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Direction-specific single-stranded DNA preparation mediated by chemically modified primers for pyrosequencing

A technology of pyrosequencing and chemical modification, applied in the field of direction-specific single-stranded DNA preparation, which can solve the problems of increased instrument cost, increased detection cost, and cross-contamination of samples

Active Publication Date: 2017-11-24
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The principle of the streptavidin microsphere solid-phase method is that the target DNA fragment is firstly amplified by PCR using biotin-labeled primers, and then the amplified fragment is immobilized on the streptavidin-coated microspheres, which are not biotin-labeled. The DNA chain is denatured with NaOH, the DNA chain is eluted and washed, and after removing all other reaction components, the sequencing primer is added and annealed to form a pure single-stranded DNA template. The operation steps of this process are cumbersome and inefficient, and expensive biotin The use of labeled primers and streptavidin-coated microspheres increases the cost of detection, the complex vacuum system increases the cost of the instrument, and the single-strand preparation process is also prone to cross-contamination of samples. These problems seriously affect the application of pyrosequencing and popularity

Method used

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  • Direction-specific single-stranded DNA preparation mediated by chemically modified primers for pyrosequencing
  • Direction-specific single-stranded DNA preparation mediated by chemically modified primers for pyrosequencing
  • Direction-specific single-stranded DNA preparation mediated by chemically modified primers for pyrosequencing

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Embodiment 1

[0030] Example 1. Enzyme digestion verification and principle explanation of thio primer PCR product

[0031] In order to verify the protective effect of phosphorothioation modification on PCR products, the inventors used the genome treated with bisulfite as a template, introduced phosphorothioation modification into PCR products using different PCR primers, and finally produced PCR products with different phosphorothioation modifications at the ends, such as figure 2 , including four types: a. Both ends have no phosphorothioation modification, b. Reverse phosphorothioation modification, c. Forward phosphorothioation modification, d. Both ends have phosphorothioation modification . In the figure, N represents the normal amplification product and S represents the phosphorothioation modified amplification product.

[0032] Sodium bisulfite treatment of genomic DNA:

[0033] Genomic DNA was treated with Zymo-Research’s Methylation Kit, and 1 μg of the extracted human cell lin...

Embodiment 2

[0041] Example 2, human 293T cell β-actin gene exon pyrosequencing

[0042] The operation flow of this embodiment is as follows figure 1 , including:

[0043] 1. Sodium bisulfite treatment of genomic DNA

[0044] Genomic DNA was treated with Zymo-Research’s Methylation Kit, and 1 μg of the extracted human cell line 293T genomic DNA was added to 130 μl of CT Conversion Reagent (containing sodium bisulfite). Put the sample into the PCR machine, and set the reaction program as follows: 98°C, 10min; 64°C, 2.5h; 4°C, ∞.

[0045] Put the Zymo column (Zymo-Spin(TM) IC Column) into the collection tube (Collection Tube), add 600μl M-binding solution (M-Binding Buffer), then add the reacted sample, cover tightly, and mix by inversion; Centrifuge at 12,000rpm (<10,000g) for 30s; add 100μl of M-Wash Buffer, centrifuge at 12,000rpm for 30s; add 200μl of M-Desulphonation Buffer, and let Place for 15-20min, centrifuge at 12,000rpm for 30s; add 200μl M-Wash Buffer, centrifuge at 12,000rpm...

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Abstract

The invention relates to preparation of chemical modification primer-mediated direction-specific single chain DNA (Deoxyribonucleic Acid) applied to pyrosequencing. In particular, an ordinary synthesized primer is taken as a primer in a PCR (Polymerase Chain Reaction), and the other primer can be an enzyme-digestion-resistant chemical modification primer. After PCR amplification, double chain DNA is treated by using a 5' to 3' cutting function, and an unmodified DNA single chain is digested to prepare the direction-specific single chain DNA. The single chain DNA prepared by using the method can be directly taken as a template for use in pyrosequencing, so that the cost of pyrosequencing is reduced greatly, and the complex steps in preparation of a sequencing template are reduced.

Description

technical field [0001] The invention belongs to the field of molecular biology; more specifically, the invention relates to the direction-specific single-stranded DNA preparation mediated by chemically modified primers applied to pyrosequencing. Background technique [0002] Pyrosequencing is a new sequencing technology different from the Sanger method. It uses single-stranded DNA as a template, and in the presence of DNA polymerase, an extension reaction occurs when primers complementary to the template and dNTP are added to release a corresponding amount of pyrosequencing. Phosphoric acid, under the action of a series of substrates, produces a fluorescent signal with an intensity corresponding to the number of template bases, which is finally used for detection. Compared with the Sanger method, pyrosequencing can quickly generate sequence data, and has extremely high accuracy and reproducibility. At present, pyrosequencing technology has been widely used in SNP detection,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/10
CPCC12Q1/6806C12Q1/6869C12Q2535/101C12Q2565/301
Inventor 于文强李晋李岩赵丽萍董世华
Owner FUDAN UNIV