Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1997-09-29
1999-11-02
Horlick, Kenneth R.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 71, 435 911, 435 912, 536 2532, C12Q 168, G01N 3353, C12P 1934, C07H 2100
Patent
active
059768029
DESCRIPTION:
BRIEF SUMMARY
The present invention concerns a method for the sequence-specific labelling and simultaneous sequencing of nucleic acids comprising the production of labelled nucleic acid fragments by an enzymatic labelling reaction in which a labelled deoxyribonucleoside triphosphate is attached to a nucleic acid primer molecule and the nucleic sequence is determined by means of the label.
In DNA sequencing by the enzymatic chain termination method according to Sanger one starts with a nucleic acid template from which many labelled nucleic acid fragments of various length are produced by an enzymatic extension and termination reaction in which a synthetic oligonucleotide primer is extended and terminated with the aid of polymerase and a mixture of deoxyribonucleoside triphosphates (dNTP) and chain termination molecules, in particular dideoxyribonucleoside triphosphates (ddNTP).
In this method a mixture of the deoxyribonucleoside triphosphates (dNTPs) and one dideoxyribonucleoside triphosphate (ddNTP) is used in each of four reaction mixtures. In this manner a statistical incorporation of the chain termination molecules into the growing nucleic acid chains is achieved and after incorporation of a chain termination molecule the DNA chain cannot be extended further due to the absence of a free 3'-OH group. Hence numerous DNA fragments of various length are formed which, from a statistical point of view, contain a chain termination molecule at each potential incorporation site and end at this position. These four reaction mixtures which each contain fragments ending at a base due to the incorporation of chain termination molecules are separated according to their length for example by polyacrylamide gel electrophoresis and usually in four different lanes and the sequence is determined by means of the labelling of these nucleic acid fragments.
Nowadays DNA sequencing is carried out with automated systems in which usually a non-radioactive label, in particular a fluorescent label, is used (L. M. Smith et al, Nature 321 (1986), 674-679; W. Ansorge et al, J. Biochem. Biophys. Meth. 13 (1986), 315-323). In these automated systems the nucleotide sequence is read directly during the separation of the labelled fragments and entered directly into a computer.
In the automated methods for sequencing nucleic acids non-radioactive labelling groups can either be introduced by means of labelled primer molecules, labelled chain termination molecules or as an internal label via labelled dNTP. In all these known labelling methods the sequencing reactions are in each case carried out individually in a reaction vessel so that always only one single sequence is obtained with a sequencing reaction.
A method for sequencing nucleic acids is described in the International Patent Application WO 93/03180 in which nucleic acid fragments are determined after incorporation of at least one non-radioactively labelled dNTP as an internal label wherein the internal label is incorporated into the nucleic acid fragments in the absence of chain termination molecules.
The simultaneous determination of two nucleic acid sequences using two differently labelled primer molecules in a single reaction vessel was described by Wiemann et al (Anal. Biochem. 224 (1995) 117-121). However, a disadvantage of this method is that dyelabelled oligonucleotide primers have to be used which are expensive or/and complicated to produce. This is a particular disadvantage when sequencing longer gene sections using the walking primer strategy in which different primers have to be used for each sequencing reaction. Moreover the use of labelled primers sometimes impedes the hybridization of the primer with the nucleic acid template which can lead to inaccurate sequencing results.
In order to avoid this disadvantage one could carry out two sequencing reactions each with an unlabelled primer and one of two differently labelled dNTP in two separate reaction vessels, mix both products, apply them simultaneously to a gel and determine the sequences. However, a disadvantage of this method is t
REFERENCES:
patent: 5173411 (1992-12-01), Tabor et al.
patent: 5266466 (1993-11-01), Tabor et al.
patent: 5302509 (1994-04-01), Cheeseman
Wiemann S. et al. Simultaneous DNA sequence on both strands with two dyes. Genome Mapping and Sequencing p. 271, 1993.
Kambara, H. et al. Real time automated simultaneous double stranded DNA sequencing using wto color fluorophore labeling. Bio/Technology vol. 9 pp. 648-651, 1991.
Voss, H. et al. New procedure for automated DNA sequencing with Multiple Internal Labeling by fluroescent dUTP. Meth. Mol. Cell. Bio. vol. 3. pp. 30-34, 1992.
Voss, H. et al. Automated Low Redundancy Large Scale DNA Sequencing by Primer Walking. BioTechniques. vol. 15, No. 4, pp. 714-721, 1993.
Lee, L.G. et al. DNA sequencing with dye-labeled terminators and T7 DNA polymerase: effect of dyes and dNTPs on incorporation of dye-terminators and probability analysis of termination fragments. Nucleic Acid Research. vol. 20, No. 10, pp. 2471-2483, 1992.
-Smith, L.M. et al Fluroescence detection in automated DNA sequence analysis Nature vol. 321, No. 6071 pp., 1986.
Analytical Biochemistry, vol. 234, Feb. 10, 1996, pp. 166-174, "Doublex Fluorescent DNA Sequencing: Two Independent Sequences Obtained Simultaneously in One Reaction with Internal . . . ".
Analytical Biochemistry, vol. 224, Jan. 1, 1995, pp. 117-121, "Simutaneous On-line DNA sequencing of both strands with two fluorescent dyes".
International Application No. WO 93/03180 published Feb. 18, 1993.
International Application No. WO 93/06243 published Apr. 1, 1993.
International Application No. WO 89/03432 published Apr. 20, 1989.
International Application No. WO 94/01447 published Jan. 20, 1994.
International Application No. WO 92/15712 published Sep. 17, 1992.
Ansorge Wilhelm
Stegemann Josef
Voss Hartmut
Wiemann Stefan
Europasches Laboratorium fur Molekularbiologie (EMBL)
Horlick Kenneth R.
Siew Jeffrey
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