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A method for constant temperature amplification of oligonucleotide library applied to DNA data storage

An oligonucleotide and isothermal amplification technology, which is applied in the field of isothermal amplification of oligonucleotide libraries, can solve the problems of complicated preliminary design and chemical modification, accelerated DNA decay speed, and reduced DNA data storage time, etc., so as to prolong DNA Data storage time, the effect of reducing DNA decay rate and improving purity

Active Publication Date: 2021-04-20
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, this technology has the following disadvantages: 1) Precise temperature and cycle control are required, and heat is bound to be released during this process, which limits the application of DNA data storage in the future (because heat is also a factor that affects CPU performance) important factor); 2) The PCR instrument, which can accurately control the temperature and cycle, is required, which relatively increases the cost; 3) The temperature of the PCR process is relatively high, and the high temperature will speed up the decay rate of DNA and reduce the DNA data. Storage time; 4) The PCR process has a certain sequence preference, which will cause some data loss; 5) The PCR product is a double-stranded product, and its 5' end has no phosphate group
Although the stoichiometric oligonucleotide library purification method (SNOP) introduced by Zhang, DY, etc. can achieve simultaneous purification, the preliminary design and chemical modification are more complicated.

Method used

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  • A method for constant temperature amplification of oligonucleotide library applied to DNA data storage
  • A method for constant temperature amplification of oligonucleotide library applied to DNA data storage
  • A method for constant temperature amplification of oligonucleotide library applied to DNA data storage

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Embodiment 1: oligo pool is carried out PCR amplification to increase the total number of molecules of oligo pool

[0083] ①PCR system and procedures

[0084] table 3

[0085] Component 50μL Final Conc. h 2 o

21.5 5хQ5 Reaction buffer 10 1х dNTPs (2.5mM) 4 0.2mM F1 (100μM) 2 4μM R1 (100μM) 2 4μM Oligo pool (0.044ng / μL) 10 0.44ng / 50μL Q5 0.5

[0086] F1 is a sequence in which the 1-5 bases at the 5' end are modified with sulfur on the basis of the sequence shown in SEQ ID NO:3, such as C*T*A*CTCCCACTCGTCTATCT; (Lambda exonuclease is 5' non-phosphorylated The modified oligonucleotide has a particularly weak cutting ability. In order to prevent its degradation, the present invention preferably performs sulfur modification on the base at the 5' end of the sequence of F1, * indicates the modified base);

[0087] R1 is the sequence for modifying the phosphate group at the 5' end on the basis of the...

Embodiment 2

[0092] Example 2: Lambda Exonuclease degrades back to single-stranded DNA oligo pool

[0093] (1) Reaction system

[0094] Table 4

[0095] Component 30μL Final Conc. h 2 o

15.5 - 10хLambda exonuclease reaction buffer 3 1х PCR product(above)(69ng / μL) 10 23ng / μL Lambda Exonuclease (5U / μL) 1.5 0.25U / μL

[0096] After incubating at 37° C. for 3 h, EDTA was added to make the final concentration to 10 mM to terminate the reaction.

[0097] (2) Purify and recover the product through column purification according to the operation steps of the instructions of Eastep Gel and PCR Cleanup Kit.

[0098] (3) Dissolve in 40 μL DNase / RNase-free H2O.

[0099] (4) 12% polyacrylamide gel electrophoresis to verify ssDNA (stained with SYBR Gold for 20 minutes), and at the same time, add a known amount of standard DNA and measure the concentration of ssDNA by grayscale analysis. The results of polyacrylamide gel electrophoresis are shown in ...

Embodiment 3

[0102]Example 3: Capture probe captures ssDNA

[0103] The number of molecules of the corresponding capture probe (complementary to the sequence of the primer 2 region and the specific barcode region) is calculated as the average number of molecules in Example 2 (the number of molecules of the capture probe is converted from a certain concentration provided by the manufacturer, N=c*v*N A , c is the concentration of the capture probe, v is the volume to be added to the capture probe) to capture ssDNA, the ssDNA above the average number of molecules is not captured and will be in a free state, while the ssDNA below the capture probe will be all captured, Excess corresponding capture probes are free. After the capture is completed, polymerase polymerase, and then the free ssDNA and capture probes are degraded by exonuclease I. In this way, the number of DNA molecules of various types in the library tends to the average number, and the concentration of various types of DNA is sh...

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Abstract

The invention relates to the field of biotechnology. The invention develops a method for equalizing the concentration of different types of oligonucleotides in an oligonucleotide library and amplifying them at normal temperature, which can be applied to DNA data storage. This method can equalize the concentration of different kinds of oligonucleotides, and can also improve the purity of oligonucleotide fragments. At the same time, combined with the strand displacement amplification technology, the oligonucleotide library can be amplified at a constant temperature, and the amplified product is ssDNA with a phosphate group, which provides greater convenience for the subsequent next-generation sequencing library preparation . At the same time, the Nickase required in the SDA reaction has a longer recognition sequence, which makes the encoding of data simpler. Moreover, the amplification method of the present invention is a linear amplification—the method of amplifying with the most original template, so compared with PCR, its mutation will not be amplified, and multiple rounds of amplification can be performed.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to an oligonucleotide library constant temperature amplification method applied to DNA data storage. Background technique [0002] Oligo pool is an important reactant in genomics, biophysics, bionic biology and biotechnology applications; oligo pool can be used as primers and probes in the amplification, enrichment, detection and sequencing of biological nucleic acids. Researchers use Oligo pool to achieve gene assembly and synthesis; use it as a probe for SNP genotyping; as scientists discover that DNA can be used as a storage medium to store information on a large scale, researchers use chip synthesis Oligo pool combined with DNA data storage Store various information in DNA. [0003] At present, people are generating data at an exponential growth rate, and its growth rate has exceeded the growth rate of storage hard disks, making human beings have higher demands on s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6844C12Q1/6806C12N15/10C40B50/06
CPCC12N15/1093C12Q1/6806C12Q1/6844C40B50/06C12Q2531/113C12Q2565/125C12Q2563/143C12Q2563/149C12Q2521/101C12Q2521/319C12Q2521/301C12Q2531/119
Inventor 齐浩郜艳敏
Owner TIANJIN UNIV
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