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  Complexity management of genomic dna by locus specific amplificationUSPTO Application #: 20060068415Title: Complexity management of genomic dna by locus specific amplification Abstract: The present invention provides for novel methods and kits for reducing the complexity of a nucleic acid sample to interrogate a collection of target sequences. In one embodiment complexity reduction can be accomplished by extension of a locus specific capture probe followed by amplification of the extended capture probe using common primers. The locus specific capture probes may be attached to a solid support. Multiple DNA sequences may be amplified simultaneously to produce a reduced complexity sample. The invention further provides for analysis of the above sample to interrogate sequences of interest such as polymorphisms. The amplified sample may be hybridized to an array, which may be specifically designed to interrogate the desired fragments for the presence or absence of a polymorphism. (end of abstract) Agent: Affymetrix, Inc Attn: ChiefIPCounsel, Legal Dept. - Santa Clara, CA, US Inventors: Keith W. Jones, Michael Shapero, Weiwei Liu USPTO Applicaton #: 20060068415 - Class: 435006000 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic Acid The Patent Description & Claims data below is from USPTO Patent Application 20060068415. Brief Patent Description - Full Patent Description - Patent Application Claims
FIELD OF THE INVENTION [0001] The invention relates to enrichment and amplification of a collection of target sequences from a nucleic acid sample and methods of analyzing amplified product. In some embodiments target sequences are amplified by extension of a locus-specific primer followed by amplification of the extended locus-specific primer with a generic pair of primers. In some embodiments the locus-specific primers are attached to a solid support and extension takes place on the solid support. In some embodiments the invention relates to the preparation of target for array based analysis of genotype. The present invention relates to the fields of molecular biology and genetics. BACKGROUND OF THE INVENTION [0002] The past years have seen a dynamic change in the ability of science to comprehend vast amounts of data. Pioneering technologies such as nucleic acid arrays allow scientists to delve into the world of genetics in far greater detail than ever before. Exploration of genomic DNA has long been a dream of the scientific community. Held within the complex structures of genomic DNA lies the potential to identify, diagnose, or treat diseases like cancer, Alzheimer disease or alcoholism. Exploitation of genomic information from plants and animals may also provide answers to the world's food distribution problems. [0003] Recent efforts in the scientific community, such as the publication of the draft sequence of the human genome in February 2001, have changed the dream of genome exploration into a reality. Genome-wide assays, however, must contend with the complexity of genomes; the human genome for example is estimated to have a complexity of 3.times.10.sup.9 base pairs. Novel methods of sample preparation and sample analysis that reduce complexity may provide for the fast and cost effective exploration of complex samples of nucleic acids, particularly genomic DNA. [0004] Single nucleotide polymorphisms (SNPs) have emerged as the marker of choice for genome wide association studies and genetic linkage studies. Building SNP maps of the genome will provide the framework for new studies to identify the underlying genetic basis of complex diseases such as cancer, mental illness and diabetes. Due to the wide ranging applications of SNPs there is still a need for the development of robust, flexible, cost-effective technology platforms that allow for scoring genotypes in large numbers of samples. SUMMARY OF THE INVENTION [0005] The present invention provides for novel methods of sample preparation and analysis comprising managing or reducing the complexity of a nucleic acid sample by amplification of a collection of target sequences using target specific capture probes. In some embodiments the extended capture probes are attached to a solid support; in some embodiments the extended capture probes are in solution. In some embodiments the amplified collection of target sequences is analyzed by hybridization to an array that is designed to interrogate sequence variation in the target sequences. In some embodiments the amplified collection of target sequences is analyzed by hybridization to an array of tag probes. [0006] In one embodiment a method of amplifying a collection of target sequences from a nucleic acid sample is disclosed. A collection of capture probes is generated. The collection comprised a plurality of different species of primers wherein each species comprises a first common sequence and a 3' variable region that is specific for a target sequence in the collection of target sequences. Each target sequence is represented by at least one species of primer which hybridizes to the target sequence and the collection of capture probes is attached to a solid support so that the 3' end of the capture probes is available for extension. The nucleic acid sample is fragmented and an adapter that has a second common sequence is ligated to the fragments. Fragmentation in some embodiments is by one or more restriction enzymes. The adapter-ligated fragments are hybridized to the collection of capture probes and the capture probes are extended using the hybridized adapter-ligated fragments as template for extension and thereby incorporating the target sequence and the second common sequence into the 3' end of the extended capture probe. The extended capture probes are then amplified using first and second common sequence primers. [0007] In some embodiments the capture probes are attached to the solid support through a covalent interaction. In another embodiment there is a tag sequence in the capture probes that is unique for each species of capture probe and the capture probes are attached to the solid support by hybridization to a collection of tag probes that are covalently attached to the solid support. In some embodiments each species of capture probe is attached to the solid support in a discrete location. [0008] In another embodiment the extended capture probes are released from the solid support prior to amplification. Prior to releasing the extended capture probes from the solid support nucleic acids that are not covalently attached to the solid support may be removed. [0009] In another embodiment the extended capture probes are enriched prior to amplification. In some embodiments capture probes are enriched by incorporation of labeled nucleotides into the extended capture probes followed by isolation of labeled capture probes by affinity chromatography. In some embodiments capture probes are labeled with biotin and avidin, streptavidin or an anti-biotin antibody, which may be monoclonal, may be used to isolate extended capture probes. In another embodiment extended capture probes are made double stranded and single stranded nucleic acid in the sample is digested by, for example a nuclease, such as, for example Exonuclease I. In another embodiment the extended capture probes are circularized prior to amplification and uncircularized nucleic acid in the sample is digested by, for example, a nuclease, such as, for example, Exonuclease III. In some embodiments the extended capture probes are circularized by hybridizing an oligonucleotide splint to the extended capture probes so that the 5' and 3' ends of extended capture probes are juxtaposed and then ligating the ends of the extended capture probes. [0010] In one embodiment a method of genotyping one or more polymorphic locations in a sample is disclosed. An amplified collection of target sequences from the sample is prepared and hybridized to an array designed to interrogate at least one polymorphic location in the collection of target sequences. The hybridization pattern is analyzed to determine the identity of the allele or alleles present at one or more polymorphic location in the collection of target sequences. [0011] In another embodiment a method for analyzing sequence variations in a population of individuals is disclosed. A nucleic acid sample is obtained from each individual and a collection of target sequences from each nucleic acid sample is amplified. Each amplified collection of target sequences is hybridized to an array designed to interrogate sequence variation in the collection of target sequences to generate a hybridization pattern for each sample and the hybridization patterns are analyzed or compared to determine the presence or absence of sequence variation in the population of individuals. [0012] In another embodiment a method of amplifying a collection of target sequences from a nucleic acid sample in solution is disclosed. A collection of capture probes is generated. The collection comprised a plurality of different species of primers wherein each species comprises a first common sequence and a 3' variable region that is specific for a target sequence wherein each target sequence in a collection of target sequences is represented by at least one species of primer which hybridizes to the target sequence. The nucleic acid sample is fragmented and an adapter is ligated to the fragments so that the strand that is ligated to the 5' end of the fragment strands comprises a second common sequence and the strand that is ligated to the 3' end of the fragments lacks the second common sequence and is blocked from extension at the 3' end. The adapter-ligated fragments are hybridized to the collection of capture probes and the capture probes are extended using the hybridized adapter-ligated fragments as template for extension and thereby incorporating the target sequence and the complement of the second common sequence into the extended capture probes. The extended capture probes are then amplified with first and second common sequence primers. [0013] In one embodiment an amino group is used to block extension at the 3' end of the adapter strand. [0014] In some embodiments fragmentation of the nucleic acid sample is by digestion with one or more restriction enzymes. [0015] In another embodiment a method for genotyping one or more polymorphisms in a nucleic acid sample is disclosed. The nucleic acid sample is fragmented and an adaptor comprising a first common priming sequence is ligated to the fragments. A collection of capture probes is ligated to the fragments. The capture probes have a second common priming sequence, a tag sequence unique for each species of capture probe, a first locus specific sequence, a Type IIs restriction enzyme recognition sequence, and a second locus specific sequence. The Type IIs restriction enzyme recognition sequence is positioned so that the enzyme will cut immediately 5' of the polymorphic base in a target sequence. The capture probes are extended to generate single-stranded extension products and then amplified using the first and second common sequence primers. The the amplified product is digested with a Type IIs restriction enzyme and the fragments are extended in the presence of one or more type of labeled ddNTP. In one embodiment the extension is done is four separate reactions, one for each ddNTP and the ddNTPs may be labeled with the same label. The extended fragments are then hybridized to four separate arrays. In another embodiment the ddNTPs are differentially labeled with at least two different labels and the extension reactions may be done in less than four reactions and each reaction may be hybridized to a separate array. The arrays are arrays of tag probes that hybridize to the tag sequences in the capture probes. The hybridization pattern on each of the arrays is analyzed to determine at least one genotype. [0016] In some embodiments the ddNTPs are labeled with biotin. [0017] In another embodiment one of the common sequence primers is resistant to nuclease digestion and the sample is treated with a nuclease that cleaves 5' to 3' after the fragments are extended in the presence of labeled ddNTP. In one embodiment the primer is resistant to nuclease digestion because it contains phosphorothioate linkages. In some embodiments the nuclease is T7 Gene 6 Exonuclease. [0018] In another embodiment a method for screening for sequence variations in a population of individuals is disclosed. A nucleic acid sample from each individual is provided and the sample is amplified and genotyped by one of the method of the invention and the genotypes from the samples are compared to determine the presence or absence of sequence variation in the population of individuals. [0019] In another embodiment a kit for amplifying a collection of target sequences is disclosed. The kit has a collection of capture probes that is specific for a collection of target sequences and has a first common sequence that is common to all of the capture probes, an adapter that has a second common sequence; and a pair of first and second common sequence primers. In another embodiment the collection of capture probes in the kit is covalently attached to a solid support so that the 3' end of the capture probes is available for extension. In another embodiment the kit also provides a restriction enzyme, buffer, DNA polymerase and dNTPs. In some embodiments the restriction enzyme is a Type IIs restriction enzyme. In another embodiment the kit also contains a ligase, dNTPs, ddNTPs, buffer and DNA polymerase. In some embodiments one of the common sequence primers is resistant to nuclease digestion. [0020] In another embodiment the capture probes also have a tag sequence unique for each species of capture probe and a Type IIs restriction enzyme recognition sequence. In another embodiment the adapter has a first strand comprising a common sequence and a second strand that does not contain the complement of that common sequence and the second strand is blocked from extension at the 3' end by, for example, an amino group. [0021] In another embodiment a collection of capture probes attached to a solid support is disclosed. The solid support may be arrays, beads, microparticles, microtitre dishes or gels. Continue reading... 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