DNA amplification method

A promoter sequence and target technology, applied in the field of DNA amplification, to achieve the effect of improving the effective utilization rate, improving the utilization rate, and avoiding purification

Active Publication Date: 2016-04-06
BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods have improved the balance of trace DNA amplification to a certain extent, but the balance of MDA amplification is still the biggest challenge for trace DNA amplification.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Apply method 1 of the present invention (method based on transposon), amplify 5 picograms (pg) DNA

[0046] 1. Prepare before starting:

[0047] Primer synthesis: transposon mosaic ends and mosaic end complementary sequences

[0048] STT_Met1 (SEQ ID No: 3):

[0049] 5'-AATTAATACGACTCACTATAGGGAGATGTGTATAAGAGACAG-3'

[0050] STT_Met2 (SEQ ID No: 4): 5'-pCTGTCTCTTATACACATCT-3'

[0051] Anneal to form a double-stranded sequence containing the T7 promoter sequence: STT_ME (10pmol / ul):

[0052] STT_ME consists of 100pmol of STT_Met1 (dissolved in annealing buffer: 10mMTris-HCl (pH7.5), 1mM EDTA, 0.1mMNaCl) and 100pmol of STT_Met2 (dissolved in annealing buffer: 10mMTris-HCl (pH7.5), 1mM EDTA, 0.1mMNaCl), etc. Volume mixed annealing (94°C for 5 minutes, gradually cooling down to 25°C at 0.1°C per second). STT_ME (50pmol / ul) was diluted with water to 10pmol / ul.

[0053] 2. Experimental steps:

[0054] Assembly of transposons containing T7 promoter sequences:

[0055] S...

Embodiment 2

[0107] Using method 2 of the present invention (random primer-based method), 5 picograms (pg) of DNA were amplified.

[0108] Random primers introduce T7 promoter sequences.

[0109] phi29 DNA Polymerase: NEB:Catalog#:M0269.

[0110] NEBbuffer4: NEB:Catalog#:B7004

[0111] T7 sequence (SEQ ID No: 2) with random primers (phosphorothioate modification) at the end:

[0112] 5'-AATTAATACGACTCACTATAGGGNNNNsNsN-3' (where s stands for phosphorothioate modification.)

[0113] DNA (5pg): 3ul

[0114] NEBbuffer4: 2ul

[0115] T7 promoter sequence (500uM) with random primers (phosphorothioate modification) at the end: 1ul

[0116] 10mMdNTP: 1ul

[0117] 100XBSA: 0.4ul

[0118] ddH2O: 10.6ul

[0119] 95°C for 3min, immediately place on ice for 3min

[0120] When done add:

[0121] phi29 DNA Polymerase: 2ul

[0122] Total 20ul

[0123] React at a constant temperature of 30°C for 30 minutes, and at 65°C for 10 minutes, and place it on ice immedi...

Embodiment 3

[0147] The present embodiment is a comparative example:.

[0148] Using a method based on multiple strand displacement reactions, 5 picograms (pg) of DNA were amplified. phi29 DNA Polymerase (NEB, Catalog #: M0269S)

[0149] DNA (5pg): 3ul

[0150] phi29DNAPolymeraseReactionBuffer: 5ul

[0151] Phosphorothioate-modified 6-base random primer (500uM): 2.5ul

[0152] 10mMdNTP: 2.5ul

[0153] 100XBSA: 1ul

[0154] ddH2O: 33.5ul

[0155] 95°C for 3min, immediately place on ice for 3min

[0156] When done add:

[0157] phi29 DNA Polymerase: 2.5ul

[0158] Total 50ul

[0159] React at a constant temperature of 30°C for 16h.

[0160] The product was purified using Agencourt AMPure XP (Beckman Coulter, Inc) magnetic beads. The overview is as follows: add 1 times the volume of magnetic beads to the amplified product, place it at room temperature for 5 minutes, absorb it on a magnetic frame for 5 minutes, remove the supernatant, wash twice with 70% a...

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PUM

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Abstract

The invention provides a DNA amplification method. The DNA amplification method is characterized by comprising the step that a T7 promoter sequence is added to the target DNA tail end. According to the DNA amplification method, amplification can be conducted on DNA smaller than one nanogram or even a single cell genome DNA on the basis of trace DNA; relatively even amplification is achieved through the target DNA containing the T7 promoter sequence; an error produced in the amplification process can be effectively removed.

Description

technical field [0001] The invention relates to a DNA amplification method. Background technique [0002] With the rapid development of next-generation sequencing technology, the Thousand Genomes Project and the Cancer Genome Project have been launched one after another, and genome research is becoming more and more mature. However, due to the characteristics of the next-generation sequencing library construction itself, the amount of starting DNA required for library construction is at the microgram level. With the continuous advancement of technology, some commercial kits are now able to reduce the DNA required for library construction to nanogram levels. How to efficiently construct and sequence libraries for trace amounts of DNA (less than 1 nanogram of DNA or even single-cell genomic DNA) has become a key issue. How to effectively and balancedly amplify trace DNA has become the biggest obstacle for trace DNA sequencing. [0003] The most common way to amplify trace D...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 阮珏王开乐沈栩吕雪梅吴仲义
Owner BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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