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Genetic assays

A sequence and nucleic acid technology, applied in the field of virus recombination, which can solve the problems of inaccuracy, time-consuming, and overestimation of the recombination level.

Active Publication Date: 2016-05-04
BIO RAD LAB INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Current methods for measuring recombination rates, such as those involving gel electrophoresis and sequencing, are imprecise and / or time-consuming and often lead to overestimation of recombination levels

Method used

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  • Genetic assays
  • Genetic assays
  • Genetic assays

Examples

Experimental program
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Embodiment 1

[0289] Example 1 - Droplet cluster identification and classification

[0290] Samples may be obtained by swabbing the throat or nose of subjects suspected of being infected with a particularly virulent recombinant strain of influenza virus. Nucleic acids from the sample are extracted, bound to specific probes and / or amplification reagents, and partitioned into aqueous droplets within a water-in-oil emulsion. Virulent strains can be characterized by an A allele at the A / a locus and a B allele at the B / b locus. Primers comprising probes attached to fluorophores are designed to detect the A locus by emitting a signal with a specific color, and the B locus by emitting a signal with a different color. Although the fluorescent probes were designed for one viral allele, they also fluoresced (with reduced intensity) when they cross-reacted with other alleles. For example, a FAM-labeled probe is designed to detect the A allele, and the droplets exhibit four levels of fluorescence: th...

Embodiment 2

[0291] Example 2 - Determining recombination rates in RNA samples

[0292] RNA was taken from blood samples of cells from HIV-infected patients. The sample was partitioned into aqueous droplets in a water-in-oil emulsion. Allele-specific fluorescent probes (FAM; and HEX) were used to detect viral alleles at two distinct loci on the HIV virus. After RT-ddPCR, droplets were positive for one fluorophore, positive for both fluorophores or positive for neither fluorophore depending on the viral allele they contained at the beginning of the reaction. Viral alleles configured in trans partitioned independently into droplets. Viral alleles configured in cis tend to cosegregate into the same droplet because they are physically linked, see Figure 14 . Restriction enzyme digestion at sites between the viral alleles configured in cis abrogates copartitioning of the two viral alleles (not shown). This quantitative difference or ratio between trans-configured and cis-configured viral ...

Embodiment 3

[0293] Example 3 - Determining recombination rates in DNA samples

[0294] DNA is taken from blood samples of cells from HIV-infected patients. The sample was partitioned into aqueous droplets in an oil-water emulsion. Allele-specific fluorescent probes (FAM; and HEX) were used to detect viral alleles at two distinct loci on the HIV virus. After ddPCR, the droplets were positive for one fluorophore, positive for both fluorophores or positive for neither, depending on the viral alleles they contained at the beginning of the reaction. Viral alleles configured in trans partitioned independently into droplets. Viral alleles configured in cis tend to cosegregate into the same droplet because they are physically linked, see Figure 14 . Restriction enzyme digestion at sites between the cis-configured viral alleles abrogates copartitioning of the two viral alleles. This quantitative difference or ratio between trans-configured and cis-configured viral alleles determines the reco...

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Abstract

Provided herein are methods, compositions, systems, and kits for recombination assays, many of which involve amplification reactions such as PCR or droplet digital PCR. The methods, compositions, kits and systems are particularly useful for minimizing artifactual biases arising from recombination between viral or other genomes (e.g., any microbial genome) during the assay process. In some aspects, provided herein are methods of performing a recombination assay.

Description

[0001] cross reference [0002] This application claims the benefit of U.S. Provisional Application No. 61 / 858,311, filed July 25, 2013, and U.S. Provisional Application No. 61 / 899,027, filed November 1, 2013, each of which is incorporated herein by reference in its entirety . Background of the invention [0003] Recombination, especially viral recombination, can greatly affect both evolution and epidemiology. In viruses, the rate of recombination depends on the frequency with which co-infection occurs and the frequency of genetic exchange between different viral genomes within the infected host cell. The ability to measure recombination rates is important for understanding virus growth, virulence, and for creating attenuated strains for the development of new vaccines. [0004] Current methods for measuring recombination rates, such as those involving gel electrophoresis and sequencing, are imprecise and / or time-consuming, and often result in overestimations of recombinatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCG01N33/56983G01N2458/10C12Q1/70C12Q1/701C12N15/102C12Q1/6827C12Q1/703C12Q2531/113C12Q2537/143C12Q2537/165C12Q2563/159Y02A50/30C12Q2600/156
Inventor 乔治·卡林-诺伊曼斯维伦·特左内夫
Owner BIO RAD LAB INC
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