Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1990-01-11
1992-05-19
Griffin, Ronald W.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
536 27, 935 77, C07H 100, C07H 2104
Patent
active
051148390
DESCRIPTION:
BRIEF SUMMARY
Sequence analysis of nucleic acid fragments, especially of DNA, may be regarded as a key process in modern biological sciences and modern biotechnology. Recently, with the constant refining of sequencing techniques, the analysis even of whole complex genomes has entered the realm of the possible.
Apart from a few less popular methods, two fundamentally different methods are used particularly at the present time for sequence analysis, namely method) and (Sanger method); cf., for example, Gassen & Schafer, Sequenzbestimmung von Nucleinsauren und Proteinen, in: Gassen et al, Gentechnik, 2nd edition, Gustav-Fischer-Verlag, 1987, page 241 ff.
The Sanger method can be carried out either with single-stranded DNA that is produced especially for the purpose of sequencing, or, as has been the case for some years now, with any sufficiently pure double-stranded DNA. A general characteristic of the Sanger method remains however, inter alia, the dependence of the addition enzyme (DNA polymerases) used both on a matrix (template =DNA to be sequenced) and on a starter molecule (primer). The primer is generally a short, chemically synthesised oligonucleotide that is base-complementary over its entire length with a portion positioned 3' from the DNA section to be sequenced: ##STR1## The attachment site (hybridising site) of the primer must be so selected that under the conditions of the experiment the primer finds only the desired attachment site and not any other attachment site, in order for a legible sequencing result to be obtained. The specificity of the primer depends directly on its length and on the complexity of the DNA used in the experiment. From statistical considerations and practical experience it has been found that primers having a chain length of up to 24 bases should suffice for all possible cases.
In the sequence analysis of very long DNA (for example in genome sequencing) at present essentially two different strategies are used.
Strategy 1: The Single-primer method. In this method the DNA to be sequenced is divided to a greater or lesser extent into random fragments or partially organised fragments (depending on the process variant) and then the fragments are inserted into the same vector. After cell transformation DNA preparations of individual clones are prepared and subjected to sequence analysis. Since all the DNA preparations differ at most by the inserted DNA, a single primer, which is, for example, hybridised directly adjacent to the insertion site on the vector DNA, can in principle be used for all the required sequence analyses. There are, however, considerable disadvantages weighing against this advantage: sections of the original DNA are very often sequenced far more frequently than is necessary, and partial sequencing results that have been arranged in an overall sequence using suitable computer programmes, since, for example, certain sections of the original DNA to be analysed do not clone readily.
Strategy 2: The Multi-primer method. In this case the DNA to be sequenced is analysed not by the shotgun method, as in Strategy 1, but by a controlled progression. The section of the sequence to have been reliably decoded in an experiment is used to select a primer-attachment site for the next experiment and so on. The advantage of this method is especially the fact that unnecessary multiple sequence analyses like those in Strategy 1 are avoided. The essential reason why this method is not the only one used for the analysis of relatively long DNA sections probably lies in the limitation imposed by the methods of chemical DNA synthesis that are currently available.
There has been no lack of attempts to alleviate the particular disadvantages of the processes described above, for example by means of "multiplex sequencing" in Strategy 1 and the simultaneous progressive sequencing from different sites in Strategy 2. One way of eliminating altogether the essential disadvantage of Strategy 2 is, theoretically, to provide a bank of all possible primers. This is not possible in practice using direct chemic
REFERENCES:
patent: 4863848 (1989-09-01), Blocker et al.
Alberts et al. (1983) Molecular Biology of the Cell, Garland Publishing, Inc.: New York, pp. 185-187.
Szybalski (1990) Gene, vol. 90, pp. 177-178.
Studier (1989) Proc. Natl. Acad. Sci., vol. 86, pp. 6917-6921.
Gesellschaft fur biotechnologische Forsching mbH
Griffin Ronald W.
Kunz Gary L.
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