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Method, kit and use for synthesizing DNA nanosphere complementary chain and sequencing method

A technology of nanospheres and complementary chains, applied in the field of sequencing, can solve the problems of low sequencing signal, affecting sequencing quality, and low replacement efficiency, and achieve the effect of increasing copy number, increasing sequencing signal and quality

Active Publication Date: 2022-03-22
MGI TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the low replacement efficiency of the first sequencing strand, which affects the DNB copy number, the sequencing signal is low
In addition, after a strand is sequenced through a long cycle (cycle), some sequencing enzymes cannot be completely eluted on the sequencing strand, and the remaining scar (scar) after excision of dNTP with fluorescence blocking will affect the displacement effect of Phi29 DNA polymerase, Thus affecting the copy number of DNB, making the fluorescent signal low, and also affecting the quality of sequencing

Method used

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  • Method, kit and use for synthesizing DNA nanosphere complementary chain and sequencing method
  • Method, kit and use for synthesizing DNA nanosphere complementary chain and sequencing method
  • Method, kit and use for synthesizing DNA nanosphere complementary chain and sequencing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] In this example, taking the BGISEQ500 sequencing platform as an example, the IP-P primer is used as a modified primer to demonstrate the PE100 sequencing effect of the present invention.

[0085] 1) Synthesize a modified one-strand sequencing primer (IP-P primer in Table 1), and dilute it with 5×SSC (sodium citrate buffer) to a working solution concentration of 1 μM, as shown in Table 2 below, in which the mother solution 20 ×SSC needs to use H 2 O for dilution.

[0086] Table 2

[0087] components Final concentration 100 μM IP-P or 100 μM IP1 1μm 20×SSC 5×

[0088] 2) Mix the two primers according to the ratio of IP-P:IP1=1:20, as shown in Table 3 below:

[0089] table 3

[0090] components volume 1 μM IP-P 10mL 1 μM IP1 200mL

[0091] 3) DNB (DNA nano Ball, DNA nanoball) was prepared using DNB preparation kit (BGI Corporation).

[0092] 4) DNB was loaded onto the surface of the chip using a DNB loading k...

Embodiment 2

[0107] In this example, taking the BGISEQ500 sequencing platform as an example, using IP-U primers, the PE100 sequencing effect of the present invention is demonstrated.

[0108] 1) Synthesize a modified one-strand sequencing primer (IP-U primer), and dilute it with 5×SSC to a concentration of 1 μM in the working solution, as shown in Table 6 below, where the mother solution 20×SSC needs to use H 2 O for dilution.

[0109] Table 6

[0110] components Final concentration 100 μM IP-U or 100 μM IP1 1μM 20×SSC 5×

[0111] 2) Mix the two primers according to the ratio of IP-U:IP1=1:20, as shown in Table 7 below:

[0112] Table 7

[0113] components volume 1 μM IP-U 10μL 1 μM IP1 200μL

[0114] 3) DNB was prepared using a DNB preparation kit (BGI Corporation).

[0115] 4) DNB was loaded onto the surface of the chip using a DNB loading kit (BGI Corporation), and the mixed primers were hybridized to the DNB.

[0116] 5) ...

Embodiment 3

[0134] In this example, taking the BGISEQ500 sequencing platform as an example, according to the ratio of the modified primer (IP-P) to the normal primer (IP1) of 1:10, the PE100 sequencing effect of the present invention is demonstrated. Refer to Example 1 for the specific steps, and the results are shown in Table 11. The results show that the experiment was carried out at a ratio of 1:10, and the result was close to that of the ratio of 1:20, and a good PE100 result could also be achieved.

[0135] Table 11

[0136]

[0137]

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Abstract

A method for synthesizing complementary strands of DNA nanospheres, a kit and its use, and a sequencing method, wherein the method for synthesizing complementary strands of DNA nanospheres includes: using a sequencing primer to combine with a DNA nanosphere template strand to perform one-strand sequencing, wherein one strand Sequencing primers include modified primers and normal primers. The 3' end of the modified primer includes a blocking modification so that the primer cannot be extended; the blocking modification is removed so that the changed modified primer can be extended on the DNA nanoball template strand under the action of the polymerase ; hybridize multiple displacement amplification primers to the DNA nanoball template strand after one-strand sequencing, and perform a strand displacement reaction to synthesize the complementary strand of the DNA nanosphere template strand. In this method, specially modified primers are mixed with normal one-strand sequencing primers. Without affecting the quality of one-strand sequencing, part of the primers hybridized to the DNA nanospheres cannot be extended. Before MDA, the modification on the specially modified primers is removed. , so that these primers are used to synthesize complementary strands and increase the copy number of complementary strands of DNA nanospheres.

Description

technical field [0001] The invention relates to the technical field of sequencing, in particular to a method for synthesizing complementary chains of DNA nanospheres, a kit and use thereof, and a sequencing method. Background technique [0002] Multiple Displacement Amplification (MDA) technology is widely used in whole-genome amplification, and it is currently the whole-genome amplification method with the widest coverage of the entire genome and the smallest amplification bias at each site. At present, this method is also used in the field of high-throughput sequencing, and DNA nanoball (DNB) complementary chain synthesis is performed based on the principle of MDA (Rongqin Ke, et.al., US20160237488 A1) to achieve the sequencing of DNB paired ends. [0003] In paired-end sequencing, after the sequencing of one strand of DNB is completed, its complementary strand needs to be generated, and then its complementary strand should be sequenced. Therefore, the generation of compl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12Q1/6869C12Q1/6806
CPCC12Q1/6869C12Q1/6806C12Q2531/119C12Q2537/143C12Q2535/122C12Q2537/1376
Inventor 王卉徐讯温晴唐国鑫邢承美章文蔚徐崇钧陈奥杨晋
Owner MGI TECH CO LTD
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