Simultaneous sequencing of nucleic acids

Details Simultaneous sequencing of nucleic acids Simultaneous sequencing of nucleic acids

1994-02-02

1998-02-03

Chambers, Jasemine C.

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

435 914, 435 9141, 4351723, 536 242, C12P 1934, C12Q 168, C12N 1509, C07H 2104

Patent

active

057143188

DESCRIPTION:

BRIEF SUMMARY
This application is a National Stage Application of PCT Application No. PCT/EP93/01376, filed Jun. 1, 1993, which claims priority from German Application No. P 42 18152.6, filed Jun. 2, 1992.
The present invention concerns a new method for the simultaneous sequencing of nucleic acids as well as a method for converting an already existing gene bank into a modified gene bank on which a simultaneous sequencing of nucleic acids can be carried out.
The sequence of nucleic acids is usually determined either by chemical DNA sequencing (Maxam-Gilbert technique) or by the enzymatic chain termination method (Sanger technique). In the Maxam-Gilbert technique a base-specific cleavage of the nucleic acid to be sequenced is carried out with the aid of certain chemicals. In the Sanger technique an enzymatic polymerization reaction is carried out using the nucleic acid to be sequenced as a template and a DNA polymerase, e.g. the Klenow fragment of E. coli DNA polymerase, of T4 DNA polymerase or of T7 DNA polymerase. 2',3'-dideoxynucleoside-5'-triphosphates (ddNTP) are used as a substrate for the enzyme as chain terminating molecules in addition to the normal deoxynucleoside triphosphates (dNTP). Incorporation of these dideoxy compounds into a freshly synthesized DNA strand causes a termination of the polymerization reaction.
The sequence of the individual steps in the procedure is essentially the same in both of the aforementioned techniques: Firstly a high molecular DNA to be sequenced is broken down into several smaller fragments whose sequence can then be determined. This fragmentation can for example be achieved by specific cleavage with restriction enzymes or also by unspecific cleavage with DNase I or by ultrasonic treatment of the DNA.
The actual determination of the sequence is carried out on the smaller fragments, the principle of both sequencing procedures being that a population of sequencing products of different length whose nucleotide sequence derives from that of the DNA to be sequenced is generated by a chemical or enzymatic reaction. One end of these sequencing products is identical for the entire population and the other end is a variable end with one in each case of the four possible bases of DNA. The sequencing products of different length are then separated from one another by separation according to size in general by gel electrophoresis in very thin, high resolution denaturing polyacrylamide gels.
The determination of the sequence is generally carried out by direct analysis of these sequence gels. In this case the sequencing products must have a direct label e.g. by incorporation of radioisotopes such as .sup.32 P or .sup.35 S or biotinylated or fluorescent-labelled nucleotides.
The disadvantage of a direct labelling of the sequencing products is that always only one single DNA fragment can be processed in each case in each base-specific sequencing reaction. This disadvantage can be eliminated by a simultaneous sequencing of nucleic acids according to the so-called multiplex technique (see e.g. EP-A 0 303 459). This technique allows the simultaneous processing of several, in general up to 50 different DNA fragments in each base-specific sequencing reaction. For this the DNA fragments to be sequenced are incorporated into different cloning vectors. These vectors differ in each case in that oligonucleotide sequences that are specific for the vector used are located to the left and right of the cloning site. All vectors carry the same cleavage site for a restriction enzyme outside these oligonucleotide sequences with which the cloned DNA fragments together with the neighbouring regions which are specific for them can be again cleaved from the vector. A Maxam-Gilbert sequence reaction is carried out on this fragment mixture. After gel-electrophoretic separation of the sequencing products, the bands are transferred from the sequence gel onto a nylon membrane and are immobilized there. By hybridizing this membrane with hybridization probes that in each case are specific for only one vector it is possible

REFERENCES:
patent: 4942124 (1990-07-01), Church
Sambrook et al (1989) In Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, pp. 8.23-8.25.
New England Biolabs 1992 Catalog, .COPYRGT. 1992, p. 158.
Maniatis et al. (1983) In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY 1 page.

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