Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1995-11-20
1998-08-25
Marschel, Ardin H.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 911, 436501, 536 221, 536 231, 536 241, 536 243, 536 2431, 536 2432, 536 2433, 536 253, 935 76, 935 77, C12Q 168
Patent
active
057982104
DESCRIPTION:
BRIEF SUMMARY
This application is a 371 filing of PCT/FR94/00345, filed Mar. 28, 1994.
The present invention relates to the synthesis of 2'-deoxy-ribonucleoside 5' triphosphates substituted at the 3' position of the deoxyribose corresponding to the four nucleotide bases A, T, C and G and their use in a novel sequencing method for nucleic acids.
The 3' position is esterified by a specific derivative conferring on each nucleotide specific fluorescent properties.
The chemical synthesis of such a compound has already been described for the nucleotide dATP by Safarti et al., J. Biol. Chem. (1990), 265, pp. 18902-18906.
The existence of such a compound as well the existence of related compounds containing a fluorescent ester with different spectral properties {Hiraksuka (1982), J. Biol. Chem, 257, pp 13354-13358} suggests to a scientist skilled in the art that it is possible to esterify at position 3' "reporter" molecules which may be used for a sequencing procedure which is the subject of the present invention.
These esterified nucleoside triphosphates (3'-RT-dNTP) are substrates of the DNA or RNA polymerases which, when they are incorporated, lead to polynucleotide chain termination.
They lend themselves to three distinct reactions, namely incorporation by a nucleic acid polymerase into a growing chain, deprotection of the 3' hydroxyl of the deoxyribose making possible the incorporation of the next 3'-RT-dNTP. These properties can be used in a new non-radioactive method not based on the use of a gel to determine a nucleotide sequence or detect mutations in a DNA sequence.
One of the essential properties of these esterified nucleoside triphosphates is the in situ reversibility of this esterification making possible the restoration of the free 3' hydroxy groups, the polynucleotide chain being capable of undergoing further elongation on incubation with a mixture of dNTPs and DNA polymerase.
Since each nucleotide ester has fluorescent properties specific for a given base it is possible after removal and characterization of this fluorescent label to determine which of the nucleotide bases has been inserted.
The repetition of the procedure thus provides a method for the determination of a sequence of nucleotides or the detection of point mutations in a nucleotide sequence or the search for variants or finally the diagnosis of the presence of a particular oligonucleotide sequence in a sample.
The standard sequencing procedures were introduced about 15 years ago by Sanger, F., Nicklen, S and Coulson, A. R. {(1977) Proc. Natl. Acad. Sci. USA 74 pp. 5463-5467}, on the one hand, and by Maxam, A. M. and Gilbert, W {(1977) Proc. Natl. Acad. Sci. USA 74, pp. 560-564} on the other.
The dideoxy sequencing of Sanger is now widely used and is still the method of choice for determining a nucleotide sequence starting from a single stranded DNA matrix.
In brief, this method is the following: during the 4 enzymatic chain elongations the dideoxynucleotides are inserted at random in the place of the corresponding deoxynucleotide. The sequencing reactions generate a complex mixture which is then resolved by electrophoresis on polyacrylamide gel.
This method is relatively complex as regards the steps following the incorporation of the dideoxynucleotides, in particular with respect to the resolution on gel on the one hand and data acquisition, on the other.
In fact, a large diversity of products is generated by the elongation.
Various improvements were then made to this procedure with the objective of simplifying and reducing the experimental manipulations involved.
For example, in the patent application EP-O 531 169 A1, the authors developed a procedure making it possible to simplify and refine the electrophoresis step by using the techniques of pulsed field electrophoresis.
Other more recent improvements in the sequencing procedures have been described in the literature. We will mention two of them: Kieffer-Higgins, Science, 240, pp. 185-188 (1988)};
This procedure involves the formation of artificial genomes from a mixture of fragments inserted in
REFERENCES:
Sarfati et al. (1991) Tetrahedron Letters, vol. 32, No. 36, pp. 4699-4702.
Woodward et al. (1991) Biochemisry, vol. 30, pp. 422-430.
Canard Bruno
Sarfati Simon
Institut Pasteur
Marschel Ardin H.
LandOfFree
Derivatives utilizable in nucleic acid sequencing does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Derivatives utilizable in nucleic acid sequencing, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Derivatives utilizable in nucleic acid sequencing will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-35213