Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1996-04-29
1998-06-09
Campbell, Eggerton A.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435 6, 536 231, C12P 1934, C12Q 168, C07H 2102
Patent
active
057632278
DESCRIPTION:
BRIEF SUMMARY
This application is a 371 of PCT/EP94/03547, filed on Oct. 28, 1994.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to the sequencing of deoxyribonucleic acid.
2. Brief Description of the Related Art
The methods used for DNA sequencing today are mainly the Maxam-Gilbert technique and the Sanger method. In the Sanger method, the DNA to be sequenced--single-stranded DNA, or DNA that has been made single-stranded--is polymerised enzymatically to form a double strand. For that purpose, a short region of the DNA first has to be made double-stranded, since DNA polymerases need a short double-stranded region having a free 3'-hydroxyl end for the incorporation of nucleotides (template/primer dependency of the enzymatically catalysed incorporation of nucleotides). According to the shortmer concept (EP-B-89 906 358.0), the short double-stranded region is synthesised by hybridising two or more shortmers adjacent to one another on, as template, the single-stranded DNA to be sequenced, and then binding or ligating the shortmers together to form a primer. Starting from the short double-stranded region, the DNA strand to be sequenced is then successively enzymatically complemented with DNA polymerase. As soon as the whole sequence has been read in order to evaluate the analysis, a primer attachment site at the 3' end of the DNA sequence just read is again selected for the next sequencing operation. Shortmers are again hybridised and ligated at that primer attachment site. The new primer is used for subsequent sequencing.
On the one hand, primers that are as long as possible are of interest, in order to enable a DNA strand that is to be sequenced to be addressed individually using such a primer. On the other hand, the size of the shortmer banks to be used for the production of the primers increases dramatically as the primer length increases. It has been found, however, that it is possible for DNA strands that arc to be sequenced to be added sufficiently accurately with primers from 12 to 18 nucleotides in length.
In addition, it has been found that the classic shortmer concept is incapable of improvement in the following respect: high-value sequencing protocols start from an excess of primer over the DNA strand to be sequenced. If the primer is present in a ratio of 1:1 or less with respect to the DNA strand to be sequenced, then, in experiments, the reading distances observed are generally shorter than would be expected with an excess of primer. If, accordingly, in the case of the shortmer concept hybridisation and ligation are carried out once only and the primer ligation product is not melted off and then hybridised and ligated again, then at the very most a 1:1 stoichiometry of primer to DNA strand to be sequenced is achieved, despite the presence or an excess of the shortmer used.
SUMMARY OF THE INVENTION
That prior art can now be improved in accordance with an embodiment of the invention by means of a process for the production of a primer for consecutive DNA sequencing, in which at least two shortmers are hybridised adjacent to one another using a template and are then ligated with one another to form the primer, the process being characterised in that which the number of nucleotides is at least 6 and is not greater than approximately twice the total number of nucleotides of all the shortmers used, and unreacted shortmers and recovered.
Primer: By "primer" there is to be understood in the present context a short strand of DNA with the aid of which the DNA strand to be sequenced is made double-stranded over a correspondingly short region.
Template: In contrast to the DNA strand that is to be sequenced, there is to be understood by "template" in the present context a single strand of nucleotides on which a primer can be produced, which primer is used for the sequencing of a DNA strand only after it has been separated from the template.
Shortmer: There are to be understood by "shortmers" oligonucleotides that can be ligated to form a primer; cf. EP-B-89 906 358.0.
BRIEF DESCRIPTIO
REFERENCES:
Khrapko et al, FEB 256(1,2): 118-122 (1989).
Szybalski et al, Gene 90: 177-178 (1990).
Campbell Eggerton A.
Gesellschaft fur Biotechnologische Forschung mbH (GBF)
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