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Overlap extension PCR method capable of connecting multiple fragments

An overlapping extension and multi-fragment technology, which is applied in recombinant DNA technology, DNA preparation, etc., can solve the problems of low enzyme digestion efficiency and high requirements for the original template quantity of multi-directional ligation, so as to reduce mutations, reduce the number of reaction cycles, and high-efficiency amplification. Increased effect

Inactive Publication Date: 2010-12-01
SOUTHERN MEDICAL UNIVERSITY
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AI Technical Summary

Problems solved by technology

Another approach is to introduce restriction sites at both ends of the primers when performing three-way and multi-way ligation, but this will generally introduce mutations (point mutations or insertion mutations) at the same time, and it is also necessary to estimate the ligation ratio between templates, Also, due to the limited number of bases added, it will inevitably lead to low enzyme digestion efficiency, and the requirement for the amount of the original template used for multi-directional connection is high.

Method used

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  • Overlap extension PCR method capable of connecting multiple fragments
  • Overlap extension PCR method capable of connecting multiple fragments
  • Overlap extension PCR method capable of connecting multiple fragments

Examples

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example 1

[0026] The equine infectious anemia virus infectious clone is inserted into the fluorescent tag IRES-EGFP to illustrate. The inserted base size is 1.3 kbp, and the total amplification length of SOE-PCR is 4.16 kbp. The vector PLG3-8 is in 7097 nt. And 8288nt contains single restriction sites XbaI and Xhol, the short fragment length is about 1.2kbp after double restriction treatment of PLG3-8. See the description figure 1 .

[0027] 1. Primer design: According to the published nucleotide sequence of the EIAV (GENBANK AF327878) vaccine strain and the full sequence of the vaccine strain obtained in the laboratory, two pairs of primers were designed using oligo5.0 to amplify the 3′-end LTR insertion position of the PLG3-8 plasmid For the two genes, the expected amplified fragments are 1.8kb (amplified by primers Penv1 and Penv2) and 1.1kb (amplified by PLTR1 and PLTR2). At the same time, a pair of primers was designed for the inserted sequence IRES-EGFP vector p-IRES-EGFP, and the a...

example 2

[0055] The Lentiviral Equine Infectious Anemia Virus (EIAV) infectious clone PLG3-8 strain and pIRES-EGFP were used for multi-fragment ligation.

[0056] 1. Primer design: design primer pairs, as shown in Table 2.

[0057] Table 2 Primers used in gene amplification

[0058]

[0059] 2. Subfragment template preparation

[0060] Plasmid PCR: ampicillin resistance screening vector plasmid EIAV infectious clone PLG3-8 and the pIRES-EGFP full-length gene vector to obtain amplification templates. Amplification using high-fidelity polymerase, using primers Eiav 1-1 and Eiav 1-2 to amplify the EIAV infectious clone PLG3-8 long nucleic acid sequence of about 2.1kb, referred to as fragment 1; using primers Eiav 2-1 and Eiav 2-2 amplified the nucleic acid sequence of the EIAV infectious clone PLG3-8env3' with a length of about 1.4 kb, referred to as fragment 2. The primers Eiav 3-1 and Eiav 3-2 were used to amplify the EIAV infectious clone PLG3- The nucleic acid sequence of about 1.8 kb in th...

example 3

[0086] We explored the amplification under the conditions of diluting the inner primers under different conditions, and performed a single-tube amplification connection for fragments 1, 2, 3 and 4.

[0087] 1. See Table 3 for primer sequence information

[0088] Table 3 Primers used in gene amplification

[0089]

[0090] 2. Subfragment template amplification Using the primer pairs in Table 3, fragments 1, 2, 3, and 4 were respectively amplified as subfragment templates. See Example 2 for amplification information.

[0091] 3. Fragments 1, 2, 3 and 4 under different dilution primers are connected

[0092] Add 10μM primers Eiav 1-1 and Eiav 4-2 each 1μl, and add equal amounts of primers Eiav1-2 and Eiav 2-1, Eiav 2-2 and Eiav 3-1, Eiav 3-2 and Eiav 3-2 in each lane. Eiav 4-1, the amount is 1 / 40, 1 / 80, 1 / 120, 1 / 160, 1 / 200 of the amount of the long fragment primers on both sides; the reaction system is: Ex Tag DNA polymerase 0.30μl, Ex Tag Buffer 5μl , DNTP 8μl (10mM), add estimated amou...

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Abstract

The invention provides an SOE-PCR method capable of effectively connecting multiple DNA fragments. In the method, completely complementary splicing area primers are adopted, non-symmetrically weighting primers (i.e. the molar ratio of the primers positioned at two tail ends of a target-length fragment and the primer positioned on the target-length fragment splicing area is 40-120:1) are introduced during overlap extension, thus realizing the purpose that finishes connection of multiple fragments is finished in the same reaction. Meanwhile, the invention also has the advantages that template amount is not required to optimize, and amplification specificity is high.

Description

Technical field [0001] The invention relates to an in vitro recombination method of DNA, in particular to overlap extension PCR. Background technique [0002] Overlap extension PCR (SOE-PCR) was established in 1989 by Horton et al. This technology uses primers with complementary ends to make PCR products form overlapping strands, so that in the subsequent amplification reaction, through the extension of the overlapping strands, the amplified fragments from different sources are overlapped and spliced ​​to achieve in vitro gene recombination. SOE-PCR technology can obtain products that are difficult to obtain by the method of restriction endonuclease digestion, and is convenient and fast. It has unique advantages in site-directed mutation, deletion of gene fragments, and chimeric connection of multiple coding sequences. . [0003] However, conventional SOE-PCR technology also has problems such as the reaction efficiency being restricted by the number and proportion of the initial ...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 赵卫钟志成黎诚耀朱利熊建英万成松张玲
Owner SOUTHERN MEDICAL UNIVERSITY
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