摘要:
Various methods, systems and devices for optical detection and analysis of polymers, such as polynucleotides, using nanopores, e.g., for determining sequences of nucleic acids, are provided herein. In certain variations, methods and systems for determining a nucleotide sequence of a polynucleotide, which include measuring mixed FRET signals as a polynucleotide translocates through a nanopore and determining a nucleotide sequence of the polynucleotide from the mixed FRET signals, are provided.
摘要:
The present disclosure provides methods, devices and systems that enable simultaneous multiplexing amplification reaction and real-time detection in a single reaction chamber. An aspect of the present disclosure provides a method for assaying a presence or absence of at least one target nucleic acid molecule. In some embodiments of aspects provided herein, at least one signal is produced upon binding of probes to the target nucleic acid molecule and/or the limiting primer. In some embodiments of aspects provided herein, a reaction mixture comprises a plurality of template nucleic acid molecules and the probes specifically bind to a plurality of target nucleic molecules as amplification products of the plurality of template nucleic acid molecules.
摘要:
The present disclosure provides methods and devices for simultaneous identification of a plurality of target nucleic acid sequences in a single sample chamber that includes an addressable array of nucleic acid probes attached to a solid surface. Addressable signals can be generated and measured, in real-time, upon hybridization of target sequences at the individual probe locations within the array while the temperature of the system is varied. Such generated signals, as a function temperature, can then be used to compute the properties of nucleic acid hybridization at each addressable location which is ultimately utilized to estimate the sequence of the target nucleic acids. In particular, an integrated semiconductor biosensor array device can be used to measure the addressable signals.
摘要:
The present invention relates to a method which prevents undesirable binding of ddNTPs to double stranded polynucleotides when in the presence of a polymerase. Such methods may be used to prevent the appearance of false positives in methods employing ddNTPs, e.g. in sequence detection methods. The present invention also provides a method of avoiding a false Tm reading or false FRET effects (such as false positive quenching), for example in a melting curve analysis method. In particular a method is provided in which a target nucleotide sequence in a test polynucleotide is detected using a method in which a double stranded molecule is generated which may or may not comprise two labels depending on whether the target sequence is present in which the presence of the two labels is determined, preferably by performing a melting curve analysis.
摘要:
The present invention is directed to methods for identifying the presence of one or more target nucleotide sequences in a sample that involve a nuclease-ligation reaction. In some embodiments, the ligation products formed in the nuclease-ligation process of the present invention are subsequently amplified using a polymerase chain reaction. The ligated product sequences or extension products thereof are detected, and the presence of one or more target nucleotide sequences in the sample is identified based on the detection.
摘要:
Interaction with a protein is detected by using an RNA probe containing the following sequences; (i) a complementary strand sequence to a DNA barcode sequence, (ii) a sequence of a first stem portion, (iii) a sequence of a second stem portion complementary to the first stem portion for hybridizing with the first stem portion to form a double-stranded stem, and (iv) a sequence of a loop portion contained in RNA for linking the first and second stem portions.
摘要:
The present invention provides a composition of matter comprising: (a) a first part comprising at least one first nucleic acid primer that comprises (i) at least one first energy transfer element; and (ii) a nucleic acid sequence which is complementary to a nucleic acid sequence in at least a portion of a nucleic acid of interest; (b) a second part comprising at least one second nucleic acid primer that comprises: (i) at least one second energy transfer element; and (ii) a nucleic acid sequence which is identical to a nucleic acid sequence in at least a portion of said nucleic acid of interest; wherein said first nucleic acid primer does not comprise said second energy transfer element, and wherein said second nucleic acid primer does not comprise said first energy transfer element; wherein the first energy transfer element is capable of acting as an energy donor and the second energy transfer element is capable of acting as an energy acceptor, or the first energy transfer element is capable of acting as acting as an energy acceptor and the second energy transfer element is capable of acting as an energy donor; and wherein the sequences of the first nucleic acid primer and the second nucleic acid primer are selected such that if the nucleic acid of interest is present in the sample, said first and second nucleic acid primers will be incorporated into different complementary strands of a double stranded amplicon in sufficient proximity for energy transfer to occur between the first energy transfer element and the second energy transfer element of the incorporated primers; (c) a sample suspected of containing said nucleic acid of interest; and (d) reagents for carrying out nucleic acid strand extension.
摘要:
The present invention relates to methods for detection of nucleotide polymerase activity and methods of detecting compounds that modulate nucleotide polymerase activity, by detecting product formation of the nucleotide polymerase to be tested based on determination of close proximity of two labeled nucleotide probes able to bind the product of the nucleotide polymerase. It is preferred that proximity dependent energy transfer, such as förster resonance energy transfer, between said labeled nucleotide probes is determined. The invention further provides kits comprising components for carrying out the inventive methods for detection of nucleotide polymerase activity.