and blood were allowed to react for approximately five minutes. Next, the sample was' centrifuged (×500 g) for approximately 2 minutes and the supernatant was removed. Some of the supernatant was left (e.g., on the walls of the vessel) to facilitate suspension. The pellet was then vortexed for approximately 5-10 seconds. An extraction solution, which contains chaotrope and detergent (Qiagen), was then added (e.g., 500 μl), the sample was vortexed again for approximately 5-10 seconds, and the solution was allowed to react for five minutes at room temperature.
Next, a GFX column, which are pre-packed columns containing a glass fiber matrix, was placed under vacuum (e.g., a Microplex 24 vacuum system) and the extracted solution containing the DNA was transferred to the column (e.g., in 500 μl aliquots). Once all of the sample has been passed through the column, the vacuum was allowed to continue for approximately 5 minutes. Subsequently, a wash solution (Tris-EDTA buffer in 80% ethanol) was added (e.g., approximately 500 μl) under vacuum. Once the wash solution had been drained from the column, the vacuum was allowed to continue for approximately 15 minutes. The GFX columns containing the DNA were then placed into sterile microfuge tubes but the lids were kept open.
Elution buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) was then added to the column (e.g., approximately 100 μl of buffer that was heated to approximately 70° C.) and the buffer was allowed to react with the column for approximately 2 minutes. Then, the tubes containing the columns were centrifuged at ×5000 g for approximately 1.5 minutes. After centrifugation, the column was discarded and the microfuge tube containing the isolated DNA was stored at −20° C. The example below describes the design and optimization of primers that allowed for the inventive high-throughput multiplex PCR technique, described herein.
Sets of primers for PCR amplification were designed for every exon of the following genes: Cystic Fibrosis Transmembrane Reductase (CFTR), Beta-hexosaminidase alpha chain (HEXA), PAH, Alpha globin-2 (HBA2), Beta globin (BBB), Glucocerebrosidase (GBA), Galactose-1-phosphae uridyl transferase (GALT), Medium chain acyl-CoA dehydrogenase (MCAD), Protease inhibitor 1 (PI), Factor VIII, FMR1, and Aspartoacylase (ASPA). The primers were designed from sequence information that was available from GenBank or from sequence information obtained from Ambry Genetics Corporation. Information regarding mutations or polymorphisms was obtained from The Human Gene Mutation Database.
One of the primers in each primer set contained a GC-clamp. It was discovered that the addition of a GC-clamp significantly altered the melting profile of the DNA extension product. Further, proper positioning of the GC-clamp served to level the melting profile. It was desired to position the GC-clamp so that a single melting domain across the fragment was created. Proper positioning of the GC-clamp was oftentimes needed to prevent the GC-clamp from masking the presence of a mutation or polymorphism (e.g., if the mutation or polymorphism is too close to the GC-clamp). Software was also used to optimize primer design. For example, many primers were designed with the aid of Primer Premiere 4.0 and 5.0 and appropriate positioning of the GC-clamps was determined using WinMelt software from BioRad. To maintain sensitivity of the test, the primers were designed to anneal at a minimum of 40 base pairs either upstream or downstream of the nearest known mutation in the intronic region of the genes.
Although multiplex PCR can be technically difficult when using the quantity of primers described herein, it was discovered that almost all of the PCR artifacts disappeared when salt concentration, temperature, primer selection, and primer concentration were carefully optimized. Optimization was determined for each primer set alone and in combination with other primer sets. Optimization experiments were conducted using Master Mix from Qiagen and a Thermocyler from MJ Research. The conditions for thermal cycling were 5 minutes @ 95° C. for the initial denaturation, then 30 cycles of: 30 seconds @ 94° C., 45 seconds @ 48-68° C., and 1 minute @ 72° C. A final extension was performed at 72° C. for 10 minutes.
In addition to primer compatibility, primers were selected to facilitate identification of extension products by electrophoresis. To optimize primer design in this regard, separate PCR reactions were conducted for each individual set of primers and the extension products were separated by the inventive DNA separation technique, described above. Identical parameters were maintained for each assay and the migration behavior for each extension product was analyzed (e.g., compared to a standard to determine a Rf value for each fragment). An Rf value is a unit less value that characterizes a fragment's mobility relative to a standard under set conditions. In many primer optimization experiments, for example, the generated extension products were compared to a standard extension product obtained from amplification of the first exon of the PAH (phenylalanine hydroxylase) gene. A measurement of the distance of migration of each band in comparison to the distance of migration of the first exon of PAH was recorded and the Rf value was calculated according to the following:
By conducting these experiments, it was verified that the selected primers did not produce extension products that overlapped on the gel. Optimal primer selection was obtained when optimal PCR parameters were maintained and the extension products produced dissimilar Rf values. Finally, the multiplex PCR was tested with all sets of primers and it was verified that few artifacts were created during amplification. Embodiments of the invention include the primers provided in the Tables and sequence listing provided herein and methods of using said primers and/or groups of primers. The example below describes an experiment that verified that the embodiments described herein effectively screen multiple loci present on a plurality of genes in a single assay.
Two independent PCR reactions were conducted to demonstrate that multiple loci on a plurality of genes can be screened in a single assay using an embodiment of the invention. In a first reaction, seven different loci from four different genes were screened and, in the second reaction, eight different loci from four different genes were screened. The primers used in each multiplex reaction are provided in Table 1.
The amplification was carried out in 25 μl reactions using a 2× Hot Start Master Mix, which contains Hot Start Taq DNA Polymerase, and a final concentration of 1.5 mM MgCl2 and 200 μM of each dNTP (commercially available from Qiagen). In each reaction, 12.50 of Hot Start Master Mix was mixed with 1 of μlgenomic DNA (approximately 200 ng genomic DNA), which was purified from blood using a commercially available blood purification kit (Pharmacia or Amersham). Primers were then added to the mixture (0.5 μM final concentration of each primer). Then, ddH2O was added to bring the final volume to 25 μl.
Thermal cycling for the Multiplex #1 reaction was performed using the following parameters: 15 minutes @ 95° C. for 1 cycle; 30 seconds @ 94° C., 1 minute (4) 53° C., 1 minute and 30 seconds (4) 72° C. for 35 cycles; and 10 minutes @ 72° C. for 1 cycle. Thermal cycling for the Multiplex #2 reaction was performed using the following parameters: 15 minutes (4) 95° C. for 1 cycle; 30 seconds @ 94° C., 1 minute @49° C., 1 minute and 30 seconds @ 72° C. for 35 cycles; and 10 minutes @ 72° C. for 1 cycle.
After the amplification was finished, approximately 5 μl of each PCR product was mixed with 5 μlof non-denaturing gel loading dye (70% glycerol, 0.05% bromophenol blue, 0.05% xylene cyanol, 2 mM EDTA). The DNA in the two reactions was then separated on the basis of melting behavior on separate denaturing gels. Each gel was a 16×16 cm, 1 mm thick, 7M urea, 8% acrylamide/bis(37.5:1) gel composed in 1.25×TAE (50 mM Tris, 25 mM acetic acid, 1.25 mM EDTA). Separation was conducted for 4 hours at 150 V on the Dcode system (BioRad) and the temperature ranged from 51° C. to 63° C. with a temperature ramp rate of 3° C./hour. Subsequently, the gels were stained in 1 μg/ml ethidium bromide in 1.25×TAE for 3 minutes and destained in 1.25×TAE buffer for 20 minutes. The gels were then photographed using the Gel Doc 1000 system from BioRad.
The primers in Table 1 were selected and manufactured because they produced extension products with very different Rf values and the extension products were clearly resolved by separation on the basis of melting behavior. Although some bands were more visible than others on the gel, seven distinct bands were observed on the gel loaded with extension products generated from the Multiplex 1 reaction and eight distinct bands were observed on the gel loaded with extension products generated from the Multiplex 2 reaction. These results verified that the described method effectively screened multiple loci on a plurality of genes in a single assay. The example below describes another experiment that verified that the embodiments described herein can be used to effectively screen multiple loci present on a plurality of genes in a single assay.
Experiments were conducted to differentiate extension products generated from wild-type DNA and extension products generated from mutant DNA. Samples of genomic DNA that had been previously identified to contain mutations or polymorphisms were purchased from Coriell Cell Repositories. The mutation or polymorphism that was analyzed in this experiment was the delta-F508 mutation of the CFTR gene. This mutation is a 3 by deletion in exon 10 of the CFTR gene. Other loci analyzed in these experiments included the Fragile X gene, exon 17; Fragile X gene, exon 3; Factor VIII gene exon 2; and the Factor VIII gene, exon 7. Both the known mutant and a control wild-type for CFTR exon 10 were amplified within a multiplex reaction and individually. PCR amplification was conducted as described in EXAMPLE 3; however, 0.25 μM (final concentration) of each primer was used. The primers used in these experiments were CFTR 10 (SEQ. ID. Nos. 294 and 312), FragX 17 (SEQ. ID. Nos. 305 and 323), FragX 3 (SEQ. ID. Nos. 304 and 322), Factor VIII 7 (SEQ. ID. Nos. 301 and 319) and Factor VIII 2 (SEQ. ID. Nos. 298 and 316). The numbers following the abbreviations represent the exons probed.
The DNA templates that were analyzed included known wild-type genomic DNA, known mutant genomic DNA, mixed wild-type genomic DNA from various subjects, and mixed wild-type and mutant genomic DNA. Approximately 200 ng of genomic DNA was added to each reaction. The mixed wild-type and mutant DNA sample had approximately 100 ng of each DNA type. Thermal cycling was carried out with a 15-minute. step at 95° C. to activate the Hot Start Polymerase, followed by 30 cycles of 30 seconds at @ 94 C, 1 minute at @ 53° C., 1 minute and 30 seconds at @ 72° C.; and 72° C. for 10 minutes.
After amplification, approximately 5 μl of the PCR product was mixed with 5 μl of non-denaturing gel loading dye (70% glycerol, 0.05% bromophenol blue, 0.05% xylene cyanol, 2 mM EDTA). The samples were then separated on a 16×16 cm, 1 mm thick, 6M urea, 6% acrylamide/bis (37.5:1) gel in 1.25×TAE (50 mM Tris, 25 mM acetic acid, 1.25 mM EDTA) for 5 hours at 130 V using the Dcode system (BioRad). The temperature ranged from 40° C. to 50° C. at a temperature ramp rate of 2° C./hour. The gels were then stained in 1 μg/ml ethidium bromide in 1.25×TAE for 3 minutes and destained in 1.25×TAE buffer for 20 minutes. The gels were then photographed using the Gel Doc 1000 system from BioRad.
The resulting gel revealed that the lane containing the extension products generated from the wild-type DNA using the CFTR10 primers had a mobility commensurate to the wild-type DNA standard, as did the extension products generated from the other primers and the wild-type DNA. That is, a single band appeared on the gel in these lanes. The lane containing the extension products generated from the template having the F508 mutant, on the other hand, showed 2 bands. One of the bands had the same mobility as the extension products generated from the wild-type or DNA standard and the other band migrated slightly faster. These two populations of bands represent the two populations of homoduplexes (i.e., wild-type/wild-type and mutant/mutant). The top band is the wild-type homoduplex and the lower band is the mutant F508 homoduplex. Similarly, the lane that contained the wild-type/mutant DNA mix exhibited two populations of extension products, one representing the wild-type homoduplex population and the other representing the mutant homoduplex. Since F508 is a 3 by deletion it failed to form heteroduplex bands in sufficient quantity to be visible on the gel. Thus, this experiment demonstrated that the described method effectively screened multiple genes, in a single assay, and detected the presence of a polymorphism in one of the screened genes. The example below describes an experiment that demonstrated that an improved sensitivity can be obtained by mixing a plurality of DNA samples.
This example describes two experiments that verified that an improved sensitivity of detection can be obtained by (1) mixing the DNA samples from a plurality of subjects prior to amplification or by (2) mixing amplification products before separation on the basis of melting behavior. In these experiments, PCR amplifications of exon 9 of the GBA gene (Glucocerebrosidase gene) were used. DNA samples known to contain a mutation in exon 9 of the GBA gene were purchased from Coriell Cell Repositories. These DNA samples contain a homozygous mutation in exon 9 of the GBA gene (the N370S mutation).
In a first experiment, single amplification of exon 9 was performed in a 25 μl reaction. A Taq PCR Master Mix (containing Taq DNA Polymerase and a final concentration of 1.5 mM MgCl2 and 200 μM dNTPs)(Qiagen) was mixed with 0.5 μM (final concentration) of primers (SEQ. ID. Nos. 309 and 327). The template genomic DNAs analyzed in this experiment included wild-type DNA, mutant DNA, and various mixtures of wild-type and mutant DNA. For the single non-mixed reactions, approximately 200 ng of genomic DNA was used for amplification. In the mixed samples, approximately 200 ng of DNA was again used, however, the percentage of wild-type to mutant genomic DNA varied. Thermal cycling was performed according to the following parameters: 10 minutes @ 94° C.; 30 cycles of 30 seconds @ 94° C., 1 minute@ 44.5° C., and 1 minute and 30 seconds @ 72° C.; and 10 minutes @ 72° C.
In the second experiment, the amplification products were mixed prior to separation on the basis of melting behavior. Amplification of both wild-type and mutant (N370S) exon 9 of the GBA gene was performed using 25 μl reactions, as before. The Taq Master Mix obtained from Qiagen was mixed with 200 ng of genomic DNA and 0.5 μM final concentration of both primers (SEQ. ID. Nos. 309 and 327). PCR was carried out for 30 cycles with an annealing temperature of 56° C. for 1 minute. The denaturation and elongation steps were 94° C. for 30 seconds and 72° C. for 1 minute and 30 seconds. Final elongation was carried out at 72° C. for 10 minutes. The extension products obtained from the single amplification of wild-type GBA exon 9 was then mixed with the extension products obtained from the single amplification of the mutant GBA exon 9. Next, the pooled DNA was subjected to denaturation at 95° C. for 10 minutes and cooled on ice for 5 minutes, then heated to 65° C. for 5 minutes and cooled to 4° C. This denaturation and annealing procedure was performed to facilitate the formation of heteroduplex DNA.
Once the extension products from both experiments were in hand, approximately 5 μl of both the prior to PCR mixture and post PCR mixture were loaded on 16×16 cm, 1 mm thick gels (7M Urea/8% acrylamide (37.5:1) gel in 1.25×TAE) using the gel loading dye and the Dcode system (BioRad), described above. The DNA on the gel was then separated at 150 V for 5 hours and the temperature was uniformly raised 2° C./hour throughout the run starting at 50° C. and ending at 60° C.
The gel was stained in 1 μg/ml ethidium bromide in 1.25×TAE buffer for 3 minutes and destained in buffer for 20 minutes.
It should be noted that the GBA gene has a pseudo gene, which was co-amplified by the procedure above. An extension product generated from this psuedo gene migrated slightly faster than the extension product generated from the true expressed gene on the gel. In all lanes, the band representing the extension product generated from the psuedo gene was present. Then next fastest band on the gel was the extension product generated from the GBA exon 9 wild-type allele. The extension product generated from the mutant GBA exon 9 allele comigrated with the wild-type allele and was virtually indistinguishable on the basis of melting behavior due to the single base difference.
The heteroduplexes formed in the mixed samples were easily differentiated from the homoduplexes. The samples mixed prior to PCR showed both homoduplexes (wild-type and mutant) along with heteroduplexes, which appeared higher on the gel. Thus, by mixing samples, either prior to amplification or prior to separation on the basis of melting behavior an improved sensitivity of detection was obtained. Since homoduplex bands no longer need to be resolved to identify a mutation or polymorphism, only the heteroduplex bands need to be resolved, the throughput of diagnostic analysis was greatly improved. The example below describes experiments that verified that the embodiments taught herein can be used to effectively screen multiple genes in a plurality of subjects, in a single assay, for the presence or absence of a polymorphism or mutation.
Two experiments were conducted to verify that multiple genes from a plurality of subjects can be screened in a single assay for the presence or absence of a genetic marker (e.g. a polymorphism or mutation) that is indicative of disease. These experiments also demonstrated that an improved sensitivity of detection could be obtained by mixing DNA samples either prior to generation of extension products or prior to separation on the basis of melting behavior.
In both experiments, five different extension products were generated from three different genes in a single reaction vessel. The five different extension products were generated using the following primers: Factor VIII 1 (SEQ. ID. Nos. 297 and 315); GBA 9 (SEQ. ID. Nos. 309 and 327); GBA 11 (SEQ. ID. Nos. 332 and 333); GALT 5 (SEQ. ID. Nos. 334 and 335), and GALT 8 (SEQ. ID. Nos. 336 and 337). Abbreviations are: Glucocerebrosidase (GBA) and Galactose-1-phosphate uridyl transferase (GALT). The numbers following the abbreviations represent the exons probed.
Extension products were generated for each experiment in 25:1 amplification reactions using Qiagen's 2× Hot Start Master Mix (Contains Hot Start Taq DNA Polymerase, and a final concentration of 1.5 mM MgCl2 and 200 :M of each dNTP). To each reaction, 12.5 μl of Hot Start Master Mix was added to 1 μl of genomic DNA (approximately 200 ng genomic DNA for the mutant DNA sample and the wild-type DNA sample), which was purified from human blood using Pharmacia Amersham Blood purification kits. For the experiment in which the DNA samples from a plurality of subjects were mixed prior to generation of the extension products, approximately 100 ng of wild-type genomic DNA was mixed with approximately 100 ng of mutant N370S genomic DNA. In both experiments, primers were added to achieve a final concentration of 0.5 :M for each primer and a final volume of 25 μl was obtained by adjusting the volume with ddH2O.
Thermal cycling for both experiments was performed using the following parameters: 15 minutes @ 95° C. for 1 cycle; 30 seconds @ 94° C., one minute @ 57° C., and one minute 30 seconds @ 72° C. for 35 cycles; and 10 minutes @ 72° C. for 1 cycle. After amplification, the extension products generated from the wild-type and mutant templates (the un-mixed samples) were separated from the PCR reactants using a PCR Clean Up kit (Qiagen). Then, approximately 10 μL of the wild-type and mutant DNA were removed from each tube and gently mixed in a single reaction vessel. This preparation was then denatured at 95° C. for 1 minute and rapidly cooled to 4° C. for 5 minutes. Finally, the preparation was brought to 65° C. for an additional 1.5 minutes. The extension products generated from the mixed sample (wild-type DNA and mutant DNA mixed prior to amplification) were stored until loaded onto a denaturing gel.
Next, approximately 10 μl of the unmixed sample was combined with 10 μl of loading dye and approximately 5:1 of the mixed sample was combined with 5:1 of loading dye. The loading dye was composed of 70% glycerol, 0.05% bromophenol blue, 0.05% xylene cyanol, and 2 mM EDTA). The samples in loading dye were then loaded on separate 16×16 cm, 1 min thick, 7M urea, 8% acrylamide/bis(37.5:1) gels in 1.25×TAE (50 mM Tris, 25 mM acetic acid, 1.25 mM EDTA). The DNA was separated on the basis of melting behavior for 5 hours at 150 V on the Dcode system (BioRad). The temperature ranged from 56° C. to 68° C. at a temperature ramp rate of 2° C./hr. The gels were then stained in 1 μg/ml ethidium bromide in 1.25×TAE for 3 minutes and destained in 1.25×TAE buffer for 20 minutes. The gels were photographed using the Gel Doc 1000 system (BioRad).
In all lanes of the gel, 5 extension products generated from three different genes were visible in the following order from top to bottom: Factor VIII 1, GBA 9, GBA 11, GALT 8, and GALT 5. Two different extension products were generated from the GBA 9 primers, as described above. The less intense band below the homoduplex bands corresponded to an extension product generated from the pseudogene. In the lanes loaded with extension products generated from only the wild-type or mutant DNA template, it was difficult to distinguish the wild type homoduplex from the N370S mutant homoduplex. In the lane loaded with the extension products generated from the mixed DNA templates (wild-type and mutant DNA mixed prior to amplification) and the lane loaded with extension products (generated from wild type and mutant DNA separately) that were mixed after amplification, heteroduplex bands were easily visualized. These experiments verified that multiple genes can be screened in a plurality of individuals in a single assay and that a single nucleotide mutation or polymorphism can be detected. Further, these experiments demonstrate that screening a plurality of DNA samples in a single reaction vessel or adding a control DNA before or after amplification greatly improves the sensitivity of detection. By practicing the methods taught in this example, the throughput of diagnostic screening can be drastically improved and the cost of identifying genetic traits can be significantly reduced. The example below describes approaches to screen multiple genes in a plurality of subjects, in a single assay, for the presence or absence of a polymorphism or mutation using DHPLC.
Multiple genes in a plurality of subjects, in a single assay, can be screened for the presence or absence of a polymorphism or mutation using a DHPLC separation approach. For example, five different extension products can be generated using the following primers: Factor VIII 1 (SEQ. ID. Nos. 297 and 315); GBA 9 (SEQ. ID. Nos. 309 and 327); GBA 11 (SEQ. ID. Nos. 332 and 333); GALT 5 (SEQ. ID. Nos. 334 and 335), and GALT 8 (SEQ. ID. Nos. 336 and 337). Abbreviations are: Glucocerebrosidase (GBA) and Galactose-1-phosphate uridyl transferase (GALT). The numbers following the abbreviations represent the exons probed. The extension products can be generated in 25:1 amplification reactions using Qiagen's 2× Hot Start Master Mix (Contains Hot Start Taq DNA Polymerase, and a final concentration of 1.5 mM MgCl2 and 200 μM of each dNTP).
To each reaction, 12.5 μl of Hot Start Master Mix is added to 1 μl of genomic DNA (approximately 200 ng genomic DNA for the mutant DNA sample and the wild-type DNA sample), which is purified from human blood using Pharmacia Amersham Blood purification kits. By another approach, the DNA samples from a plurality of subjects can be mixed prior to generation of the extension products. In this case, approximately 100 ng of wild-type genomic DNA is mixed with approximately 100 ng of mutant N370S genomic DNA. Primers are added to achieve a final concentration of 0.5 μM for each primer and a final volume of 25 μl is obtained by adjusting the volume with ddH2O.
Thermal cycling is performed using the following parameters: 15 minutes @ 95° C. for 1 cycle; 30 seconds @ 94° C., one minute @ 57° C., and one minute 30 seconds @ 72° C. for 35 cycles; and 10 minutes @ 72° C. for 1 cycle. After amplification, the extension products generated from the wild-type and mutant templates (if un-mixed samples) are separated from the PCR reactants using a PCR Clean Up kit (Qiagen). Then, approximately 10 :L of the wild-type and mutant DNA are removed from each tube and gently mixed in a single reaction vessel. This preparation is then denatured at 95° C. for 1 minute and rapidly cooled to 4° C. for 5 minutes. Finally, the preparation is brought to 65° C. for an additional 1.5 minutes. The extension products generated from the mixed sample (wild-type DNA and mutant DNA mixed prior to amplification) can be stored until loaded onto a DHPLC column.
Next, the extension products are loaded on to a 50×4.6 mm ion pair reverse phase HPLC column that is equilibrated in degassed Buffer A (0.1 M triethylamine acetate (TEAA) pH 7.0) at 56° C. A linear gradient of 40%-50% of degassed Buffer B (0.1 M triethylamine acetate (TEAA) pH 7.0 and 25% acetonitrile) is then performed over 2.5 minutes at a flow rate of 0.9 ml/min at 56° C., followed by a linear gradient of 50%-55.3% Buffer B over 0.5 minutes, and finally a linear gradient of 55.3%-61% Buffer B over 4 minutes. U.V. absorption is monitored at 260 nm, recorded and plotted against retention time.
When the loaded sample is un-mixed extension products, the extension products generated from only the wild-type or mutant DNA template, it is difficult to distinguish the wild type homoduplex from the N370S mutant homoduplex. When the loaded sample is the mixed extension products, the extension products generated from the mixed DNA templates (wild-type and mutant DNA mixed prior to amplification), or the extension products (generated from wild type and mutant DNA separately) that were mixed after amplification, heteroduplex elution behavior is detected. By practicing the methods taught in this example, the throughput of diagnostic screening can be drastically improved and the cost of identifying genetic traits can be significantly reduced. The example below describes an approach that was used to diagnostically screen patient samples for the presence or absence of polymorphisms or mutations on genes associated with HNPCC.
Sets of primers for PCR amplification were designed for every exon of the MLH1 and MSH2 genes. The primers were designed from sequence information that was available from GenBank or from sequence information obtained from Ambry Genetics Corporation. Information regarding mutations or polymorphisms was obtained from The Human Gene Mutation Database.
Primer sets and PCR stacking groups were designed for optimal sensitivity for TTGE, as described above. DNA from one individual was amplified with each primer set in a separate reaction, then stacked in average groups of three fragments/gel for gel analysis. Preferably, one of the primers in each primer set contained a GC-clamp. It was discovered that the addition of a GC-clamp significantly altered the melting profile of the DNA extension product. Further, proper positioning of the GC-clamp served to level the melting profile. It was desired to position the GC-clamp so that a tight single melting domain across the fragment was created. Proper positioning of the GC-clamp was often times needed to prevent the GC-clamp from masking the presence of a mutation or polymorphism (e.g., if the mutation or polymorphism is too close to the GC-clamp). Software was also used to optimize primer design. For example, many primers were designed with the aid of Pruner Premiere 4.0 and 5.0 and appropriate positioning of the GC-clamps was determined using WinMelt software from BioRad. To maintain sensitivity of the test, the primers were designed to anneal at a minimum of 40 base pairs either upstream or downstream of the nearest known mutation in the intronic region of the genes.
Optimization was determined for each primer set. Optimization experiments were conducted using Hotstart Master Mix from Qiagen and a Thermocyler from MJ Research. Resulting PCR conditions for all fragments were 15 minutes @ 95° C. for the initial denaturation, then 35 cycles of 30 seconds @ 94° C., 30 seconds @ 46-62° C., and 30 seconds @ 72° C. A final extension was performed at 72° C. for 10 minutes. Approximately 15 ul PCR reactions contained 7.5 ul Qiagen 2× Hotstart Master Mix, 50-200 ng genomic DNA, sense and antisense primer for each fragment at a final concentration of 0.5-1 μM. Prior to gel loading and stacking of gel groups PCR samples were heated and re-annealed to provide best heteroduplex formation. PCR product was heated to 95° C. for 5 min, 50° C. for 10 min, then brought to 4° C.
PCR products (approximately 4-8 μl each depending on signal strength) were then assembled for groups of equal melting characteristics and mixed with loading dye consisting of 70% glycerol, 0.05% bromophenol blue, 0.05% xylene cyanol, 2 mM EDTA). DNA was separated on denaturing gels (7 M urea, 8% acrylamide/bis(37.5:1) in 50 mM Tris, 25 mM acetic acid, 1.25 mM EDTA) for 3-5 hours at 125 V or 150 V on the Dcode system. (Biorad). Temperature ranged from 45.5° C. to 64° C. with ramp rates of 1.0-1.5° C./hr depending on gel groups. The gels were stained in 1 :g/ml ethidium bromide in 1.25×TAE for 3 minutes and destained in 1.25×TAE buffer for 20 minutes. The gels were photographed using the Gel Doc 1000 system (BioRad). Table 2 below lists the primers used in this assay. TABLE D shows the TTGE gel grouping (MLH or MSH stacking group) and temperatures used for TTGE separation (under “Run group”). TABLE D also names the extension products generated from the various primer sets employed and the positions of each fragment on the gel after separation (listed in order). Previous experiments, described above, have demonstrated that extension products generated from primers that are any number between 1-75 nucleotides upstream or downstream from the primers listed in TABLE A (e.g., the primer sets listed in Table 2) can be grouped and efficiently separated in accordance with rules set forth herein. Preferably, the primers listed in Table 2 are used to generate extension products that are grouped according to TABLE D and are separated on the basis of melting behavior (e.g., TTGE). In Table 2, the notation “(*)-” indicates the presence of a GC-rich clamp sequence, the sequence of which is given at the bottom of the Table.
In particular embodiments of the invention, primers used to amplify DNA regions from patient samples are labeled with fluorescent tags. Fluorescently tagged primers are used to detect the presence of PCR products without chemical staining as well as the origins of a product when two or more reaction products are mixed and analyzed in the same gel lane.
In this example, fluorescently labeled primers that detect the presence of absence of polymorphisms in the CTFR gene were employed. Exon 10 of the CFTR gene was amplified with a primer set that detects the entire exon using a PCR protocol similar to that of Example 8. PCR was performed as described in Example 8 with a primer set that was modified with Texas Red (primers were obtained from MWG Biotech), and a second primer set that was modified with Oregon Green (also from MWG). Extension products were analyzed on TTGE side by side after being forced into a heteroduplex against themselves or by mixing with a control DNA. The extension products were analyzed on TTGE and the common mutation for deltaF508 and polymorphism M470V was observed.
Results revealed the same banding pattern on TTGE for each individual fragment regardless of the modification state of the primer. Results also indicate the homozygous state of the DNA samples if the samples were mixed with wildtype DNA, which appears as a visually apparent heterozygous banding pattern (
This example demonstrates that the use of fluorescently labeled primers allows one to rapidly identify the presence or absence of polymorphisms in an analyzed gene without staining or autoradiography and to rapidly differentiate the identity of individual extension products that are mixed and segregated on the same lane of a TTGE gel.
In one embodiment of the invention, the techniques described above in Example 8 can be used to screen DNA samples isolated from patient blood samples for mutations associated with HNPCC. In some embodiments of the invention, if a DNA sample generates a positive result in the assay, the existence of one or more mutations associated with HNPCC is confirmed with DNA sequencing of the relevant exons. Table E provides primer pairs to be used for the sequencing of each exon of the MSH2 and MLH1 genes, including first and second choices in some instances. A protocol for PCR-based sequencing reactions using these primers, as well as the primer sequences themselves, are also provided. Using the primers, the primer pairings and the protocol provided, a person with skill in the art is able to sequence any or all of the exons of the MSH2 and MLH1 genes and confirm the existence of HNPCC-related or other mutations in the coding sequences of these genes.
Using a protocol similar to that of Example 8, the HNPCC assay is performed with primers that have been modified with a fluorescent label for visualization on a fluorescent imager. In this Example, the short primer (without the GC clamp sequence) of each primer pair listed in Table 2 is modified by the addition of a fluorescent label such as Texas Red (absorption peak 595 nm, emission peak 615 nm) or Oregon Green (absorption peak 496 nm, emission peak 524 nm) (primers are obtained from MWG Biotech). The GC clamp primer is used in the unmodified form.
Primer sets and PCR stacking groups are designed for optimal sensitivity for TTGE, as described in Example 8. In particular embodiments, DNA from one individual is amplified with each primer set in a separate reaction, then stacked in average groups of three fragments/gel for gel analysis. PCR conditions for all fragments are as follows: 15 minutes @ 95° C. for the initial denaturation, then 35 cycles of: 30 seconds @ 94° C., 30 seconds @ 47-58.5° C., and 30 seconds @ 72° C. A final extension is performed at 72° C. for 10 minutes. The approximately 15 ul PCR reactions contain 7.5 ul Qiagen 2× Hotstart Master Mix, 50-200 ng genomic DNA, sense and antisense primers for each fragment at a final concentration of 0.5-1 uM. Prior to gel loading and stacking of gel groups, PCR samples are heated and re-annealed to provide best heteroduplex formation. Each PCR product is heated to 95° C. for 5 min, 50° C. for 10 min, then brought to 4° C. PCR products (approximately 4-8 μl each depending on signal strength) are then assembled into groups of products with equal melting characteristics and mixed with loading dye consisting of 70% glycerol, 0.05% bromophenol blue, 0.05% xylene cyanol, 2 mM EDTA). DNA is separated on denaturing gels (7 M urea, 8% acrylamide/bis(37.5:1) in 50 mM Tris, 25 mM acetic acid, 1.25 mM EDTA) for 3-5 hours at 125 V or 150 V on the Dcode system. (Biorad). Temperature ranges from 45° C. to 67° C. are used with ramp rates of 1.0-1.5° C./hr, depending on gel groups. The gels are imaged on a fluorescent image, and images are captured in the respective channel. Gels can also be photographed using the Versadoc 1000 system (BioRad).
Resulting images show extension products in the respective channel, e.g. presenting as a red pattern for Texas Red modified primers, and as a green pattern for Oregon Green modified primers.
Moreover, since the labeled extension products fluoresce in different spectra, this method allows for the simultaneous visualization of multiple DNA samples at once. For example, if one sample of primer has been previously amplified with Texas Red modified primers and the another with Oregon Green modified primers. one can multiplex the same extension product from 2 or more different DNA samples at the gel stage of the process.
In a specific embodiment, DNA from one individual is amplified with each primer set in separate reactions, using short primers labeled with the Texas Red fluorescent tag. DNA from another individual is amplified with primer sets labeled with the Oregon Green fluorescent tag. Prior to gel loading and stacking of gel groups, Texas Red tagged extension product and Oregon Green tagged extension product are mixed at equal ratios, and re-annealed to provide heteroduplex formation. Mixed PCR products are heated to 95° C. for 5 min, 50° C. for 10 min, then brought to 4° C.
The PCR products (approximately 4-8 μl of each depending on signal strength) are then assembled into groups of products with equal melting characteristics and mixed with loading dye. DNA is separated on denaturing gels, and gels are imaged on a fluorescent imager. Images for each gel are captured in both channels, after which they are overlayed for viewing of both colors. Whenever the extension products have identical sequence, the banding pattern appears as yellow on the overlay image. If one extension product is missing, the other extension product will be visible (red or green). Moreover, since all products are forced into a heteroduplex, any one homozygous mutation appear as a heterozygous pattern after having been mixed with wildtype sequence. The heterozygous pattern may present as a distinct pattern of 2 yellow, 1 red and 1 green band, or as a compressed yellow pattern of all 4 bands, depending on the specific melting temperature shift of each duplex. Most importantly, this mandatory heteroduplex formation of every fragment in the assay facilitates homozygous detection. This provides an advantage over conventional TTGE, since the homozygous mutations can be the most difficult to resolve on gel. In addition, the cost for analyzing samples is reduced because each gel is loaded with a multiple number of DNA samples.
As noted above, heteroduplexes have one or more mismatched base pairs between the two strands comprising the duplex. Creating heteroduplexes in the TTGE samples permits a greater difference in melting temperatures between PCR products with different sequences than would be seen between homoduplexes differing in sequence by only one or a few bases. Heteroduplex formation assists with the melting temperature (Tm) calculations in various Tm calculating software programs, such as the Bio-Rad Winmelt software. In order to get efficient and sensitive TTGE PCR fragments, it is helpful to have the regions of sensitivity be linear within 0.1° C. Consistent predictions of Tm ranges within that level of specificity are difficult to obtain. By increasing the difference in melting temperature of double stranded PCR products in a sample through the formation of heteroduplexes, the need for precise melting temperature predictions is reduced.
Another aspect of the invention involves the importance of analysis consistencies in the laboratory. In TTGE, SSCP, DGGE, or any other denaturing assay, the primary determinant for the detection of an abnormality is the mobility shift of the fragment. Even if the assay works technically, the shift may be so slight that it is only apparent if it is known that there is a mutation on the input DNA. Mobility shifts should be visually significant in order to be detected every single time. By creating multicolor heteroduplex under denaturing conditions, color change is added to the visual criteria whereby the mutation can be detected. This additional visual criteria increases the sensitivity of the assay.
Although the invention has been described with reference to embodiments and examples, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.
61
ACAGTGGTGA ACCGCATCGC GGCGGGGGAA GTTATCCAGC GGCCAGCTAA TGCTATCAAA
121
GAGATGATTG AGAACTGgta cggagggagt cgagccgggc tcacttaagg gctacgactt
3121
GTGATTGTTA AAGAGGGAGG CCTGAAGTTG ATTCAGATCC AAGACAATGG CACCGGGATC
3181
AGGgtaagta aaacctcaaa gtagcaggat gtttgtgcgc ttcatggaag agtcaggacc
7441
GATATTGTAT GTGAAAGGTT CACTACTAGT AAACTGCAGT CCTTTGAGGA TTTAGCCAGT
7501
ATTTCTACCT ATGGCTTTCG AGGTGAGgta agctaaagat tcaagaaatg tgtaaaatat
10921
AAACAGCTGA TGGAAAGTGT GCATACAGgt atagtgctga cttcttttac tcatatatat
15301
TTTACAACAT AGCCACGAGG AGAAAAGCTT TAAAAAATCC AAGTGAAGAA TATGGGAAAA
15361
TTTTGGAAGT TGTTGGCAGg tacagtccaa aatctgggag tgggtctctg agatttgtca
18301
GTACACAATG CAGGCATTAG TTTCTCAGTT AAAAAAgtaa gttcttggtt tatgggggat
18541
TATTCGCTCC ATCTTTGGAA ATGCTGTTAG TCGgtatgtc gataacctat ataaaaaaat
20941
TAGCCTTCAA AATGAATGGT TACATATCCA ATGCAAACTA CTCAGTGAAG AAGTGCATCT
21001
TCTTACTCTT CATCAACCgt aagttaaaaa gaaccacatg ggaaatccac tcacaggaaa
24001
CCTTGAGAAA AGCCATAGAA ACAGTGTATG CAGCCTATTT GCCCAAAAAC ACACACCCAT
24061
TCCTGTACCT CAGgtaatgt agcaccaaac tcctcaacca agactcacaa ggaacagatg
26821
GCACCCCACA AAGCATGAAG TTCACTTCCT GCACGAGGAG AGCATCCTGG AGCGGGTGCA
26881
GCAGCACATC GAGAGCAAGC TCCTGGGCTC CAATTCCTCC AGGATGTACT TCACCCAGgt
32161
CAACAAGTCT GACCTCGTCT TCTACTTCTG GAAGTAGTGA TAAGGTCTAT GCCCACCAGA
32221
TGGTTCGTAC AGATTCCCGG GAACAGAAGC TTGATGCATT TCTGCAGCCT CTGAGCAAAC
32281
CCCTGTCCAG TCAGCCCCAG GCCATTGTCA CAGAGGATAA GACAGATATT TCTAGTGGCA
32341
GGGCTAGGCA GCAAGATGAG GAGATGCTTG AACTCCCAGC CCCTGCTGAA GTGGCTGCCA
32401
AAAATCAGAG CTTGGAGGGG GATACAACAA AGGGGACTTC AGAAATGTCA GAGAAGAGAG
32461
GACCTACTTC CAGCAACCCC AGgtatggcc ttttgggaaa agtacagcct acctccttta
35281
GATGTGGAAA TGGTGGAAGA TGATTCCCGA AAGGAAATGA CTGCAGCTTG TACCCCCCGG
35341
AGAAGGATCA TTAACCTCAC TAGTGTTTTG AGTCTCCAGG AAGAAATTAA TGAGCAGGGA
35401
CATGAGGgta cgtaaacgct gtggcctgcc tgggatgcat agggcctcaa ctgccaaggt
46681
AACCACTCCT TCGTGGGCTG TGTGAATCCT CAGTGGGCCT TGGCACAGCA TCAAACCAAG
46741
TTATACCTTC TCAACACCAC CAAGCTTAGg taaatcagct gagtgtgtga acaagcagag
48781
CCAATTTTGG TGTTCTCAGG TTATCGgtaa gtttagatcc ttttcacttc tgaaatttca
54001
CACCGCTCTT TGACCTTGCC ATGCTTGCCT TAGATAGTCC AGAGAGTGGC TGGACAGAGG
54061
AAGATGGTCC CAAAGAAGGA CTTGCTGAAT ACATTGTTGA GTTTCTGAAG AAGAAGGCTG
54121
AGATGCTTGC AGACTATTTC TCTTTGGAAA TTGATGAGgt gtgacagcca ttcttatact
55021
GATTGACAAC TATGTGCCCC CTTTGGAGGG ACTGCCTATC TTCATTCTTC GACTAGCCAC
55081
TGAGgtcagt gatcaagcag atactaagca tttcggtaca tgcatgtgtg ctggagggaa
55381
GAATTGGGAC GAAGAAAAGG AATGTTTTGA AAGCCTCAGT AAAGAATGCG CTATGTTCTA
55441
TTCCATCCGG AAGCAGTACA TATCTGAGGA GTCGACCCTC TCAGGCCAGC AGgtacagtg
57001
CTGTGGAACA CATTGTCTAT AAAGCCTTGC GCTCACACAT TCTGCCTCCT AAACATTTCA
57061
CAGAAGATGG AAATATCCTG CAGCTTGCTA ACCTGCCTGA TCTATACAAA GTCTTTGAGA
57121
GGTGTTAAat atggttattt atgcactgtg ggatgtgttc ttctttctct gtattccgat
121
GCTTCGTGCG CTTCTTTCAG GGCATGCCGG AGAAGCCGAC CACCACAGTG CGCCTTTTCG
181
ACCGGGGCGA CTTCTATACG GCGCACGGCG AGGACGCGCT GCTGGCCGCC CGGGAGGTGT
241
TCAAGACCCA GGGGGTGATC AAGTACATGG GGCCGGCAGg tgagggccgg gacggcgcgt
5281
CAAAGAATCT GCAGAGTGTT GTGCTTAGTA AAATGAATTT TGAATCTTTT GTAAAAGATC
5341
TTCTTCTGGT TCGTCAGTAT AGAGTTGAAG TTTATAAGAA TAGAGCTGGA AATAAGGCAT
5401
CCAAGGAGAA TGATTGGTAT TTGGCATATA AGgtaattat cttccttttt aatttactta
GTGTTGTGGGTGTTAAAATGTCCGCAGTTGATGGCCAGAGACAGGTTGGAGTTGGGTATGTGGATTCCATA
CAGAGGAAACTAGGACTGTGTGAATTCCCTGATAATGATCAGTTCTCCAATCTTGAGGCTCTCCTCATCCA
GATTGGACCAAAGGAATGTGTTTTACCCGGAGGAGAGACTGCTGGAGACATGGGGAAACTGAGACAGgtaa
7921
AGAAAAAAAG CTGACTTTTC CACAAAAGAC ATTTATCAGG ACCTCAACCG GTTGTTGAAA
7981
GGCAAAAAGG GAGAGCAGAT GAATAGTGCT GTATTGCCAG AAATGGAGAA TCAGgtacat
9781
AGAACTCTTA TCAGATGATT CCAACTTTGG ACAGTTTGAA CTGACTACTT TTGACTTCAG
9841
CCAGTATATG AAATTGGATA TTGCAGCAGT CAGAGCCCTT AACCTTTTTC AGgtaaaaaa
11821
AAGTGTAAAA CCCCTCAAGG ACAAAGACTT GTTAACCAGT GGATTAAGCA GCCTCTCATG
11881
GATAAGAACA GAATAGAGGA GAGgtatgtt attagtttat actttcgtta gttttatgta
24601
GAAGCTTTTG TAGAAGATGC AGAATTGAGG CAGACTTTAC AAGAAGATTT ACTTCGTCGA
24661
TTCCCAGATC TTAACCGACT TGCCAAGAAG TTTCAAAGAC AAGCAGCAAA CTTACAAGAT
24721
TGTTACCGAC TCTATCAGGG TATAAATCAA CTACCTAATG TTATACAGGC TCTGGAAAAA
24781
CATGAAGgta acaagtgatt ttgttttttt gttttccttc aactcataca atatatactt
40441
GATCTTCGTT CTGACTTCTC CAAGTTTCAG GAAATGATAG AAACAACTTT AGATATGGAT
40501
57901
AAACCTTCAT TTGATCCTAA TCTCAGTGAA TTAAGAGAAA TAATGAATGA CTTGGAAAAG
57961
AAGATGCAGT CAACATTAAT AAGTGCAGCC AGAGATCTTG gtaagaatgg gtcattggag
61561
TTCGTGTAAC CTGTAAGGAA GAAAAAGTCC TTCGTAACAA TAAAAACTTT AGTACTGTAG
61621
ATATCCAGAA GAATGGTGTT AAATTTACCA ACAGgtttgc aagtcgttat tatattttta
65821
TTCTTTAAAT GAAGAGTATA CCAAAAATAA AACAGAATAT GAAGAAGCCC AGGATGCCAT
65881
TGTTAAAGAA ATTGTCAATA TTTCTTCAGg taaacttaat agaactaata atgttctgaa
69901
GATGTGTTAG CTCAGCTAGA TGCTGTTGTC AGCTTTGCTC ACGTGTCAAA TGGAGCACCT
69961
GTTCCATATG TACGACCAGC CATTTTGGAG AAAGGACAAG GAAGAATTAT ATTAAAAGCA
70021
TCCAGGCATG CTTGTGTTGA AGTTCAAGAT GAAATTGCAT TTATTCCTAA TGACGTATAC
70081
TTTGAAAAAG ATAAACAGAT GTTCCACATC ATTACTGgta aaaaacctgg tttttgggct
71221
TATGGGAGGT AAATCAACAT ATATTCGACA AACTGGGGTG ATAGTACTCA TGGCCCAAAT
71281
TGGGTGTTTT GTGCCATGTG AGTCAGCAGA AGTGTCCATT GTGGACTGCA TCTTAGCCCG
71341
AGTAGGGGCT GGTGACAGTC AATTGAAAGG AGTCTCCACG TTCATGGCTG AAATGTTGGA
71401
AACTGCTTCT ATCCTCAGgt aagtgcatct cctagtccct tgaagataga aatgtatgtc
73141
ATAATCATAG ATGAATTGGG AAGAGGAACT TCTACCTACG ATGGATTTGG GTTAGCATGG
73201
GCTATATCAG AATACATTGC AACAAAGATT GGTGCTTTTT GCATGTTTGC AACCCATTTT
73261
CATGAACTTA CTGCCTTGGC CAATCAGATA CCAACTGTTA ATAATCTACA TGTCACAGCA
73321
CTCACCACTG AAGAGACCTT AACTATGCTT TATCAGGTGA AGAAAGgtat gtactattgg
75601
TGTAATAGAG TGTGCTAAAC AGAAAGCCCT GGAACTTGAG GAGTTTCAGT ATATTGGAGA
75661
ATCGCAAGGA TATGATATCA TGGAACCAGC AGCAAAGAAG TGCTATCTGG AAAGAGAGgt
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US05/20721 | 6/14/2005 | WO | 00 | 6/21/2007 |
| Number | Date | Country | |
|---|---|---|---|
| 60579773 | Jun 2004 | US |
