Creating seamless junctions independent of restriction sites in PCR cloning
A method is described for the efficient cloning of any given DNA sequence into any desired location without the limitation of naturally occurring restriction sites. The technique employs the polymerase chain reaction (PCR) combined with the capacity of the type-IIS restriction endonuclease (ENase) E...
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Published in | Gene Vol. 168; no. 1; pp. 31 - 35 |
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Main Authors | , |
Format | Journal Article |
Language | English |
Published |
Netherlands
Elsevier B.V
02.02.1996
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Subjects | |
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Abstract | A method is described for the efficient cloning of any given DNA sequence into any desired location without the limitation of naturally occurring restriction sites. The technique employs the polymerase chain reaction (PCR) combined with the capacity of the type-IIS restriction endonuclease (ENase)
Eam1104I to cut outside its recognition sequence. Primers that contain the
Eam1104I recognition site (5′-CTCTTC) are used to amplify the DNA fragments being manipulated. Because the ENase is inhibited by site-specific methylation in the recognition sequence, all internal
Eam1104I sites present in the DNA can be protected by performing the PCR amplification in the presence of 5-methyl-deoxycytosine (
m5dCTP). The primer-encoded
Eam1104I sites are not affected by the modified nucleotides (nt) since the newly synthesized strand does not contain any cytosine residues in the recognition sequence. In addition, the ENase's ability to cleave several bases downstream from its recognition site allows the removal of superfluous, terminal sequences from the amplified DNA fragments, resulting in 5′ overhangs that are defined by the nt present within the cleavage site. Thus, the elimination of extraneous nt and the generation of unique, non-palindromic sticky ends permits the formation of seamless junctions in a directional fashion during the subsequent ligation event. |
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AbstractList | A method is described for the efficient cloning of any given DNA sequence into any desired location without the limitation of naturally occurring restriction sites. The technique employs the polymerase chain reaction (PCR) combined with the capacity of the type-IIS restriction endonuclease (ENase) Eam1104I to cut outside its recognition sequence. Primers that contain the Eam1104I recognition site (5'-CTCTTC) are used to amplify the DNA fragments being manipulated. Because the ENase is inhibited by site-specific methylation in the recognition sequence, all internal Eam1104I sites present in the DNA can be protected by performing the PCR amplification in the presence of 5-methyldeoxycytosine (m5dCTP). The primer-encoded Eam1104I sites are not affected by the modified nucleotides (nt) since the newly synthesized strand does not contain any cytosine residues in the recognition sequence. In addition, the ENase's ability to cleave several bases downstream from its recognition site allows the removal of superfluous, terminal sequences from the amplified DNA fragments, resulting in 5' overhangs that are defined by the nt present within the cleavage site. Thus, the elimination of extraneous nt and the generation of unique, non-palindromic sticky ends permits the formation of seamless junctions in a directional fashion during the subsequent ligation event. A method is described for the efficient cloning of any given DNA sequence into any desired location without the limitation of naturally occurring restriction sites. The technique employs the polymerase chain reaction (PCR) combined with the capacity of the type-IIS restriction endonuclease (ENase) Eam1104I to cut outside its recognition sequence. Primers that contain the Eam1104I recognition site (5′-CTCTTC) are used to amplify the DNA fragments being manipulated. Because the ENase is inhibited by site-specific methylation in the recognition sequence, all internal Eam1104I sites present in the DNA can be protected by performing the PCR amplification in the presence of 5-methyl-deoxycytosine ( m5dCTP). The primer-encoded Eam1104I sites are not affected by the modified nucleotides (nt) since the newly synthesized strand does not contain any cytosine residues in the recognition sequence. In addition, the ENase's ability to cleave several bases downstream from its recognition site allows the removal of superfluous, terminal sequences from the amplified DNA fragments, resulting in 5′ overhangs that are defined by the nt present within the cleavage site. Thus, the elimination of extraneous nt and the generation of unique, non-palindromic sticky ends permits the formation of seamless junctions in a directional fashion during the subsequent ligation event. A method is described for the efficient cloning of any given DNA sequence into any desired location without the limitation of naturally occurring restriction sites. The technique employs the polymerase chain reaction (PCR) combined with the capacity of the type-IIS restriction endonuclease (ENase) Eam1104I to cut outside its recognition sequence. Primers that contain the Eam1104I recognition site (5'-CTCTTC) are used to amplify the DNA fragments being manipulated. Because the ENase isinhibited by site-specific methylation in the recognition sequence, all internal Eam1104I sites present in the DNA can be protected by performing the PCR amplification in the presence of 5-methyl-deoxycytosine(m5dCTP). The primer-encoded Eam1104I sites are not affected by the modified nucleotides (nt) since the newly synthesized strand does not contain any cytosine residues in the recognition sequence. In addition,the ENase's ability to cleave several bases downstream from its recognition site allows the removal of superfluous, terminal sequences fromthe amplified DNA fragments, resulting in 5' overhangs that are defined by the nt present within the cleavage site. Thus, the elimination of extraneous nt and the generation of unique, non-palindromic sticky ends permits the formation of seamless junctions in a directional fashion during the subsequent ligation event. |
Author | Sorge, Joseph A. Padgett, Kerstein A. |
Author_xml | – sequence: 1 givenname: Kerstein A. surname: Padgett fullname: Padgett, Kerstein A. – sequence: 2 givenname: Joseph A. surname: Sorge fullname: Sorge, Joseph A. email: joe_sorge@stratagene.com |
BackLink | https://www.ncbi.nlm.nih.gov/pubmed/8626061$$D View this record in MEDLINE/PubMed |
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Cites_doi | 10.1016/0378-1119(91)90480-Y 10.1016/0378-1119(91)90345-C 10.1093/nar/22.15.3259 10.1093/nar/22.17.3640 10.1006/abio.1994.1445 10.1093/nar/19.5.1081 10.1126/science.2833816 10.1016/0378-1119(89)90359-4 10.1093/nar/17.12.4895 |
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Keywords | Km DTT E Taq 5-methyl-dCTP lacI Recombinant DNA type-IIS restriction endonuclease bp kb cDNA m5dCTP oligo Pfu ss amplimer Eam1104I ntm SOE ENase (R) βGal XGal u IPTG PCR |
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Mol. Biol. Res. contributor: fullname: Russek – volume: 17 start-page: 4895 year: 1989 ident: 10.1016/0378-1119(95)00731-8_BIB14 article-title: Precise gene fusion by PCR publication-title: Nucleic Acids Res. doi: 10.1093/nar/17.12.4895 contributor: fullname: Yon |
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SubjectTerms | 5-methyl-dCTP amplimer Base Sequence Cloning, Molecular - methods Deoxycytidine - analogs & derivatives Deoxycytidine - pharmacology Deoxyribonucleases, Type II Site-Specific - metabolism DNA Primers - chemistry Eam1104I Electrophoresis, Polyacrylamide Gel Genetic Vectors - genetics Methylation Molecular Sequence Data Polymerase Chain Reaction Recombinant DNA type-IIS restriction endonuclease |
Title | Creating seamless junctions independent of restriction sites in PCR cloning |
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