A noncommercial polymerase chain reaction–based method to approach one hundred percent recombinant clone selection efficiency
Molecular cloning is an important procedure in molecular biology, but this is often a rate-limiting step and can be very time-consuming, possibly due to low ligation efficiency. Here, we describe a simple polymerase chain reaction (PCR)-based strategy to approach 100% selection efficiency. The post-...
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Published in | Analytical biochemistry Vol. 382; no. 1; pp. 75 - 76 |
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Main Authors | , , , , , , |
Format | Journal Article |
Language | English |
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Elsevier Inc
01.11.2008
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Abstract | Molecular cloning is an important procedure in molecular biology, but this is often a rate-limiting step and can be very time-consuming, possibly due to low ligation efficiency. Here, we describe a simple polymerase chain reaction (PCR)-based strategy to approach 100% selection efficiency. The post-ligation mixture containing the recombinant was subjected to insert-specific primer-mediated PCR amplification using a high-fidelity DNA
Pfu polymerase generating a plasmid containing staggered nicks. The PCR mixture was then digested with endonuclease DpnI, which digests the methylated and hemimethylated parental DNA template. The nicked vector was transformed into XL1 blue supercompetent cells where the nicks were repaired, thus amplifying and selecting only the newly amplified recombinant clones. |
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AbstractList | Molecular cloning is an important procedure in molecular biology, but this is often a rate-limiting step and can be very time-consuming, possibly due to low ligation efficiency. Here, we describe a simple polymerase chain reaction (PCR)-based strategy to approach 100% selection efficiency. The post-ligation mixture containing the recombinant was subjected to insert-specific primer-mediated PCR amplification using a high-fidelity DNA Pfu polymerase generating a plasmid containing staggered nicks. The PCR mixture was then digested with endonuclease DpnI, which digests the methylated and hemimethylated parental DNA template. The nicked vector was transformed into XL1 blue supercompetent cells where the nicks were repaired, thus amplifying and selecting only the newly amplified recombinant clones. Molecular cloning is an important procedure in molecular biology, but this is often a rate-limiting step and can be very time-consuming, possibly due to low ligation efficiency. Here, we describe a simple polymerase chain reaction (PCR)-based strategy to approach 100% selection efficiency. The post-ligation mixture containing the recombinant was subjected to insert-specific primer-mediated PCR amplification using a high-fidelity DNA Pfu polymerase generating a plasmid containing staggered nicks. The PCR mixture was then digested with endonuclease DpnI, which digests the methylated and hemimethylated parental DNA template. The nicked vector was transformed into XL1 blue supercompetent cells where the nicks were repaired, thus amplifying and selecting only the newly amplified recombinant clones. |
ArticleNumber | 75 |
Author | Sheldon, David G. Myers, Tamara T. Shareef, Mohammed M. Gross, Jessica L. Ahmed, Mansoor M. Dancea, Horatiu C. Griggs, Wendy W. |
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BackLink | https://www.ncbi.nlm.nih.gov/pubmed/18674510$$D View this record in MEDLINE/PubMed |
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References | Lund, Duch, Pedersen (bib3) 1996; 24 Cline, Braman, Hogrefe (bib4) 1996; 24 Sambrook, MacCallum (bib1) 2001 Rusche, Howard-Flanders (bib2) 1985; 13 Sambrook (10.1016/j.ab.2008.07.003_bib1) 2001 Rusche (10.1016/j.ab.2008.07.003_bib2) 1985; 13 Cline (10.1016/j.ab.2008.07.003_bib4) 1996; 24 Lund (10.1016/j.ab.2008.07.003_bib3) 1996; 24 |
References_xml | – volume: 24 start-page: 3546 year: 1996 end-page: 3551 ident: bib4 article-title: PCR fidelity of Pfu DNA polymerase and other thermostable DNA polymerases publication-title: Nucleic Acids Res. contributor: fullname: Hogrefe – volume: 13 start-page: 1997 year: 1985 ident: bib2 article-title: Hexamine cobalt chloride promotes intermolecular ligation of blunt end DNA fragments by T4 DNA ligase publication-title: Nucleic Acids Res. contributor: fullname: Howard-Flanders – volume: 24 start-page: 800 year: 1996 end-page: 801 ident: bib3 article-title: Increased cloning efficiency by temperature-cycle ligation publication-title: Nucleic Acids Res. contributor: fullname: Pedersen – year: 2001 ident: bib1 article-title: Molecular Cloning: a Laboratory Manual contributor: fullname: MacCallum – year: 2001 ident: 10.1016/j.ab.2008.07.003_bib1 contributor: fullname: Sambrook – volume: 13 start-page: 1997 year: 1985 ident: 10.1016/j.ab.2008.07.003_bib2 article-title: Hexamine cobalt chloride promotes intermolecular ligation of blunt end DNA fragments by T4 DNA ligase publication-title: Nucleic Acids Res. doi: 10.1093/nar/13.6.1997 contributor: fullname: Rusche – volume: 24 start-page: 3546 year: 1996 ident: 10.1016/j.ab.2008.07.003_bib4 article-title: PCR fidelity of Pfu DNA polymerase and other thermostable DNA polymerases publication-title: Nucleic Acids Res. doi: 10.1093/nar/24.18.3546 contributor: fullname: Cline – volume: 24 start-page: 800 year: 1996 ident: 10.1016/j.ab.2008.07.003_bib3 article-title: Increased cloning efficiency by temperature-cycle ligation publication-title: Nucleic Acids Res. doi: 10.1093/nar/24.4.800 contributor: fullname: Lund |
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SubjectTerms | Base Sequence Cell Line, Tumor Cloning, Molecular - methods DNA, Recombinant - genetics DNA, Recombinant - isolation & purification Genetic Vectors - genetics Humans Polymerase Chain Reaction - methods |
Title | A noncommercial polymerase chain reaction–based method to approach one hundred percent recombinant clone selection efficiency |
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