The invention provides a novel array method for nucleic acid sequence detection with improved specificity which allows for detection of genetic variation, from simple SNPs (where the variation occurs at a fixed position and is of limited allelic number) to more complex sequence variation patterns (such as with multigene families or multiple genetic strains of an organism where the sequence variation between the individual members is neither fixed nor consistent). The array is comprised of short, synthetic oligonucleotide probes attached to a solid surface which are hybridized to single-stranded targets. Single stranded targets can be produced using a method that employs primers modified on the 5′ end to prohibit degradation by a 5′-exonuclease that is introduced to degrade the unprotected strand. The invention further provides for printing buffers/solutions for the immobilization of oligonucleotide probes to an array surface. The invention also provides hybridization and wash buffers and conditions to maximize hybridization specificity and signal intensity, and reduce hybridization times.

The invention provides a novel array method for nucleic acid sequence detection with improved specificity which allows for detection of genetic variation, from simple SNPs (where the variation occurs at a fixed position and is of limited allelic number) to more complex sequence variation patterns (such as with multigene families or multiple genetic strains of an organism where the sequence variation between the individual members is neither fixed nor consistent). The array is comprised of short, synthetic oligonucleotide probes attached to a solid surface which are hybridized to single-stranded targets. Single stranded targets can be produced using a method that employs primers modified on the 5′ end to prohibit degradation by a 5′-exonuclease that is introduced to degrade the unprotected strand. The invention further provides for printing buffers/solutions for the immobilization of oligonucleotide probes to an array surface. The invention also provides hybridization and wash buffers and conditions to maximize hybridization specificity and signal intensity, and reduce hybridization times.

The present invention provides methods, compositions, and systems for enriching compositions for polymerase enzyme complexes. In particular, the methods, compositions, and systems of the present invention remove free polymerases from the compositions using one or more purification steps, including protease treatment, thus enriching the compositions for polymerases complexed with a template nucleic acid.

The present invention provides methods, compositions, and systems for enriching compositions for polymerase enzyme complexes. In particular, the methods, compositions, and systems of the present invention remove free polymerases from the compositions using one or more purification steps, including protease treatment, thus enriching the compositions for polymerases complexed with a template nucleic acid.

Methods of producing single-stranded deoxyribonucleic acids (ssDNAs) are provided. Aspects of the methods include generating a double stranded deoxyribonucleic acid (dsDNA) and then selectively degrading one strand of the dsDNA to produce a ssDNA. ssDNAs produced using methods of the invention find use in a variety of applications, including genomic modification applications. Also provided are compositions, e.g., kits, that find use in practicing various embodiments of the invention.

Methods of producing single-stranded deoxyribonucleic acids (ssDNAs) are provided. Aspects of the methods include generating a double stranded deoxyribonucleic acid (dsDNA) and then selectively degrading one strand of the dsDNA to produce a ssDNA. ssDNAs produced using methods of the invention find use in a variety of applications, including genomic modification applications. Also provided are compositions, e.g., kits, that find use in practicing various embodiments of the invention.

The present invention provides for novel methods and compositions for nucleic acid sequence detection. Unique, identifying cleavage fragments from probes, bound to target nucleic acids, are produced during PCR by the 5′-nuclease activity of the polymerase. The identity of the targets can be determined by identifying the unique cleavage fragments.

The present invention provides for novel methods and compositions for nucleic acid sequence detection. Unique, identifying cleavage fragments from probes, bound to target nucleic acids, are produced during PCR by the 5′-nuclease activity of the polymerase. The identity of the targets can be determined by identifying the unique cleavage fragments.

A multiplex assay for nucleic acid detection includes a substrate, a sample, and a fluorophore-labeled oligonucleotide. The substrate has a plurality of physically separated assay locations, each of which includes a nucleotide-targeting enzyme configured to cleave nucleic acids, a guide ribonucleic acid (gRNA), and a quencher-labeled oligonucleotide. A portion of the sample is distributed to each assay location. The gRNA recognizes target nucleic acid in the sample, thereby activating the nucleotide-targeting enzyme to cleave nucleic acids, including the quencher-labeled oligonucleotide. The fluorophore-labeled oligonucleotide is subsequently added to each assay location, which facilitates identification of a presence of the target nucleic acid in the sample via detection of unquenched light emitted by the fluorophore in one or more of the plurality of assay locations.

A multiplex assay for nucleic acid detection includes a substrate, a sample, and a fluorophore-labeled oligonucleotide. The substrate has a plurality of physically separated assay locations, each of which includes a nucleotide-targeting enzyme configured to cleave nucleic acids, a guide ribonucleic acid (gRNA), and a quencher-labeled oligonucleotide. A portion of the sample is distributed to each assay location. The gRNA recognizes target nucleic acid in the sample, thereby activating the nucleotide-targeting enzyme to cleave nucleic acids, including the quencher-labeled oligonucleotide. The fluorophore-labeled oligonucleotide is subsequently added to each assay location, which facilitates identification of a presence of the target nucleic acid in the sample via detection of unquenched light emitted by the fluorophore in one or more of the plurality of assay locations.