Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1990-10-11
1992-10-27
Wax, Robert A.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
4351723, 4353201, 4351721, 536 27, 935 11, 935 6, 935 18, 935 10, C12P 1934, C07H 1512, C07H 1700
Patent
active
051588773
DESCRIPTION:
BRIEF SUMMARY
This invention relates to a method of synthesising double stranded DNA (ds-DNA), and to ds-DNA produced by such a method. ds-DNAs produceable by a method in accordance with the invention include synthetic genes coding for the transactivator of human immunodeficiency virus, to which the invention also relates.
Examples of ds-DNA and in particular total gene synthesis are becoming increasingly common as the reliability of oligonucleotide synthesis and the efficiency of assembly methods continue to improve. Gene synthesis is now an invaluable tool for the molecular biologist because of the total control it affords over restriction sites, codon usage and subsequent genetic manipulation and expression of the gene. This is particularly true where the gene is refractory to manipulation because of a lack of useful restriction sites or because it is derived from a spliced RNA, as is true in the case of the transactivator (TAT) protein of HIV1.
Methods of oligonucleotide synthesis have been reviewed extensively elsewhere (Gait, M. J. (1984) In M. J. Gait (ed), Oligonucleotide Synthesis: a Practical Approach, IRL Press, Oxford pp. 1-22). A number of different methods for the assembly of oligonucleotides have been described which divide into two main groups. In the first pioneered by Khorana and co-workers (Khorana, H. G. (1979) Science 203, 614-625), both strands of the desired sequence are divided such that adjacent pairs of complementary oligomers possess short (4-7 base) cohesive ends. The oligomers are then synthesised, kinased and annealed in pairs prior to ligation in a duplex corresponding to the intact gene. The ends of the gene are also endowed with cohesive ends to allow subsequent cloning of the gene in an appropriate vector. A recent development has been the successful solid phase assembly of a gene for cow colostrum trypsin inhibitor (Hostomsky, Z., Smrt, J., Arnold, L., Tocik, Z. and Paces, V. (1987) Nucelic Acids Research 15, 489-4856). The essential feature of these approaches is that both strands of the duplex are synthesised in their entirety.
The second strategy is based on the use of longer oligomers that share a complementary 3, end (Rossi, J. J., Kierzek, R., Huang, T., Walker, P. A. and Itakura, K. (1982) J. Biol. Chem. 257, 9226). Annealing a pair of such oligomers results in a short duplex region with two long single stranded extensions. Treating this partial duplex with Klenow fragment of DNA polymerase I in the presence of all four dNTP's results in the conversion of this structure to a complete duplex with blunt ends. This method has been successfully applied to the synthesis of a gene for Eglin C (Rink, H., Liersch, M., Sieber, P. and Meyer, F. (1984) Nucleic Acids Research 12, 6369-6387) and could in theory be extended to the construction of larger genes. It is attractive in that it reduces the amount of oligonucleotide synthesis required, but it has its drawbacks in that rearrangements and deletions are not uncommon.
A method of synthesising lengths of ds-DNA, particularly synthetic genes, has now been developed which reduces the amount of oligonucleotide synthesis required in the Khorana method but which does not have all the drawbacks of the Klenow fragment method.
According to a first aspect of the invention, there is provided a method of synthesising double stranded DNA, the method comprising preparing hybrid DNA containing a single stranded portion and a double stranded portion and carrying out in vivo gap repair on the hybrid DNA.
The single stranded portion may comprise at least 10, 20, 50 or 100 nucleotides.
The hybrid DNA (which may be a vector such as a plasmid) may be prepared by synthesising a single strand of DNA and introducing the single strand into double stranded DNA.
The single strand can conveniently be provided with double stranded ends and subsequently introduced into a double stranded vector. The double stranded ends of the single strand may be sticky, to correspond with restriction endonuclease cuts (natural or synthesised) in the ds-DNA, and will therefore for pr
REFERENCES:
patent: 4657858 (1987-04-01), Davison, Jr.
Narang et al. 1987 Biochem. Biophys. Res. Commun. 134,407-411.
Rink et al. 1984, Nucleic Acids Res. 12, 6369-6387.
Balland et al. 1985, Biochimie, 67,725-736.
Aldovini et al 1986, Proc. Nat'l. Acad. Sci USA 83,6672-6676.
Arya et al. 1985, Trans-Activator Gene of Human T-Lymphotropic Virus Type III (HTLV-III), in: Kulstad. R (ed.) 1986, Aids:Papers from Science, 1982-1985, American Soc. Adv. Sci. Washington, D.C., pp. 553-563.
Arentzen et al. 1984, Nuc. Acids Res. 12, 777-787.
Roberts, R. J. 1987, Nuc. Acids Res. 15 (Suppl.), pp. 189-217.
Ratner et al. 1985 Nature 313, 277-284.
Adams Sally E.
Edwards Richad M.
British Bio-technology Limited
Low Christopher G. F.
Wax Robert A.
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