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Single molecule label and application thereof

A single-molecule and labeling technology, applied in the field of molecular biology, can solve the problems of inability to effectively trace the sequencing read length, complicated operation steps, and high equipment requirements, and achieve the effect of simple and efficient introduction method, ingenious conception, and remarkable functions

Active Publication Date: 2018-11-09
深圳海普洛斯医学检验实验室
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, none of the above existing technologies can effectively trace the source of sequencing reads, and the operation steps are complicated, requiring high equipment requirements, making it difficult to apply and promote

Method used

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  • Single molecule label and application thereof
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  • Single molecule label and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] (1) A tag, the nucleotide sequence of which is shown in SEQ ID NO.1:

[0044] SEQ ID NO.1 is Oligo: -CATAG / dU / NNNNNNNNCTATGT 3’

[0045] Phosphorylation modification at the 5' end of Oligo ( And the five bases at the 5' end are reverse complementary to the 3' end, forming a stem-loop structure after annealing, and the label Barcode (that is, random sequence Poly dNTP) of the overhanging thymine at the 3' end, in the formed stem-loop structure, The first unpaired base near the 5' end is uracil (U).

[0046] (2) Sample processing:

[0047] If the sample to be processed is gDNA, use the fragmentation enzyme of NEB company to fragment the gDNA of 2 μg sample, and select the DNA fragment of 100-250 bp by the method of magnetic bead double screening or gel cutting, the fragmentation enzyme treatment conditions: 37°C, 43min , dissolve the fragment-selected DNA with 30 μL deionized water;

[0048] If the processed sample is cfDNA, it can be directly used in the subsequen...

Embodiment 2

[0074] (1) A tag, the nucleotide sequence of which is shown in SEQ ID NO.1:

[0075] SEQ ID NO.1 is Oligo: -CATAG / dU / NNNNNNNNCTATGT 3’

[0076] Phosphorylation modification at the 5' end of Oligo ( And the five bases at the 5' end are reverse complementary to the 3' end, forming a stem-loop structure after annealing, and the barcode of the overhanging thymine label at the 3' end, in the formed stem-loop structure, the first one near the 5' end The unpaired base is uracil (U).

[0077] (2) Sample processing:

[0078] If the sample to be processed is gDNA, use the fragmentation enzyme of NEB company to fragment the gDNA of 2 μg sample, and select the DNA fragment of 100-250 bp by the method of magnetic bead double screening or gel cutting, the fragmentation enzyme treatment conditions: 37°C, 43min , dissolve the fragment-selected DNA with 30 μL deionized water;

[0079] If the processed sample is cfDNA, it can be directly used in the subsequent experimental process.

[0...

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Abstract

The invention provides a single molecule label and application thereof. The label is any one selected from a group consisting of a stem ring structure formed by annealing of one oligo, a structure formed by two annealed oligos with both ends in complementary pairing, and a structure formed by two annealed oligos with one ends in complementary pairing, wherein the 3' terminal of the label extends while the 5' terminal of the label is phosphorylated; and a ring structure not in complementary pairing is modified by uracil. According to the invention, single molecule coding and labeling of both terminals of DNA are realized through selection of a specific structure and a modified label and cooperative utilization of a simple and efficient introduction method, the original source of the read length of sequencing is more effectively located, and manpower and material resources are saved; and the single molecule label of the invention has promotion and application prospects and value.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a single molecule label and its application. Background technique [0002] As a very important experimental technology, DNA sequencing has been widely used in the field of biological research, especially in the genetic testing industry. From the initial end-termination method to the relatively mature second-generation sequencing technology (Next-Generation Sequencing). Compared with first-generation sequencing, the current next-generation sequencing has lower cost, higher throughput, and faster speed. [0003] The basic principle of Illumina sequencing is sequencing by synthesis. During the library construction process, the genomic DNA needs to be randomly fragmented, and specific sequencing adapters (Adaptors) are added to both ends of the fragmented genome, and then PCR amplification of the library is performed. Due to the randomness of the final PCR amplification and fragme...

Claims

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Application Information

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IPC IPC(8): C12Q1/6876C12Q1/6806C40B40/06
CPCC12Q1/6806C12Q1/6876C40B70/00C12Q2563/185C12Q2525/301C12Q2525/173C12Q2521/525C12Q2531/113C12Q2521/531
Inventor 张晓妮屈宏越许明炎
Owner 深圳海普洛斯医学检验实验室