摘要:
A method is described whereby new cDNA or RNA sequences can be introduced into or substituted for cDNA in any chosen position without specific sequence requirements using the polymerase chain reaction (PCR). The method entails the use of primers which are complementary to the 3' and 5' ends of the desired sequence to be inserted as well as to the 3' and 5' ends of the chosen site of insertion in the acceptor molecule. The desired sequence is amplified by PCR such that single stranded fragments are produced. The single stranded fragment of the desired sequence is then annealed to a single stranded acceptor molecule at the site of insertion and extended to produce a double stranded molecule. The double stranded molecule is then separated into two strands which are identical except that one of the strands contains the desired sequence inserted at the chosen site. A second double stranded molecule is then generated.
摘要:
A method comprises loading a sample portion into a sample chamber which comprises means for minimizing diffusion of the sample portion, subjecting the sample portion to an amplification step, and determining whether the sample portion contains at least one molecule of a target nucleic acid. If the sample portion contains a single molecule of the target nucleic acid, the sample portion would attain a detectable concentration of the target nucleic acid after a single round of amplification. Also, a microfluidic device comprising a sample portion and a sample chamber comprising means for minimizing diffusion of the sample portion. Also, a microfluidic device comprising a sample chamber and an amplification targeting reagent positioned in the first sample chamber.
摘要:
A microfluidic device comprising a first sample chamber and a first sample portion positioned in the first sample chamber. If the first sample portion contains a single molecule of a target nucleic acid, the first sample portion would attain a detectable concentration of the target nucleic acid after a single round of amplification. Also, a microfluidic device comprising a first sample chamber and at least one amplification targeting reagent positioned in the first sample chamber. If a sample portion which comprises at least a single molecule of a target nucleic acid is positioned in the first sample chamber, the sample portion would attain a detectable concentration of the target nucleic acid after a single round of amplification.
摘要:
A microfluidic device comprising a first surface portion and a first sample retaining element, which have differing affinities to a fluid, and a method comprising supplying a sample to such a device. In some embodiments, the differing affinity is a result of plasma, ion embedding, surface charging, chemical, optical, electronic and/or electromagnetic treatment. Also, a microfluidic device comprising at least one microcapillary device having a sample retaining element, at least one surface of which exhibits hydrophobicity, hydrophilicity, electromagnetic force exertion and electrostatic force exertion. Also, a microfluidic device comprising a first element having a hydrophilic pattern comprising at least a first sample retaining element. Also, a method comprising supplying a sample to a channel between a first element and a second element, and inducing in the first element at least one hydrophilic pattern by electrets or by internal or external electrodes to provide a charged surface.
摘要:
A method comprises loading a sample portion into a sample chamber which comprises means for minimizing diffusion of the sample portion, subjecting the sample portion to an amplification step, and determining whether the sample portion contains at least one molecule of a target nucleic acid. If the sample portion contains a single molecule of the target nucleic acid, the sample portion would attain a detectable concentration of the target nucleic acid after a single round of amplification. Also, a microfluidic device comprising a sample portion and a sample chamber comprising means for minimizing diffusion of the sample portion. Also, a microfluidic device comprising a sample chamber and an amplification targeting reagent positioned in the first sample chamber.
摘要:
A method comprising subjecting one or more sample portion(s) to a single amplification step, thereby amplifying a single molecule in the sample portion to a detectable level, and, in some embodiments, then determining whether the sample portion contains at least one molecule of the target nucleic acid. In some embodiments, the sample portion is in a porous sample structure, or in a sample chamber which comprises means for minimizing diffusion of the sample portion, or in a sample chamber which is inside a microcapillary device, or in a sample retaining means.
摘要:
A miniaturized assembly is provided whereby a fluid sample can be divided into a plurality of sample portions in retaining wells and the sample fluid can be displaced from open ends of the wells while simultaneously being sealed in the wells. A method of dividing a fluid sample using the assembly is also provided.
摘要:
A method comprising loading a sample into a microfluidic device which comprises plural sample chambers, subdividing the sample into a plurality of sample portions, such that respective sample portions are positioned in each of a plurality of the sample chambers, and subjecting the sample portions loaded into the respective sample chambers to at least a first amplification step. Each of the sample chambers has a respective volume such that if a sample portion positioned in the sample chamber comprises at least one molecule of a target nucleic acid, the target nucleic acid would attain a detectable concentration in the sample chamber after a single round of amplification.
摘要:
A method comprises loading a sample portion into a first porous structure, subjecting the sample portion to an amplification step, and determining whether the sample portion contains at least one molecule of a target nucleic acid. If the sample portion contains a single molecule of the target nucleic acid, the sample portion would attain a detectable concentration of the target nucleic acid after a single round of amplification. Also, a microfluidic device comprising a porous sample structure and a sample portion positioned in the porous sample structure. Also, a microfluidic device comprising a porous sample structure and an amplification targeting reagent positioned in the porous sample structure.
摘要:
Methods of detecting and quantifying genomic nucliec acid molecule sequences are provided using the simultaneous amplification of a plurality of discrete nanoliter-sized samples. A miniaturized closed assembly is also provided for carrying out amplification of a nucleic acid molecule by polymerase chain reaction in multiple nanoliter-sized samples. Methods of filling miniaturized sample chambers are also provided as are methods for determining the number of template molecules in a sample by conducting replicate nucleic acid sequence amplification reactions on a set of terminally diluted samples and counting the number of positive amplification reactions. The methods can be used to detect a single starting nucleic acid target molecule.