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Kits for Modification of Nucleic Acids

A kit and polynucleotide technology, applied in the field of kits for modifying nucleic acids, can solve the problems of cumbersome, difficult procedures, waste of DNA, etc.

Active Publication Date: 2019-03-12
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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  • Kits for Modification of Nucleic Acids
  • Kits for Modification of Nucleic Acids
  • Kits for Modification of Nucleic Acids

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0475] Assemble the following reaction mixture:

[0476]

[0477] *In some embodiments, two different pMEDS transposon ends that each additionally display an arbitrary sequence that differs in the corresponding 5' portion of the transferred transposon end, i.e., 5' of the transferred transposon end sequence ( Figure 4 ).

[0478] After mixing, the reactions were incubated at 37°C for 1 hour. Stop the reaction 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), mix 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 fractions using LMP agarose. The gel was stained with SYBR Gold and DNA was visualized with non-UV light. The gel shee...

Embodiment 2

[0482] Using different EZ-Tn5 TM Size range of 5'-tagged DNA fragment transposition products by Tn5 transposase concentration.

[0483] Add Tn5 hyperactive 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 phage T7D111 target DNA (with a size of approximately 39 Kbp) and 1 micromole of pMEDS transposon ends in TA buffer in a final reaction mixture volume of 50 microliters at 37 °C. Incubate for 1 hour.

[0484] The reaction was 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 of proteinase K. After mixing and incubation at 50 °C for 10 min, 10 microliter aliquots were run on a 1% agarose gel in TAE buffer at 100 volts for 1 h. Gels were stained with SYBR Gold and photographed ...

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 control in a buffer containing 1×TA buffer, 1 μg of phage T7D111 target DNA, and 0.4 units / μl of hyperactive Incubate the 50 μl reaction with 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 T7D111 target DNA was fragmented into DNA that migrated on the gel in siz...

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Abstract

The present invention provides kits for generating a library of tagged DNA fragments of target DNA comprising a plurality of transposome complexes, wherein at least one first transposome complex comprises a highly active Tn5 transposase and comprises a Tn5 at least one first polynucleotide comprising a Tn5-type transposon end sequence and a first tag sequence, and a second transposome complex comprising a highly active Tn5 transposase and comprising a Tn5-type transposon end sequence and a second tag sequence at least one second polynucleotide of, and b) a DNA polymerase having strand displacement activity, a DNA polymerase lacking 3' to 5' exonuclease activity and a DNA polymerase having 3' to 5' exonuclease activity Blend of DNA polymerases, DNA polymerase composition with 5' to 3' exonuclease activity, ligase and DNA polymerase lacking both strand displacement activity and 5' to 3' exonuclease activity , or at least one of ligase and ligation oligonucleotides. This kit can be used to generate 5' and 3' tagged DNA fragments for use in a variety of procedures.

Description

[0001] This application is a divisional application of the Chinese invention patent application with the filing date of November 24, 2009, the application number of 200980152461.8, and the title of the invention "Transposon end composition and method for modifying nucleic acid". This application claims U.S. Provisional Application Serial No. 61 / 108,321, filed October 24, 2008, 61 / 108,326, filed October 24, 2008, 61 / 108,329, filed October 24, 2008, 2009 61 / 155,431, filed February 25, 2009, U.S. Provisional Application Serial No. 61 / 152,868, filed February 16, 200, and 61 / 184,530, filed June 5, 2009, by reference Each of them is incorporated herein as a whole. technical field [0002] The present invention relates to methods, compositions and kits for generating libraries of tagged DNA fragments from target DNA using transposases and transposon end compositions. The resulting ssDNA fragments can be used as templates, eg, in a variety of applications including, eg, high-throughp...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/34C07H21/00C12N9/00
CPCC12N9/1252C12N9/22C12N9/93C12N15/1065C12N15/1093C12P19/34C12Q1/6806C12N15/10C12N15/66C12Q2525/155C12Q2535/122C12Q2565/518C12Q2521/301C12Q2521/507C12Q2525/191
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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