Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1997-08-28
2000-07-18
Horlick, Kenneth R.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 911, 436 94, C12Q 168, C12P 1934, G01N 3300
Patent
active
060905506
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to automated DNA sequencing.
The most commonly used DNA sequencing technique used at the present time is due to Sanger et al, and was first discussed in Sanger, F., Nicklen, S. and Coulson, A. R. (1977): DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74; 5463-5467. Variations of Sanger's technique have revolutionised genome analysis by enabling rapid and reasonably accurate determination of unknown DNA sequences.
In the Sanger technique, the unknown DNA to be sequenced (known as the "template") is put into solution, and the DNA is denatured, or split into its separate strands, by heating. To the solution is added a short artificially created DNA sequence known as a "primer", the primer corresponding to a small section of the template which is already known. When the solution of denatured template and primer molecules is cooled, the primer adheres to its complementary sequence on the template. Also added to the solution are appropriate polymerase molecules, along with molecules forming the building blocks for the required extension reaction. As the extension reaction proceeds, the bound primer is extended along the length of the template, gradually building up, base by base, an elongate sequence which is the complement of that of the template.
There are four types of building block molecules used in the reaction, each corresponding to one of the bases, A, C, G and T. Specifically, the building blocks are deoxynucleotides known as dATP,dCTP,dGTP and dTTP. Any one of these may be referred to as dNTP. Left to itself, the copying reaction would continue until either the required dNTPs are exhausted, or some extraneous event occurs to stop the reaction. In the Sanger method, a certain proportion of the building blocks are replaced by dideoxynucleotides, namely ddATP, ddCTP, ddGTP and ddTTP (generically ddNTP). If the continuing reaction happens to make use of a dideoxynucleotide from solution, rather than a deoxynucleotide, the molecule binds in the usual way, but all further reaction along that strand is inhibited. Since there is only a relatively small concentration of dideoxynucleotides with the deoxynucleotides, random chance dictates when the reaction on any given chain will be stopped.
Since all copies start at the same position (with the primer), and the stopping position is substantially random, the reaction creates a large number of fragments of cloned DNA for each possible terminating position.
A single vessel charged with template, primer, polymerase, dNTP's and ddNTP's, along with some housekeeping reagents, will give rise to a set of fragments representing each base position in the sequence. Using a single reaction vessel, of course, produces a mixture of all the fragments. The use of four separate reaction volumes, identical except that each contains only one type of terminating ddNTP, ensures that fragments ending in only one of the bases are formed in each volume. The products of each reaction are loaded into a separate lane in a polyacrylamide gel and subject to electrophoresis, causing the fragments to move along the gel. Shorter fragments move more quickly, so the result is an array of fragments laid out along the gel, where each successive group of fragments ends with the next base in the sequence when read up the gel. To ensure that the fragments are visible, an appropriate label must be attached. One method is to attach a radiolabel to one of the dNTP's, ensuring that each fragment on the gel is marked with the radioactive label. Photographic film is placed over the gel, and the mark left on the film by the radioactive decay products are seen in the developed negative as dark bands which can be read off to give the sequence. Alternatively, a fluorescent dye may be attached to the primer, or instead to the dideoxynucleotides.
All methods based on the Sanger technique give rise to measurements which may be represented as four separate graphs, each representing the intensity of a reading from one of the bases on the gel.
The most popu
REFERENCES:
patent: 4941092 (1990-07-01), Hara et al.
patent: 4958281 (1990-09-01), Hara et al.
Genomics, vol. 19, No. 3, Feb. 1994, San Diego, US, pp. 417-424, XP000568813 Lipshutz et al.: "DNA Sequence confidence estimation".
Nature, vol. 365, Oct. 1993, London, GB, pp. 671-673, XP002001715 Larder et al.: "Quantitative detection of HIV-1 drug resistance mutations by automated DNA sequencing".
Nucleic Acids Research, vol. 20, No. 10, 1992, Oxford GB, pp. 2471-1483, XP002001716 Lee et al.: "DNA sequencing with dye labeled terminators and T7 polymerase".
Nucleic Acids Research, vol. 21, No. 19, 1993, Oxford GB, pp. 4530-4540, XP002001717 Giddings M C; Brumley R L Jr; Haker M; Smith L M: "An adaptive, object oriented strategy for base calling in DNA sequence analysis".
Collinge John
Thornley David
Horlick Kenneth R.
Imperial College of Science Technology and Medicine
LandOfFree
Automated DNA sequencing comparing predicted and actual measurem does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Automated DNA sequencing comparing predicted and actual measurem, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Automated DNA sequencing comparing predicted and actual measurem will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2034945