Primer group for constructing CDR3 region high-throughput sequencing library of human TCR[beta] and application of primer group

A sequencing library and primer set technology, applied in chemical libraries, microbial measurement/testing, combinatorial chemistry, etc., can solve the problems of repetitive influence, high operation requirements, and complicated processes, and achieve the effect of simple and fast analysis process

Active Publication Date: 2020-12-29
BEIJING IMMUPEUTICS MEDICINE TECH LTD
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows accurate and stable identification of specific types of cells called CAR (Carcinoma Antigens Responsive) proteins on cancerous tissue cell surfaces through their ability to specifically bind certain molecules found within these cells. By detecting this binding pattern from fluorescently labeled monoclonal plasma membrane protein fragments attached to DNA probes, researchers have been able to identify new treatments targeted against different forms of lung carcinomas without relying solely upon histopathology methods like cytologic examination alone.

Problems solved by technology

This patents describes various technical problem addressed in this patented text: how identify and isolate specific types of tissue-associated proteins called Tcell associated antigonucleases (TAbs) from complex mixtures containing many thousands of unique combinations of protein fragments generated within one host organism's own lineage. Current technologies involve identifying specific parts of these proteins based upon their amino acid composition, making them difficult to distinguish among normal individuals who respond well when exposed to certain agents like chemoattractants. Additionally, existing approaches require multiple steps involving analysis and interpretation, leading to increased costs and reduced efficiency.

Method used

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  • Primer group for constructing CDR3 region high-throughput sequencing library of human TCR[beta] and application of primer group
  • Primer group for constructing CDR3 region high-throughput sequencing library of human TCR[beta] and application of primer group
  • Primer group for constructing CDR3 region high-throughput sequencing library of human TCR[beta] and application of primer group

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Establishment of TCR sequencing library

[0069] The steps to build a TCR sequencing library using whole blood samples or FFPE slices are as follows:

[0070] Step 1 Isolation of PBMC cells from whole blood

[0071] PBMC cells from whole blood were isolated using Ficoll separation.

[0072] Step 2 Extract total RNA or DNA from the sample

[0073] Use the Trizol method to extract the total RNA in PBMC, use the kit to extract the total RNA of the FFPE slice, and measure the concentration of the extracted total RNA with a nanodrop one ultra-micro ultraviolet spectrophotometer. The test results show that the OD260 / 280 is between 1.8-2.0 between. An Agilent 2100 was used to determine RNA integrity.

[0074] Use the kit to extract DNA from PBMC and FFPE slices, and use nanodrop one to measure the concentration of the extracted RNA. The test results show that the OD260 / 280 is between 1.8-2.0.

[0075] Step 3 total RNA reverse transcription:

[0076] If total RN...

Embodiment 2

[0089] Example 2 Different RNA input amounts of FFPE samples and intra-batch repetition of cell line samples

[0090] Select a case of whole blood sample with good DNA extraction quality, and use 50ng, 250ng, and 500ng of DNA as the initial amount to amplify the target sequence by PCR, build a library, and sequence it. It can be seen that when the DNA quality is good, , DNA as low as 50ng can also get better quality library ( Figure 2A -C)

[0091] One cell line sample was selected, and the library was constructed twice in the same batch, and a total of 2 libraries were obtained, and the sequencing was completed. After obtaining the expression level (nrpm) of the target sequence in each library, Pearson correlation analysis was performed on the two libraries. The correlation coefficient R was above 0.9, and the consistency was very high (see image 3 ).

Embodiment 3

[0092] Repeatability between batches of embodiment 3 cell samples

[0093] One cell line sample was selected, under the same experimental conditions, the same initial amount of library construction (10ng) was constructed, two libraries were obtained in different batches, two sequencing data were performed on the machine, and a total of two sequencing data were obtained. Obtain the expression level (nrpm) of the target sequence in each library, and perform Pearson correlation analysis on the two libraries. The correlation coefficient R is above 0.9, and the consistency is extremely high (see Figure 4 ).

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Abstract

The invention belongs to the technical field of biology, particularly relates to a primer group for constructing a CDR3 region high-throughput sequencing library of human TCR[beta] and an applicationof the primer group, and especially relates to the primer group required for constructing the CDR3 region high-throughput sequencing library of the human TCR[beta] and a library preparation method. The primer group provided by the invention can be used in a comprehensive, efficient, convenient and high-sensitivity TCR detection and analysis method, and has the following characteristics of high compatibility to various samples, high standardization and automation of an experimental operation process, and simple, convenient and rapid analysis process.

Description

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Claims

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Application Information

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Owner BEIJING IMMUPEUTICS MEDICINE TECH LTD
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