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Method for constructing high-flux and low-cost Fosmid library, label and label joint used in method

A tag and library technology, applied in the field of nucleic acid sequencing, high-throughput and low-cost Fosmid library construction, can solve the problems of laborious and time-consuming purification steps and loss of DNA, so as to reduce sequencing costs, reduce purification steps, and save library construction. effect of time

Active Publication Date: 2012-04-11
WUXI QINGLAN BIOLOGICAL SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The purification steps are laborious and time-consuming, and the more purification steps, the more DNA is lost

Method used

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  • Method for constructing high-flux and low-cost Fosmid library, label and label joint used in method
  • Method for constructing high-flux and low-cost Fosmid library, label and label joint used in method
  • Method for constructing high-flux and low-cost Fosmid library, label and label joint used in method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] 1. Use four 96-well plates, respectively named plate No. 1, plate No. 2, plate No. 3, and plate No. 4. Each well contains 40 ul of 50 ng / ul Fosmid sample, and use the E210 model breaker produced by Covaris to break , the interrupt parameters are as follows:

[0060]

[0061] 2. Purify after interruption, take out 40ul from each well of the four 96-well plates into a new 96-well plate, recover and purify with Backman’s Ampure magnetic beads, and finally dissolve the samples in each well in 45ul ultrapure water, and take 42ul carry out the subsequent reaction.

[0062] 3. For end repair, configure the enzyme digestion reaction system in each well of four 96-well plates according to the following table:

[0063]

[0064] Place on a PCR instrument at 20°C for 30 minutes, Ampure magnetic beads are purified and dissolved in 22ul ultrapure water, and 17.7ul is taken out from each well and placed in a new 96-well plate for the following reaction.

[0065] 4. Add "A" bas...

Embodiment 2

[0090] Illumina HiSeq Sequencing

[0091] According to the Q-PCR detection results, eight PCR products with a fragment size of 800 bp were combined in equal amounts and sequenced using the index PE101+8+101 cycle program. For details, see the Illumina HiSeq operating instructions. The results of the sequencing run are as follows:

[0092]

[0093] It can be seen from the results in the table that the deviation of the inserted fragment is less than 50bp, which meets the requirements, and the total data output is greater than 10G, indicating that the library was successfully built.

Embodiment 3

[0095] Result analysis

[0096] The sequencing results produced by Illumina HiSeq are a series of DNA sequences. By searching the adapter tag sequence, positive and negative primer tag sequences, and primer sequences in the sequencing results, the sequence information of the sample corresponding to each primer tag is established.

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Abstract

Based on a test platform of the Illumina Company at present and aiming at the characteristic of a small section of a Fosmid library, a plurality of libraries are mixed together to form one library; furthermore, a purifying step is omitted, so a new Fosmid library preparation flow is established. The Fosmid library is constructed successfully; the Fosmid library is successfully used for sequencing; therefore, the library construction time is saved and the sequencing cost is reduced.

Description

technical field [0001] The invention relates to the technical field of nucleic acid sequencing, in particular to the technical field of high-throughput and low-cost Fosmid library construction. In addition, the present invention also relates to labeling technology and a method for constructing a label library in the same reaction system for multiple samples. The method of the present invention is particularly suitable for the second generation sequencing technology, especially the solexa sequencing technology. Background technique [0002] DNA sequencing technology has played a vital role in advancing molecular biology since its invention [1] . In the past 30 years, as one of the most important biomedical research methods, the data output capacity of DNA sequencing technology has grown exponentially [2] . From the early manual sequencing of Frederick Sanger, and the first-generation automated sequencer developed based on the Sanger method, to the current next-generation ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C40B40/06C40B50/06
CPCC40B50/06C12N15/1065C40B40/06
Inventor 樊帆张俊青胡帅星王博孔淑娟程玲
Owner WUXI QINGLAN BIOLOGICAL SCI & TECH
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