Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1994-04-18
1999-06-15
Myers, Carla J.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 911, 435 912, 435 915, 436 94, C12Q 168, C12P 1934
Patent
active
059121181
DESCRIPTION:
BRIEF SUMMARY
DESCRIPTION
The present invention concerns a method for the non-radioactive sequencing of nucleic acids.
In general two methods are known for DNA sequencing, namely the chemical degradation method according to Maxam and Gilbert (A. M. Maxam and W. Gilbert, Proc. Natl. Acad. Sci. USA 74 (1977), 560 and A. M. Maxam and W. Gilbert, Methods in Enzymol. 65 (1980), 499) and the enzymatic chain termination method (F. Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977), 5463-5467).
In the Maxam-Gilbert method labelled DNA molecules are modified chemically in a base-specific manner, a partial chain termination is effected and the fragments obtained in this way are separated according to size and the sequence is determined by means of the labelling.
In the method according to Sanger many labelled nucleic acid fragments are produced of different length starting from a DNA template by enzymatic elongation or extension of a synthetic oligonucleotide primer with the aid of polymerase and a mixture of deoxyribonucleoside triphosphates and chain terminating molecules, in particular dideoxyribonucleoside triphosphates. In this procedure a mixture of a particular deoxyribonucleoside triphosphate (dNTP) and a corresponding dideoxyribonucleoside triphosphate (ddNTP) is usually used together with the three other deoxyribonucleoside triphosphates in each of four mixtures. In this manner a statistical incorporation of the chain terminating molecules into the growing nucleic acid chain is achieved, but after incorporation of the chain terminating molecule the DNA chain cannot grow further because of the absence of a free 3'-OH group. Thus numerous DNA fragments of different length are formed which from a statistical point of view contain at least one chain terminating molecule at each possible incorporation site and end there. These four mixtures each having fragments ending specifically at a base due to the incorporation of chain terminating molecules are separated according to their length, e.g. by polyacrylamide gel electrophoresis, usually in four different lanes and the sequence is determined by means of a labelling of these nucleic acid fragments.
Up to about 1986 the DNA sequencing was carried out using radioactive (.sup.32 p or .sup.35 S) labels. The gelelectrophoretic separation of the fragments and autoradiography of the nucleotide sequence was carried out either manually or semi-automatically. Today DNA sequencing is carried out with automated systems in which 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 straight into a computer.
At present two different non-radioactive labelling systems are available for DNA sequencing according to the chain termination method. One possibility of labelling nucleic acid fragments is to use a labelled oligonucleotide primer for the extension reaction so that the nucleic acid chain carries a label at its 5' end. The other possibility is to use labelled terminators ie. chain terminating molecules so that the nucleic acid chain carries a label at its 3' end. A paper by Prober et al. (Science 238 (1987), 336-341) describes a sequencing method in which the four chain-terminating ddNTPs are each coupled to different fluorescent dyes so that all four sequencing mixtures can be separated on a single gel lane. A similar system using four different labelled primers is disclosed in GB-A 2155176.
However, in both the aforementioned methods only a single label is introduced into the nucleic acid chain to be determined which can often lead to sensitivity problems. A further disadvantage in the use of dye-labelled oligonucleotide primers, in particular in a "walking primer strategy" is that different primers have to be used for each sequencing reaction, the production of labelled primers, however, being very expensive or/and complicated. M
REFERENCES:
patent: 4795699 (1989-01-01), Innis
patent: 5124247 (1992-06-01), Ansorge
patent: 5674679 (1997-10-01), Fuller
Brumbaugh et al. Proceedings of the National Academy of Sciences. 85:5610-5614, Aug. 1988.
Prober et al. Science. 238:336-341, Oct. 1987.
Sanger et al., Proc. Natl. Acad. Sci. 74(12):5463-5467, Dec. 1977.
Maxam et al., Proc. Natl. Acad. Sci. 74(2):560-564, Feb. 1977.
Ansorge Wilhelm
Voss Hartmut
Europaisches Laboratorium fur Molekularbiologie (EMBL)
Myers Carla J.
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