Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1996-05-24
1997-12-30
Zitomer, Stephanie W.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435174, 536 221, 536 2532, C12P 1934, C12N 1100, C07H 2102, C07H 2100
Patent
active
057029253
DESCRIPTION:
BRIEF SUMMARY
This application is filed under 35 USC 371 as the National Stage of PCT/GB94/02630, filed Dec. 1, 1994, now WO 95/15395, published Jun. 8, 1995 and has priority to EPO application Ser. No. 93309597.8 filed Dec. 1, 1993. The current chemical production method is very inefficient. This invention provides an enzymatic method of preparation, which improves efficiency. The method is applicable to a wide range of compounds besides this one.
T4 polynucleotide kinase (T4 PNK) is usually associated with the phosphorylation of the 5'-OH group of an oligonucleotide, DNA or 2-deoxynucleoside-3-monophosphate (or ribo) by transfer of the gamma-phosphate group from ATP. (Analytical Biochemistry 214, 338-340, 1993). It is generally believed that for T4 PNK to phosphorylate the 5'-OH group of a nucleotide, the nucleotide must contain a 5'-OH and a 3'-phosphate group. Because the 3'-phosphate group is clearly absent from 2',3'-dideoxynucleosides and other known chain terminators, the use of T4 PNK to catalyse their phosphorylation has hitherto been considered impossible. This invention results from our surprising discovery that T4 PNK can be used to catalyse this reaction. The invention covers the use of T4 PNK and other enzymes to catalyse this and related reactions.
Known kits for sequencing nucleic acids comprise supplies of all four nucleotides, and supplies of all four 2',3'-dideoxynucleotides, and a supply of one nucleotide which has been labelled, generally radioactively labelled, to permit detection of the products after sequencing by electrophoresis. In another aspect, this invention is based on the realization that improved results can be obtained by radiolabelling the dideoxynucleotides.
Kits containing fluorescently labelled chain terminators (ddNTPs) are known but isotopically labelled ddNTPs have structures which are less likely to interfere with polymerase activity, gel mobility and do not require sophisticated equipment for detection.
In one aspect, this invention provides a method of making a nucleotide or nucleotide analogue or nucleotide adduct, having a 5'-phosphate or a 5'-thiophosphate group which method comprises reacting a starting nucleoside or nucleoside analogue or nucleoside adduct having a 5'-OH group but no 3'-phosphate group with a nucleotide phosphate or thiophosphate donor in the presence of an enzyme which catalyses the reaction. The nucleoside can be an unmodified ribo or deoxyribonucleoside e.g. 2' deoxyadenosine.
It is possible that the nucleoside, nucleoside analogue or nucleoside adduct may be non-labelled and that the phosphate donor is also non-labelled. This produces the corresponding nucleotide without the need to use chemical phosphorylating agents which may be damaging to the starting material in some circumstances.
Alternatively the nucleoside can be labelled with a detectable isotope e.g. a radioisotope such as for example .sup.3 H or .sup.14 C and then converted to the corresponding labelled nucleotide with a non-labelled phosphate donor or thiophosphate donor.
Preferably the nucleotide phosphate or thiophosphate donor is radiolabelled with a detectable isotope e.g. a radioactive isotope such as .sup.32 P or .sup.33 P or .sup.35 S, whereby the obtained nucleotide or nucleotide analogue or nucleotide adduct is radiolabelled by virtue of having a 5'-phosphate or 5'-thiophosphate group comprising .sup.32 P or .sup.33 P or .sup.35 S
The term nucleoside analogue refers to a compound which is similar to a nucleoside and is capable of performing at least some of the biochemical functions of a nucleoside, and includes monomers and multimers. There follows a non-exhaustive list of nucleoside analogues.
2-aminoadenosine
5-bromocytosine
5-methylcytosine
5-(1-propynyl)cytosine
5-(1-propynyl)uracil
5-aminoallyluracil
5-aminoallyluracil-"label"
thiouracil/thiothymine/thioguanine
aziridene derivatives
2'-O-alkyl (e.g. allyl or methyl)
2'-fluoro
2'-amino
2'-deoxy
3'-deoxy
3'-"label"
3'-fluoro
3'-amino
3'-azido
2',3'-unsaturated
Phosphorothioate
Phosphorodithioate
Hydrogen phosphonate
Methyl p
REFERENCES:
patent: 5002868 (1991-03-01), Jacobson et al.
Fontanel et al. P32 Labeling of nonnucleosidic moieties 5'-attached to oligonucleotides, Analytical Biochemistry vol. 214, No. 1, pp. 338-340, 1993.
Fontanel et al., ".sup.32 P Labeling of Nonnucleosidic Moieties 5'-Attached to Oligonucleotides," Analytical Biochemistry 214(1):338-340 (1993).
Meldrum et al., "Kinetics and Mechanism of DNA Repair. Preparation, Purification and Some Properties of Caged Dideoxynucleoside Triphosphates," Chemical Abstracts 112(23):313 (1990).
Wharton et al., "Kinetics and Mechanism of DNA Repair: An Automated Programmable Apparatus for Fast Time-Resolved Studies of the Repair of Mammalian DNA After UV Irradiation," Biochemical Journal 293(3):825-828 (1993).
Yousaf et al., "A New and Improved Method For 3'-end Labelling DNA Using
Fuller Carl
Smith Clifford
Amersham International plc
Tung Joyce
Zitomer Stephanie W.
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