Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1991 Dec 25;19(24):6757–6761. doi: 10.1093/nar/19.24.6757

Method of artificial DNA splicing by directed ligation (SDL).

E N Lebedenko 1, K R Birikh 1, O V Plutalov 1, Berlin YuA 1
PMCID: PMC329306  PMID: 1662363

Abstract

An approach to directed genetic recombination in vitro has been devised, which allows for joining together, in a predetermined way, a series of DNA segments to give a precisely spliced polynucleotide sequence (DNA splicing by directed ligation, SDL). The approach makes use of amplification, by means of several polymerase chain reactions (PCR), of a chosen set of DNA segments. Primers for the amplifications contain recognition sites of the class IIS restriction endonucleases, which transform blunt ends of the amplification products into protruding ends of unique primary structures, the ends to be used for joining segments together being mutually complementary. Ligation of the mixture of the segments so synthesized gives the desired sequence in an unambiguous way. The suggested approach has been exemplified by the synthesis of a totally processed (intronless) gene encoding human mature interleukin-1 alpha.

Full text

PDF
6757

Images in this article

6758Image
on p.6758
6759Image
on p.6759
6760Image
on p.6760

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Auch D., Reth M. Exon trap cloning: using PCR to rapidly detect and clone exons from genomic DNA fragments. Nucleic Acids Res. 1990 Nov 25;18(22):6743–6744. doi: 10.1093/nar/18.22.6743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Butkus V., Bitinaité J., Kersulyté D., Janulaitis A. A new restriction endonuclease Eco31I recognizing a non-palindromic sequence. Biochim Biophys Acta. 1985 Dec 18;826(4):208–212. doi: 10.1016/0167-4781(85)90008-9. [DOI] [PubMed] [Google Scholar]
  3. Furutani Y., Notake M., Fukui T., Ohue M., Nomura H., Yamada M., Nakamura S. Complete nucleotide sequence of the gene for human interleukin 1 alpha. Nucleic Acids Res. 1986 Apr 25;14(8):3167–3179. doi: 10.1093/nar/14.8.3167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Horton R. M., Cai Z. L., Ho S. N., Pease L. R. Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction. Biotechniques. 1990 May;8(5):528–535. [PubMed] [Google Scholar]
  5. Horton R. M., Hunt H. D., Ho S. N., Pullen J. K., Pease L. R. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene. 1989 Apr 15;77(1):61–68. doi: 10.1016/0378-1119(89)90359-4. [DOI] [PubMed] [Google Scholar]
  6. Khorana H. G. Total synthesis of a gene. Science. 1979 Feb 16;203(4381):614–625. doi: 10.1126/science.366749. [DOI] [PubMed] [Google Scholar]
  7. Kim S. C., Podhajska A. J., Szybalski W. Cleaving DNA at any predetermined site with adapter-primers and class-IIS restriction enzymes. Science. 1988 Apr 22;240(4851):504–506. doi: 10.1126/science.2833816. [DOI] [PubMed] [Google Scholar]
  8. Kimmel A. R., Berger S. L. Preparation of cDNA and the generation of cDNA libraries: overview. Methods Enzymol. 1987;152:307–316. doi: 10.1016/0076-6879(87)52035-3. [DOI] [PubMed] [Google Scholar]
  9. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  10. Scharf S. J., Horn G. T., Erlich H. A. Direct cloning and sequence analysis of enzymatically amplified genomic sequences. Science. 1986 Sep 5;233(4768):1076–1078. doi: 10.1126/science.3461561. [DOI] [PubMed] [Google Scholar]
  11. Sinha N. D., Biernat J., McManus J., Köster H. Polymer support oligonucleotide synthesis XVIII: use of beta-cyanoethyl-N,N-dialkylamino-/N-morpholino phosphoramidite of deoxynucleosides for the synthesis of DNA fragments simplifying deprotection and isolation of the final product. Nucleic Acids Res. 1984 Jun 11;12(11):4539–4557. doi: 10.1093/nar/12.11.4539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Tindall K. R., Kunkel T. A. Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase. Biochemistry. 1988 Aug 9;27(16):6008–6013. doi: 10.1021/bi00416a027. [DOI] [PubMed] [Google Scholar]
  13. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES