The present invention provides a method for detecting a target nucleic acid that discriminates the target nucleic acid from a non-target nucleic acid having a nucleotide sequence or modification state that differs from a portion of the target nucleic acid, the method comprising conducting a nucleic acid amplification reaction using a region in the non-target nucleic acid that differs from the target nucleic acid as a target region, using a region in the target nucleic acid that differs from the non-target nucleic acid as a corresponding target region, using a nucleic acid test sample as a template, and using a primer that hybridizes with both the target nucleic acid and the non-target nucleic acid, with the nucleic acid amplification reaction conducted in the presence of a molecule capable of binding specifically to the target region in the non-target nucleic acid, under temperature conditions under which the molecule can bind to the non-target nucleic acid, and then detecting the target nucleic acid on the basis of the presence or absence of an amplification product.

Methods, devices and systems for analyzing precious samples of cells, including single cells are provided. The methods, devices, and systems in various embodiments of the invention are used to assess genomic heterogeneity, which has been recognized as a central feature of many cancers and plays a critical role in disease initiation, progression, and response to treatment. The methods devices and systems are also used to analyze embryonic biopsies for preimplantation genetic diagnosis (PGD). In one embodiment, the devices, systems and methods provided herein allow for the construction of genomic and RNA-seq libraries without a pre-amplification step.

Methods, devices and systems for analyzing precious samples of cells, including single cells are provided. The methods, devices, and systems in various embodiments of the invention are used to assess genomic heterogeneity, which has been recognized as a central feature of many cancers and plays a critical role in disease initiation, progression, and response to treatment. The methods devices and systems are also used to analyze embryonic biopsies for preimplantation genetic diagnosis (PGD). In one embodiment, the devices, systems and methods provided herein allow for the construction of genomic and RNA-seq libraries without a pre-amplification step.

A method for determining sequences from sense and antisense strands of a nucleic acid, including (a) providing a nucleic acid cluster attached to a solid support, wherein the nucleic acid cluster includes a sense strand and an antisense strand of a concatemer, the concatemer including multiple copies of a sequence unit, the sequence unit including a target sequence and a primer binding site; (b) hybridizing a primer to a primer binding site in the antisense strand; (c) extending the primer along the antisense strand to determine the sequence from at least a portion of the target sequence in the antisense strand; (d) hybridizing a second primer to a primer binding site in the sense strand; and (e) extending the second primer along the sense strand to determine the sequence from at least a portion of the target sequence in the sense strand.

A method for determining sequences from sense and antisense strands of a nucleic acid, including (a) providing a nucleic acid cluster attached to a solid support, wherein the nucleic acid cluster includes a sense strand and an antisense strand of a concatemer, the concatemer including multiple copies of a sequence unit, the sequence unit including a target sequence and a primer binding site; (b) hybridizing a primer to a primer binding site in the antisense strand; (c) extending the primer along the antisense strand to determine the sequence from at least a portion of the target sequence in the antisense strand; (d) hybridizing a second primer to a primer binding site in the sense strand; and (e) extending the second primer along the sense strand to determine the sequence from at least a portion of the target sequence in the sense strand.

This invention relates to methods and compositions for amplifying nucleic acids, e.g., genomic DNA, using nicking agents. The method of amplifying nucleic acids comprising: (a) forming a reaction mixture comprising: (i) a first nucleic acid template comprising a strand having a first nicking agent recognition sequence; (ii) a second nucleic acid template comprising a strand having a second nicking agent recognition sequence; (iii) at least one primer for a target region on the first or second nucleic acid template; (iv) at least one protein having DNA polymerase domain function, wherein the domain function comprises a first domain function capable of strand displacement activity and a second domain function capable of high processivity activity, or at least one protein having DNA polymerase domain function capable of strand displacement activity and at least one protein having DNA polymerase domain function capable of high processivity activity; (v) at least one deoxynucleoside triphosphate; and (vi) a first nicking agent for recognizing the first nicking agent recognition sequence and a second nicking agent for recognizing the second nicking agent recognition sequence; (b) incubating the reaction mixture under conditions that amplifies the nucleic acid templates, wherein the domain functions capable of strand displacement activity and high processivity activity are separate from each other and capable of carrying out their activities simultaneously. In specific embodiments, the nicking agent is NB.BsrDI and the proteins having DNA polymerase domain functions are Bst 3.0 polymerase and Pfu polymerase.

This invention relates to methods and compositions for amplifying nucleic acids, e.g., genomic DNA, using nicking agents. The method of amplifying nucleic acids comprising: (a) forming a reaction mixture comprising: (i) a first nucleic acid template comprising a strand having a first nicking agent recognition sequence; (ii) a second nucleic acid template comprising a strand having a second nicking agent recognition sequence; (iii) at least one primer for a target region on the first or second nucleic acid template; (iv) at least one protein having DNA polymerase domain function, wherein the domain function comprises a first domain function capable of strand displacement activity and a second domain function capable of high processivity activity, or at least one protein having DNA polymerase domain function capable of strand displacement activity and at least one protein having DNA polymerase domain function capable of high processivity activity; (v) at least one deoxynucleoside triphosphate; and (vi) a first nicking agent for recognizing the first nicking agent recognition sequence and a second nicking agent for recognizing the second nicking agent recognition sequence; (b) incubating the reaction mixture under conditions that amplifies the nucleic acid templates, wherein the domain functions capable of strand displacement activity and high processivity activity are separate from each other and capable of carrying out their activities simultaneously. In specific embodiments, the nicking agent is NB.BsrDI and the proteins having DNA polymerase domain functions are Bst 3.0 polymerase and Pfu polymerase.

Provided is a method for synchronously sequencing a sense strand and an antisense strand of an insert DNA, including: performing two rounds of rolling circle amplification and multiple displacement amplification to obtain a DNA nano ball template including a read1 strand sequencing template and a read2 strand sequencing template; and hybridizing the read1 strand sequencing template and the read2 strand sequencing template with read1 strand sequencing primers and read2 strand sequencing primers, respectively, and simultaneously performing read1 strand sequencing and read2 strand sequencing to obtain sequences of the sense strand and the antisense strand of the insert DNA. The method of the present disclosure can perform the sequencing from both ends of the insert DNA, significantly saving the time and costs for sequencing, and increasing the sequencing throughput.

Provided is a method for synchronously sequencing a sense strand and an antisense strand of an insert DNA, including: performing two rounds of rolling circle amplification and multiple displacement amplification to obtain a DNA nano ball template including a read1 strand sequencing template and a read2 strand sequencing template; and hybridizing the read1 strand sequencing template and the read2 strand sequencing template with read1 strand sequencing primers and read2 strand sequencing primers, respectively, and simultaneously performing read1 strand sequencing and read2 strand sequencing to obtain sequences of the sense strand and the antisense strand of the insert DNA. The method of the present disclosure can perform the sequencing from both ends of the insert DNA, significantly saving the time and costs for sequencing, and increasing the sequencing throughput.

A method for determining sequences from sense and antisense strands of a nucleic acid, including (a) providing a nucleic acid cluster attached to a solid support, wherein the nucleic acid cluster includes a sense strand and an antisense strand of a concatemer, the concatemer including multiple copies of a sequence unit, the sequence unit including a target sequence and a primer binding site; (b) hybridizing a primer to a primer binding site in the antisense strand; (c) extending the primer along the antisense strand to determine the sequence from at least a portion of the target sequence in the antisense strand; (d) hybridizing a second primer to a primer binding site in the sense strand; and (e) extending the second primer along the sense strand to determine the sequence from at least a portion of the target sequence in the sense strand.