Abstract: 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.

Abstract: A method comprises loading one or more sample portions into respective sample chambers, subjecting each of the sample portions to an amplification step, for each sample portion, determining whether the sample portion contains at least one molecule of a target nucleic acid; and then quantifying a number of the sample portions which contain at least one molecule of the target nucleic acid, and/or quantifying a number of peaks indicative of the detectable concentration, and/or detecting an intensity of at least one peak indicative of a concentration of the target nucleic acid. Any of the sample portions which contains a single molecule of the target nucleic acid would attain a detectable concentration of the target nucleic acid after a single round of amplification.

Abstract: 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.

Abstract: A method comprising loading sample into at least two sample chambers, subjecting the sample portions in each sample chamber to at least a first amplification step, and determining whether each sample portion contains at least one molecule of a target nucleic acid. If a sample portion contains at least a single molecule of the target nucleic acid, it would attain a detectable concentration of the target nucleic acid after a single round of amplification. Also, a method for detecting whether at least one molecule of a first target nucleic acid is present in a first sample and detecting whether at least one molecule of a second target nucleic acid is present in a second sample. Also, a microfluidic device comprising sample chambers comprising respective ingredients which will react with respective target nucleic acids if contacted with the target nucleic acids and subjected to a round of amplification.

Abstract: 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.

Abstract: 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.

Abstract: Methods of detecting and quantifying genomic nucleic 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.

Abstract: 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.