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Primers, methods and kits for pretreatment of next-generation sequencing samples

A primer set and sequencing library technology, applied in the field of molecular biology, can solve the problems affecting the quality of the library construction, not suitable for rapid library construction, complicated operation steps, etc. Effect

Active Publication Date: 2020-08-25
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Conventional library construction methods require a relatively high concentration of starting nucleic acid, and the operation steps are cumbersome. Pretreatment is required to connect the sequencing adapter to the target gene fragment, followed by PCR amplification and enrichment, which is time-consuming and laborious, and the adapter ligation Efficiency directly affects the quality of library construction, so it is not suitable for rapid library construction of large-scale clinical samples and trace nucleic acid samples

Method used

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  • Primers, methods and kits for pretreatment of next-generation sequencing samples
  • Primers, methods and kits for pretreatment of next-generation sequencing samples
  • Primers, methods and kits for pretreatment of next-generation sequencing samples

Examples

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Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Primers for Pretreatment of Next Generation Sequencing Samples

[0063] 1. Linker sequence

[0064] The present invention designs linker sequences with high specificity, as shown in Table 1.

[0065] Table 1 Linker sequence

[0066] name Base sequence (5'→3') SEQ ID NO. A1 connector CAGTCCTAGTGACAAGTCGACTC 1 A2 connector CGCATTGACCAGTACAGTCCTAGA 2 A3 connector ACCGTGACTGTCGAACTGATCGA 3 A4 connector CACGTTCGGAATGCCTTACGTACT 4 A5 connector AGTCCTTGAATCGCTAAGCGCTCG 5 A6 connector CAAGTCGATAGCGAATCTGACCT 6 A7 connector CTTGAACGTCAGTCAAGCTCGCA 7 A8 connector CGTATGGACTTGACCTGCATGGAC 8 A9 connector AAGTCGGTCAGCCTTGAGTACCG 9 A10 connector CGGATCGAACGTACCTATGCGAA 10 A11 connector CCTAGTTCGATCCGTGCGACTGA 11 A12 connector ATTGCCATGTACGGTAACTGCATC 12 A13 connector ACATGGTCAATGCCTATGCAACTC 13 A14 connector CACTGAACTGATCCAATGCATAGG 14 A15 connec...

Embodiment 2

[0091] Example 2 Kit for Pretreatment of Next Generation Sequencing Samples

[0092] The kit described in this example has three types, all of which are adapter PCR primers composed of different linking sequences.

[0093] Wherein, the kit A described in this embodiment uses the linker sequence 1 to construct the adapter PCR primer, mainly including:

[0094] 1. Different adapter PCR primer sets for different samples

[0095] The adapter PCR primer sequence consists of adapter sequence, DNA tag sequence and connection sequence 1 from 5' to 3' end. The linker sequences selected by all linker PCR primers in the same kit are the same, and the linker sequences in this example are selected from 2 of the linker sequences (Table 1) in Example 1; the DNA tag sequences of the same primer set are the same, and the DNA tag sequences of different primer sets The sequences are different. In this embodiment, the DNA tag sequence is selected from the DNA tag sequence in Example 1 (Table 2)...

Embodiment 3

[0110] Example 3 Using the kit A in Example 2 to detect the sample

[0111] In this example, the kit A in Example 2 was used to detect 20 samples. The 20 samples were respectively derived from the lung cancer cell line NCI-H1975, the prostate cancer cell line LNcap, the gastric cancer cell line HGC-27, and the breast cancer cell line MCF. -7. The liver cancer cell line HepG2 and the negative control lymphoblastoid CCRF-HSB-2 can be purchased from the company as long as those skilled in the art know the name of the cell line. Five replicates were set up for each cell line to form 30 test samples. The sample numbers and corresponding linker PCR primers are shown in Table 7.

[0112] Table 7 Sample-corresponding adapter PCR primers

[0113]

[0114] 1. Extraction of sample DNA

[0115] Prepare 5 repetitions of the above-mentioned various spare cell lines to form 30 test samples in Table 7. Genomic DNA was extracted with BioVision's Genomic DNA Isolation Kit (product number:...

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Abstract

The invention relates to an adapter sequences, which are at least two sequences selected from SEQ ID NO:1-SEQ ID NO:20. The invention also relates to a tag adapter, which is composed of an adapter sequence and a DNA tag sequence. The invention also discloses an adapter PCR primer combination. Each adapter PCR primer is composed of the above tag adapter and a linker sequence connected to 3' end of the tag adapter. By the use of the linker sequence, the tag adapter can be directly added to two ends of a target gene fragment so as to construct a sequencing library. Through the adapter PCR primers, a sequencing adapter, a sample tag and an amplification primer can be added to two ends of the target gene fragment at a time. Then, through PCR enrichment, the sequencing library can be obtained. Therefore, the steps of genomic DNA fragmentation, purification, end repairing, purification, addition of A tail, purification and adapter connection during the conventional library construction process are avoided; the influence of various unstable factors such as repeated operations and adapter connection efficiency and the like on the quality of the constructed library is avoided; and the detection accuracy is greatly raised.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, and in particular relates to a primer, a method and a kit for the pretreatment of next-generation sequencing samples. [0002] technical background [0003] With the development of molecular biology, genetics and other disciplines, traditional Sanger sequencing can no longer fully meet the needs of research. For genome sequencing, sequencing technology with lower cost, higher throughput and faster speed is needed. Second-generation sequencing Technology came into being. The second-generation sequencing technology is also called deep sequencing and massively parallel sequencing. Its core idea is to sequence while synthesizing, that is, to determine the sequence of DNA by capturing the label of the newly synthesized end, for example, when synthesizing a new DNA complementary strand When the added dNTP catalyzes the substrate to excite fluorescence through an e...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6869C12N15/11C40B50/06
CPCC12N15/1068C12Q1/6869C12Q2525/191C12Q2531/113C12Q2535/122
Inventor 刘志明吴诗扬廖传荣
Owner SUREXAM BIO TECH