Provided herein are methods and compositions for performing PCR with primers with blocked 3′-ends that are unblocked when these primers anneal to the template. The multiplexed PCR can be used as real-time qPCR, for end-point detection or as enrichment method for next generation sequencing (NGS). Also described herein are methods and compositions to improve sensitivity of mutation-specific PCR when targeting closely-spaced mutations.

Provided herein are methods and compositions for performing PCR with primers with blocked 3′-ends that are unblocked when these primers anneal to the template. The multiplexed PCR can be used as real-time qPCR, for end-point detection or as enrichment method for next generation sequencing (NGS). Also described herein are methods and compositions to improve sensitivity of mutation-specific PCR when targeting closely-spaced mutations.

Provided is a sensing apparatus comprising a chip for integrated amplification and sequencing of a template polynucleotide in a sample. The apparatus comprises a chip with at least one ISFET in a well or chamber, amplification means for amplifying the template polynucleotide on a surface of said chip and comprising at least one heating means suitable for conducting amplification of the template polynucleotide at temperatures elevated with respect to room temperature, and sequencing means for sequencing the amplified template polynucleotide in said well or chamber. Methods of use are also provided.

Provided is a sensing apparatus comprising a chip for integrated amplification and sequencing of a template polynucleotide in a sample. The apparatus comprises a chip with at least one ISFET in a well or chamber, amplification means for amplifying the template polynucleotide on a surface of said chip and comprising at least one heating means suitable for conducting amplification of the template polynucleotide at temperatures elevated with respect to room temperature, and sequencing means for sequencing the amplified template polynucleotide in said well or chamber. Methods of use are also provided.

Methods and systems for ordering assays which detect SNPs or gene expression are provided. The methods use PCR and RT-PCR procedures. Collections of stock assays are assembled using pre- and post-manufacturing quality control procedures and made available to consumers via the Internet. In addition, custom assays are prepared upon order from the consumer and these assays are also prepared using pre- and post-manufacturing quality control procedures. The assays are then delivered to the consumer.

Methods and systems for ordering assays which detect SNPs or gene expression are provided. The methods use PCR and RT-PCR procedures. Collections of stock assays are assembled using pre- and post-manufacturing quality control procedures and made available to consumers via the Internet. In addition, custom assays are prepared upon order from the consumer and these assays are also prepared using pre- and post-manufacturing quality control procedures. The assays are then delivered to the consumer.

The present invention discloses a two-stage reaction and detection tube comprises a first tube, a second tube and a connector. The first tube comprises a detection space for placing a dipstick and a detection space for the test result. The second tube comprises a storing space for the PCR or RT-PCR reagents and the target gene segments. The connector comprises a first portion and a second portion which connect to the first tube and the second tube respectively. The connector further comprises a diversion unit, a liquid collection space, and a dipstick fixing space, where the liquid collection space is connected to the dipstick fixing space. The target gene amplification and detection could be directly processed in the same tube without any liquid transfer.

The present invention discloses a two-stage reaction and detection tube comprises a first tube, a second tube and a connector. The first tube comprises a detection space for placing a dipstick and a detection space for the test result. The second tube comprises a storing space for the PCR or RT-PCR reagents and the target gene segments. The connector comprises a first portion and a second portion which connect to the first tube and the second tube respectively. The connector further comprises a diversion unit, a liquid collection space, and a dipstick fixing space, where the liquid collection space is connected to the dipstick fixing space. The target gene amplification and detection could be directly processed in the same tube without any liquid transfer.

A method for testing target nucleic acid includes the steps from (1) through (5) below: (1) mixing a specimen containing target nucleic acid with positive control nucleic acid to obtain a specimen mixture of the specimen and the positive control nucleic acid; (2) mixing the specimen mixture with a PCR buffer solution containing a surfactant to obtain a buffer solution mixture; (3) adding a portion of the buffer mixture to a solid composition for PCR control containing DNA polymerase, positive control nucleic acid, and PCR reaction control nucleic acid; (4) adding a portion of the buffer mixture to a solid composition for PCR reaction containing DNA polymerase and one or more kinds of PCR primer pair; and (5) detecting a PCR product generated as a result of the steps (3) and (4).

A method for testing target nucleic acid includes the steps from (1) through (5) below: (1) mixing a specimen containing target nucleic acid with positive control nucleic acid to obtain a specimen mixture of the specimen and the positive control nucleic acid; (2) mixing the specimen mixture with a PCR buffer solution containing a surfactant to obtain a buffer solution mixture; (3) adding a portion of the buffer mixture to a solid composition for PCR control containing DNA polymerase, positive control nucleic acid, and PCR reaction control nucleic acid; (4) adding a portion of the buffer mixture to a solid composition for PCR reaction containing DNA polymerase and one or more kinds of PCR primer pair; and (5) detecting a PCR product generated as a result of the steps (3) and (4).