Method for synthesizing DNA nanosphere complementary chains and sequencing method

Abstract

The invention relates to a method for synthesizing DNA nanosphere complementary chains and a sequencing method. The method comprises the following steps: treating a product after one chain sequencingof a DNA nanosphere by using an enzyme, wherein a primer for one chain sequencing is modified, and after enzyme treatment, the modification is destroyed by the enzyme, so that the primer is digested;hybridizing a multiple displacement amplification primer with the product obtained in the previous step, wherein the multiple displacement amplification primer at least comprising a first primer, andthe first primer is completely or partially complementary to a primer binding region of one chain of sequencing; and under the action of polymerase with a chain displacement function, carrying out chain displacement reaction by taking the multiple displacement amplification primer and one chain as primers to generate DNA nanosphere complementary chains. According to the method disclosed by the invention, one chain of occupied primers are removed before MDA, and the position is vacated to carry out MDA primer hybridization, so that the quantity of hybridization primers is increased, and the copy number of DNB is increased.

Description

technical field [0001] The invention relates to the technical field of sequencing, in particular to a method for synthesizing complementary chains of DNA nanoballs 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, based on the principle of MDA to generate DNA nanoball (DNB) complementary strands (Rongqin Ke, et.al., US20160237488 A1) to achieve the sequencing of DNB paired ends. [0003] In the paired-end sequencing of DNB, after the first strand is sequenced, its complementary strand needs to be generated, and then its complementary strand should be sequenced. Therefore, the generation of complementary strands has a great influence on the q...

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Problems solved by technology

like figure 1 As shown, the existing method is to use the sequencing strand of the first strand and the label (Barcode) primer on the new hybrid to generate a complementary strand, and after the first strand is sequenced through a long cycle (cycle), the sequencing primer of the first strand has already occupied the position, so Only one of the MDA primers (i.e., the Barcode primer) can fully hybridize to the adapter, and the other primer is difficult to hybridize. When performing MDA, only two primers (i.e., the Barcode primer and one sequencing strand) are amplified, and the sequencing strand primer The replacement efficiency will be low, which will affect the copy number of DNB, so that the sequencing signal will be low

In addition, after a strand is sequenced through a long cycle (cycle), some sequencing enzymes are bound to the sequencing strand and the remaining scar (scar) after excision of dNTP with fluorescence blocking will affect the displacement effect of Phi29 DNA polymerase, thereby affecting the copy of DNB number, the fluorescence signal is low, and it will also affect the quality of sequencing

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