Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2002-03-29
2004-12-21
Fredman, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, 53, 53, 53, 53
Reexamination Certificate
active
06833246
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the sequencing of polynucleotides. In particular, this invention discloses methods for determining the sequence of polynucleotides arrayed on a solid support.
BACKGROUND TO THE INVENTION
Advances in the study of molecules have been led, in part, by improvement in technologies used to characterize the molecules or their biological reactions. In particular, the study of the nucleic acids DNA and RNA has benefited from developing technologies used for sequence analysis and the study of hybridization events.
An example of the technologies that have improved the study of nucleic acids, is the development of fabricated arrays of immobilized nucleic acids. These arrays consist typically of a high-density matrix of polynucleotides immobilized onto a solid support material. Fodor et al, Trends in Biotechnology (1994) 12:19-26, describes ways of assembling the nucleic acids using a chemically sensitized glass surface protected by a mask, but exposed at defined areas to allow attachment of suitably modified nucleotide phosphoramidites. Fabricated arrays may also be manufactured by the technique of “spotting” known polynucleotides onto a solid support at predetermined positions (e.g. Stimpson et al, PNAS (1995) 92:6379-6383).
A further development in array technology is the attachment of the polynucleotides to the solid support material to form single molecule arrays. Arrays of this type are disclosed in WO-A-00/06770. The advantage of these arrays is that reactions can be monitored at the single molecule level and information on large numbers of single molecules can be collated from a single reaction.
For DNA arrays to be useful, the sequences of the molecular must be determined. U.S. Pat. No. 5,302,509 discloses a method to sequence polynucleotides immobilized on a solid support. The method relies on the incorporation of 3-blocked bases A, G, C and T having a different fluorescent label to the immobilized polynucleotide, in the presence of DNA polymerase. The polymerase incorporates a base complementary to the target polynucleotide, but is prevented from further addition by the 3′-blocking group. The label of the incorporated base can then be determined and the blocking group removed by chemical cleavage to allow further polymerisation to occur. However, the need to remove the blocking groups in this manner is time-consuming and must be performed with high efficiency.
Similarly, EP-A-0640146 discloses a potymerisation-based technique for sequencing DNA. The technique again requires removal of a blocking group prior to subsequent incorporation of nucleotides.
SUMMARY OF THE INVENTION
In the general method of the invention, a target polynucleotide sequence can be determined by generating its complement using the potymerase reaction to extend a suitable primer, and characterizing the successive incorporation of bases that generate the complement. The target sequence is, typically, immobilized on a solid support. Each of the different bases A, T, G or C is then brought, by sequential addition, into contact with the target, and any incorporation events detected via a suitable label attached to the base. In contrast to the prior art methods, the present invention requires the presence of a polymerase enzyme that retains a 3′ to 5′ exonuclease fiction, which is induced to remove an incorporated labelled base after detection of incorporation. A corresponding non-labelled base may then be incorporated into the complementary strand to allow further sequence determinations to be made. Repeating the procedure allows the sequence of the complement to be identified, and thereby the target sequence also.
The use of the polymerase enzyme's exonuclease function in this way is a characteristic feature of the invention. It permits repeated incorporation of labelled bases to take place, without the requirement for separate steps of chemical cleavage or photoblcaching.
Accordingly, a method for determining the sequence of a target polynucleotide on an array, comprises the steps of:
(i) contacting the array with one or more detectably-labelled bases A, T, G and C, under conditions that permit the polymerisation reaction to occur, to thereby incorporate a labelled base into a strand complementary to the target;
(ii) removing non-incorporated bases and detecting an incorporation event;
(iii) optionally repeating steps (i) and (ii) with one or more additional labelled bases, to determine a partial sequence;
(iv) contacting the array of step (iii) with a DNA polymerase having 3′ to 5′ exonuclease activity, under conditions whereby the polymerase cleaves the labelled base(s) and incorporates corresponding non-labelled base(s); and
(v) repeating steps (i)-(iv) sequentially, to determine the sequence.
Sequencing the polynucleotides on the array makes it possible to form a spatially addressable array. This may then be used for many different applications, including genotyping studies and other characterization experiments.
The method of the present invention may be automated to produce a very efficient and fast sequence determination.
REFERENCES:
patent: 5302509 (1994-04-01), Cheeseman
patent: 6245507 (2001-06-01), Bogdanov
patent: 6555349 (2003-04-01), O'Donnell
patent: WO00/06770 (2000-02-01), None
Jacobs, et al. “Combinatorial Chemistry-Applications of Light-directed Chemical Synthesis”, Trends in Biotechnology (1994) 12:19-26.
Stimpson, et al. “Real-Time Detection of DNA Hybridization and Melting on Oligonucleotide Arrays by Using Optical Wave Guides”, PNAS (1995) 92:6379-6383.
Fredman Jeffrey
Klauber & Jackson
Solexa Ltd.
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