Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Patent
1994-02-28
1996-11-12
Jones, W. Gary
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
536 253, 435 911, 435 912, 435 6, 435 5, C07H 1900, C07H 2100, C12P 1934, C12Q 168
Patent
active
055741410
DESCRIPTION:
BRIEF SUMMARY
This application is a 371 of PCT/EP92/01932, filed Aug. 22, 1992.
Techniques for the hybridization of nucleic acid fragments to DNA or RNA and their template-dependent enzymatic extension coupled with their non-radioactive labelling, e.g. by fluorescent dyes or affinity groups, are the basis for many applications of oligonucleotides in the biomedical field. These include for example the sequencing of nucleic acids by means of labelled primers according to Sanger's dideoxy method or the use of the polymerase chain reaction (PCR) in connection with nucleic acid diagnostics. Non-radioactive labelling methods should generally be preferred for such purposes since this approach avoids problems of handling of and contamination with radioisotopes as well as the difficulties in their storage and disposal.
The introduction of non-radioactive labelling groups can in principle be achieved by chemical or enzymatic means. In this connection one has to assume that the synthesis of the oligonucleotide chains themselves is usually carried out by chemical means. Therefore a subsequent enzymatic labelling, e.g. by reacting oligonucleotides with 5-bromo-dUTP (Nucleosides & Nucleotides 8, 805-813 (1989)), biotin-dUTP (Nucleic Acids Res. 16, 4077-4095 (1988)) or digoxigenin-dUTP (Biol. Chem. Hoppe Seyler 371, 917-927 (1990)) catalysed by deoxynucleotidyl-terminal transferase is from the outset complicated by the necessity for additional process steps. Above all, it should, however, be noted that this is usually an extension with structurally modified nucleoside-5' triphosphates beyond the 3' end of the oligonucleotide chain. In this process more than one nucleotide unit is usually (depending on the template) attached automatically to the 3' end, so that oligonucleotides labelled in this way cannot be used from the outset for all template-dependent chain extensions.
Deoxyoligonucleotides can be non-radioactively labelled by chemical means by introducing appropriate groups either on the nucleobase, on the sugar residue or at the internucleotide bond (phosphate). At present this is almost exclusively carried out during or after synthesis and processing of the oligonucleotide. In this way one either substitutes the 5'-OH end of the oligonucleotide chain (Nucleic Acid Res. 14, 6227-6245 (1986); Nucleic Acids Res. 14, 7985-7994 (1986)), nucleobases at the 5' end or internal (Nucleic Acids Res. 15, 3131-3139, (1987); J. Org. Chem. 55, 5931-5933, (1990)) or 5'-terminal (Nucleic Acids Res. 18, 4355-4360 (1990)) or internucleotidic (Nucleosides & Nucleotides 10, 303-306 (1991)) phosphate groups. Most labelling reactions are carried out by reactions after chemical synthesis of the chain. In this case the reaction partner is the relatively labile oligonucleotide chain so that there is a considerable risk of side reactions. In some cases labelling groups are introduced in such a way that appropriate amidophosphite derivatives are used as reagents. Such reagents are usually less stable and can only be stored in solution for a limited period. Recently CPG carrier materials have been described which enable the introduction of groups at the 3'-OH end of an oligonucleotide chain (Belmont, Calif., p. 188).
However, the 3'-OH end is blocked by these groups against any further chain extension so that oligonucleotides labelled in this manner do not come into consideration as primers for template-dependent polymerase reactions. Up to now 5'-terminally labelled oligonucleotides have been used exclusively for all oligonucleotides which have been used for template-dependent polymerase reactions. In this case it has been assumed that, on the one hand, internal labelling groups can interfere with the hybridization and, on the other hand, that template-dependent polymerases do not extend a primer beyond the 3' end if this contains a non-biological substituent at this position.
Surprisingly we have now found that deoxyoligonucleotides are also accepted as primers by DNA polymerases and are incorporated into polynucleotides even when they contain labelling g
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Gr oger Gabriele
Klotz Margit
Markiewicz Wojciech
R osch Rudi
Seliger Hartmut
Boehringer Mannheim GmbH
Jones W. Gary
Rees Dianne
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