Transposon end compositions and methods for modifying nucleic acids

A technology of transposon and composition, applied in the field of composition and method of transposon end for modifying nucleic acid, can solve the problems of low efficiency, high cost, tediousness and the like

Active Publication Date: 2011-11-30
EPICENT TECH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

They found that an alternative method called "adapted focused acoustics" gave higher yields of fragmented DNA and that about 17% of the starting DNA consisted of fragments in the expected 200-bp size range, but even this This method is also wasteful in terms of sample or target DNA
Furthermore, the resulting DNA fragments often require size selection by gel electrophoresis and the additional step of tagging the size-selected DNA fragments, which is difficult, laborious, time-consuming and expensive
[0011] Consequently, many of the methods currently used to fragment and tag double-stranded DNA for use in next-generation sequencing waste DNA, require expensive fragmentation instruments, and procedures for fragmenting, tagging, and recovering tagged DNA fragments are difficult, tedious, laborious, time-consuming, inefficient, costly, and require relatively large amounts of sample nucleic acid
Furthermore, many of these methods produce tagged DNA fragments that do not fully represent the sequences contained in the sample nucleic acids from which they were produced

Method used

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  • Transposon end compositions and methods for modifying nucleic acids
  • Transposon end compositions and methods for modifying nucleic acids
  • Transposon end compositions and methods for modifying nucleic acids

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0474] Using EZ-Tn5 TM Transposases and EZ-Tn5 TM In vitro transposition-mediated DNA fragmentation and 5' tagging of transposon ends

[0475] The following reaction mixtures were assembled:

[0476]

[0477] * In some embodiments, two different pMEDS transposon ends ( Figure 4 ).

[0478] After mixing, the reaction was incubated at 37°C for 1 hour. The reaction was stopped with 10 microliters of stop solution (15% sucrose, 66 mM EDTA, 20 mM TRIS, pH 8.0, 0.1% SDS, 0.9% Orange G [Sigma O-7252], and 100 micrograms per milliliter of proteinase K), mixed and Heat at 50°C for 10 minutes.

[0479] DNA was analyzed by 1% agarose gel electrophoresis in TAE buffer. DNA was separated into size grades using LMP agarose. Gels were stained with SYBR Gold and DNA was visualized with non-UV light. The gel slices of LMP were incubated at 70°C for 5 minutes to liquefy the gel. After 5 minutes at 37°C, add one percent volume of Gelase TM Agarose Digestion Solution (EPICENTRE Bio...

Embodiment 2

[0482] Use different EZ-Tn5 TM Size range of transposition products of 5'-tagged DNA fragments at Tn5 transposase concentration.

[0483] Add Tn5 highly active EZ-Tn5 at a concentration of 90 units per microliter TM Transposase (EPICENTRE) was diluted to final concentrations of 45, 22.5, 11.3 and 9 units per microliter. Combine two microliters of each concentration of enzyme with 1 microgram of bacteriophage T7 D111 target DNA (with a size of approximately 39 Kbp) and 1 micromolar of pMEDS transposon ends in TA buffer in 50 microliters of final reaction mixture at 37 °C Incubate in volume for 1 hour.

[0484] Reactions were stopped with 10 microliters of stop solution containing 15% sucrose, 66 mM EDTA, 20 mM Tris / HCl pH 8.0, 0.1% SDS, 0.9% Orange G and 100 micrograms per milliliter proteinase K. After mixing and incubating for 10 minutes at 50°C, 10 microliter aliquots were electrophoresed on a 1% agarose gel in TAE buffer at 100 volts for 1 hour. Gels were stained with ...

Embodiment 3

[0487] Size range of transposition products of 5′-tagged DNA fragments using different concentrations of pMEDS transposon ends.

[0488] use T 10 E 1 Buffer 25 micromolar stocks of pMEDS transposon ends were serially diluted 2-fold, 4-fold and 8-fold. Then, 2 μl of each transposon-end dilution and no transposon-end buffer control in a buffer containing 1×TA buffer, 1 μg of phage T7 D111 target DNA, and 0.4 units / μl of high temperature at 37°C. Incubate 50 µl reactions of active Tn5 transposase for 1 hr.

[0489] Reactions were terminated as described in Example 2 and samples were analyzed by 1% agarose gel electrophoresis.

[0490] A 4-fold dilution of the 25 uM stock to give a final concentration of 0.25 micromolar pMEDS transposon ends in the reaction mixture produced good fragmentation of the target DNA and was probably the most efficient in terms of use of pMEDS transposon ends. At this concentration, most of the phage T7 D111 target DNA was fragmented into DNA that mi...

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Abstract

The present invention provides the use of transposases and transposon ends to generate extensive fragmentation and 5′-tagging of double-stranded target DNA in vitro, followed by the use of DNA polymerases to generate 5′- and 3′-tagged single DNA without PCR amplification reactions. Methods, compositions and kits for stranded DNA fragments, wherein the first label on the 5' end shows the sequence of the transferred transposon end and optionally an additional arbitrary sequence, and the second label on the 3' end shows the same The first tab shows a sequence different from the sequence. The method can be used to generate 5' and 3' tagged DNA fragments for use in a variety of processes including metagenomic analysis of DNA in environmental samples, copy number variation (CNV) analysis of DNA, and including massively parallel DNA sequencing (so-called "next generation sequencing") involves the process of comparative genome sequencing (CGS).

Description

[0001] This application claims US Provisional Application Serial Nos. 61 / 108,321 filed October 24, 2008, 61 / 108,326 filed October 24, 2008, 61 / 108,329 filed October 24, 2008, 2009 Priority to No. 61 / 155,431, filed February 25, 2009, and No. 61 / 184,530, filed June 5, 2009, each of which is hereby incorporated by reference in its entirety. Field of Invention [0002] The present invention relates to methods, compositions and kits for generating libraries of tagged DNA fragments from target DNA using transposase and transposon end compositions. The resulting ssDNA fragments can be used as templates, eg, for a variety of applications including, eg, high-throughput, massively parallel, and / or multiplex DNA sequencing. Background of the Invention [0003] There are a variety of methods and applications for which it is desirable to generate libraries of fragmented and tagged DNA molecules from double-stranded DNA (dsDNA) target molecules. Typically, the goal is to generate smaller...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/34C07H21/00C12N9/00
CPCC12N15/1093C12N15/66C12N9/22C12N9/1252C12N15/10C12Q1/6806C12N15/1065C12N9/93C12P19/34C12Q2525/155C12Q2535/122C12Q2565/518C12Q2521/301C12Q2521/507C12Q2525/191
Inventor 杰罗马·箭之撒加力·大鹿海缨·李·格鲁内瓦德猊克拉士·铠如酋
Owner EPICENT TECH CORP
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