Bladder cancer mutation gene specific primer, kit and library construction method

A technology for mutated genes and library construction, which is applied in biochemical equipment and methods, chemical libraries, microbial assay/testing, etc. It can solve problems such as poor fragment integrity, templates that cannot be covered by double-sided primers at the same time, template loss, etc.

Active Publication Date: 2019-11-19
HUNAN YEARTH BIOTECHNOLOGICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In traditional gene mutation detection methods, double-sided primers are used for amplification. Because the fragmentation of the region to be detected is random, templates with poor fragment integrity cannot be covered by double-sided primers at the same time, resulting in the loss of trace templates and affecting the detection limit.
There are also unilateral enrichment-strand circling (back-to-back primer) modes and un

Method used

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  • Bladder cancer mutation gene specific primer, kit and library construction method
  • Bladder cancer mutation gene specific primer, kit and library construction method
  • Bladder cancer mutation gene specific primer, kit and library construction method

Examples

Experimental program
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Effect test

Embodiment approach

[0092] According to a typical embodiment of the present invention, using the kit of the present invention to detect fragmented DNA of bladder cancer includes the following steps: 1) designing specific primers according to the locus or region of the gene to be detected in bladder cancer; 2) analyzing the fragmented DNA Carry out end repair and fill in and add "A"; 3) Connect adapters to the repaired products; 4) Perform PCR amplification of molecular tags and specific primers on the products connected to the adapters; 5) Perform sequencing primers on specific products introduction; 6) and detection of library products. In the construction of traditional fragmented library, PCR pre-amplification is performed on the template added with the adapter, so that the template reaches a certain amount; when PCR pre-amplification is used, a certain wrong mutation generated by the polymerase will be infinitely magnified exponentially , to increase the noise source. In the present inventio...

Embodiment 1

[0097] Example 1 Fragmented DNA for bladder cancer tumor detection gene (panel) sequencing

[0098] 1.1 Joint design

[0099] ADT-1:

[0100] 5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNCCCTGCGTGACGT*Y-3' is shown in SEQ ID NO.157.

[0101] ADT-2: 5'-P-ACGTCACGCAGGGGAGAGCCAGGGATGACTAGG-3' as shown in SEQ ID NO.158.

[0102] N in the above ADT-1 sequence can be 8 random bases, *Y is a thio-modified A / T base; P in ADT-2 is phosphorylated, and the two sequences need to be annealed into a "Y"-shaped linker . Upstream-specific primers and downstream-specific primers are located to the left and right of the detection point, respectively. The same sequence "AGATGTGTATAAGAGACAG" needs to be added to the 5' ends of the specific primers, see SEQ ID NO.159 (this sequence is a partial sequence of the universal primer). Each primer was first mixed into an upstream-specific primer set and a downstream-specific primer set at a concentration of 0.5 µM. The specific gene locus-specific pr...

Embodiment 2

[0173] According to the above-mentioned Example 1, 16 cases of T1-T2 stage bladder cancer patients were tested for bladder cancer tumor gene

[0174] 2.1 Sample preparation

[0175]Collect 16 patients with bladder cancer preoperative urine stock solution and bladder cancer surgical lesion tissue samples; extract urine supernatant cfDNA, urine precipitate and cancer tissue genomic DNA; genomic DNA was fragmented to obtain fragmented DNA.

[0176] 2.2 Library construction

[0177] The library construction was carried out according to the library construction steps in Example 1, wherein the optimal combination of the upstream specific primer set 1 and the optimal combination 1 of the downstream specific primer set in Table 1 were used for the specific primer set.

[0178] 2.3 Library quality inspection and on-machine sequencing

[0179] Library quality control and on-machine sequencing were performed on the library according to the method in Example 1.

[0180] 2.4 Experimenta...

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Abstract

The invention discloses a bladder cancer mutation gene specific primer, kit and library construction method. A mode of unilateral enrichment-single molecule specific primer (single primer) is used foramplifying sub enrichment technologies for the first time; the application scope of the primer is expanded, the use amount of an effective template is increased, especially the detection limit for detecting low-frequency mutation samples is greatly improved; and secondly, a single molecule specific primer is composed of specific sequences and sequencing sequences, the specificity and the primer use ratio of multiple PCR can be effectively provided. The bladder cancer mutation gene specific primer, kit and library construction method applies the technology to the field of bladder cancer tumormutation gene detection, prepares corresponding reagents, can detect the mutation condition of tumor genes related bladder cancer efficiently and sensitively, and provides guidance for clinical application.

Description

technical field [0001] The invention belongs to the technical field of molecular biology detection, and in particular relates to a bladder cancer tumor mutation gene-specific primer, kit and library construction method. Background technique [0002] Urinary system tumors mainly include bladder cancer, kidney cancer, urethral cancer, etc., and bladder cancer is the most common urinary system tumor. At present, the examination of bladder cancer mainly relies on imaging examination and pathological examination, etc., but generally expensive and invasive means; urine cytology examination is convenient and non-invasive, but the sensitivity is not very high; cystoscopy is the gold standard for diagnosis of bladder cancer , is also one of the main means of postoperative recurrence monitoring, but it is invasive and may cause related complications; these defects limit the early diagnosis and treatment of bladder cancer, as well as prognosis monitoring. [0003] Researchers continue...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/11C12Q1/6806C40B50/06
CPCC12Q1/6806C12Q1/6886C12Q2600/156C12Q2600/16C40B50/06C12Q2525/191C12Q2531/113
Inventor 王龙朱丽芳陆利曹曼曼徐根明潘艺赵谦
Owner HUNAN YEARTH BIOTECHNOLOGICAL CO LTD
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