Linker, primer group and kit for cfDNA library construction and library construction method
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A kit and primer set technology, applied in the field of molecular genetics, can solve the problems of limited detection sensitivity, cfDNA cannot be filled with junctions, low cfDNA content, etc., and achieve the effects of improving detection sensitivity, stable sequencing results, and low sequencing costs.
Inactive Publication Date: 2017-06-20
ANHUI ANKE BIOTECHNOLOGY (GRP) CO LTD
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However, there are two huge technical obstacles in the accurate sequencing of cfDNA: first, the cfDNA content in plasma is extremely low, and most of them are characterized by small fragment molecules, resulting in difficult extraction, large loss, and low detection sensitivity, which limits its clinical application. application on
Secondly, because cfDNA is highly fragmented, the current method of cfDNA library construction on the market, such as adding A at the end of double-stranded DNA and then adding adapters for amplification, will cause some cfDNA to fail to fill in and add adapters, resulting in cfDNA fragmentation. Attrition, so that some gene mutation sites cannot be detected, further limiting the detection sensitivity
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Embodiment 1
[0023] The invention provides a cfDNA library construction kit product, which includes:
[0024] A single-stranded linker A for connecting and labeling the denatured single-stranded cfDNA, the 3' end of the single-stranded linker A is modified with biotin;
[0025] Single-stranded ligase for ligation of single-stranded cfDNA and single-stranded adapter A and buffer for single-stranded ligase;
[0026] A double-stranded adapter B for connecting double-stranded cfDNA, said double-stranded adapter B comprising adapter B1 and adapter B2;
[0027] T4 DNA ligase for ligation of double-stranded adapter B of double-stranded cfDNA and buffer for T4 DNA ligase;
[0028] Primer CL9 for unidirectional amplification of single-stranded cfDNA and primers IS7 and IS8 for amplifying library molecules for library detection:
[0029]
[0030] Wherein, the double connector B is made by the following method:
[0031] Perform PCR on the following system, react at 95°C for 10S, and then cool d...
Embodiment 2
[0054] A method for building a cfDNA library, comprising the steps of:
[0055] (1) Dephosphorylate and denature cfDNA to obtain single-stranded cfDNA;
[0056] (a1): According to the instructions for use of the QIAamp Circulating Nucleic Acid Kit (50) kit provided by QIAGEN, use the kit to extract free cfDNA in plasma;
[0057] (b1): Add the following components to the PCR tube:
[0058]
[0059] Using a PCR instrument, first react at 37°C for 10 minutes, then react at 95°C for 2 minutes;
[0060] Take out the PCR tube, quickly put it into the ice-water mixture, keep it in the ice bath for at least 1min, take it out, centrifuge it, and store it at room temperature.
[0061] (2) Ligate the single-stranded cfDNA and the single-stranded linker A modified at the 3' end to obtain the single-stranded ligation product modified at the 3' end
[0062] (a2): Add the following components to the PCR tube:
[0063]
[0064]
[0065] Use a PCR instrument, react at 60°C for 1 h...
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Abstract
The invention belongs to field of molecular genetics, and particularly relates to a linker, a primer group and a kit for cfDNA library construction and a library construction method. The linker comprises a single-chain linker A and a double-chain linker B; the primer group comprises a sequence shown as SEQ ID NO.4-6; the kit comprises the linker and the primer group; the library construction method comprises the following steps: (1) dephosphorylating and denaturating cfDNA, to obtain the single-chain cfDNA; (2) connecting the single-chain cfDNA with a single-chain linker A with a modified 3'terminal, to obtain a single-chain connecting product with a modified 3'terminal; (3) unidirectionally amplifying a first-time connecting product, to obtain a double-chain cfDNA; (4) connecting the double-chain cfDNA with a double-chain linker B, to obtain a double-chain connecting product; (5) removing one chain with modified 3'terminal in the double-chain connecting product; and (6) performing library quality inspection. According to the linker, the primer group and the kit for cfDNA library construction and library construction method, the cfDNA is firstly denaturated so that the double chain is changed to single chain, then the single chain is connected with the linker through a single-chain connecting enzyme so as to cause almost all cfDNA to be captured, the loss of cfDNA can be avoided, and further the detection sensitivity can be improved.
Description
technical field [0001] The invention belongs to the field of molecular genetics, and in particular relates to a linker, a primer set, a kit and a method for building a cfDNA library. Background technique [0002] cfDNA, or circulating tumor DNA, is the remnants of circulating tumor cells (CTCs) and dead tumor cells in the body left in plasma during cellular metabolism. cfDNA high-throughput sequencing technology is a kind of liquid biopsy. It only needs to collect venous blood like liver and kidney function tests to accurately measure the tumor gene information in the whole body. Based on the tumor gene information, it can guide the selection of anti-tumor drugs , curative effect evaluation, prognosis judgment, and even early detection and early diagnosis. However, there are two huge technical obstacles in the accurate sequencing of cfDNA: first, the cfDNA content in plasma is extremely low, and most of them are characterized by small fragment molecules, resulting in diffic...
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