Method for improving detection of b cell immunoglobulin gene recombination

A technology of immunoglobulins and nucleotides, applied in biochemical equipment and methods, microbial determination/inspection, organic chemistry, etc., and can solve problems such as no consideration

Inactive Publication Date: 2013-06-12
韩建
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

Scientists at NIAID recognized that "current methods for examining differences in gene expression in mixtures of immune cells in the blood do not account for the wide range of variations in the proportions of the respective cell types that exist even in healthy individuals" (Mark Davis, Ph.D., NIH News( NIH News), March 8, 2010)

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  • Method for improving detection of b cell immunoglobulin gene recombination
  • Method for improving detection of b cell immunoglobulin gene recombination

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Embodiment Construction

[0026] Blood samples were obtained from Conversant Healthcare Systems, Inc., Huntsville, Alabama for figure 1 Amplifications shown in . For the first amplification reaction using the Qiagen one-step RT-PCR kit (Qiagen, Carlsbad, CA), mRNA was extracted using the Qiagen kit. Add reverse transcriptase to samples: 50°C, 40 min (30 min, min RT) for initial PCR activation at 95°C for 15 min. The enrichment cycle was performed: 94 °C, 30 seconds → 63 °C, 2 minutes → 72 °C, 30 seconds, for 15 cycles. A 2-step cycle of 94°C, 30 sec→72°C, 2 min was performed for 15 cycles, with a final extension of 10 min at 72°C. In the second amplification reaction using the Qiagen multiplex PCR kit, initial PCR activation was performed at 95 °C for 15 min, followed by a 3-step cycle: 94 °C, 30 sec → 55 °C, 30 sec → 72 °C, 30 sec, 40 cycles were performed with a final extension of 5 minutes at 72°C. Also joined the reorganization from Promega Ribonuclease inhibitors.

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Abstract

Disclosed is a method for generating primers to increase the yield of PCR products that represent the various genetic recombination events and antibodies that exist in a sample from a human or animal.

Description

[0001] This application claims priority to US Provisional Patent Application No. 61 / 318,417, filed March 29, 2010. field of invention [0002] The present invention relates to methods for identifying and quantifying B-cell immunoglobulin gene recombinations. More specifically, the present invention relates to methods of designing primers for increasing the amount of PCR-amplified products from immunoglobulin cDNA and / or RNA. Background of the invention [0003] When considering the "stagnation zone" of scientific research, immunology has become a major part of the study of the body and its states in health and disease. Scientists continue to discover links between the body's natural defenses and diseases that were once thought to have nothing to do with immunology. Cells of the immune system provide the most significant part of the inflammatory response, and inflammation can be associated with various diseases such as cardiovascular disease, kidney disease, diabetes, arthri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/00C12P19/34
CPCC12Q1/6853
Inventor 韩建
Owner 韩建
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