DNA sequencing method

Abstract

The present invention pertains to a method for determining the sequence of a polynucleotide, the method relying on the detection of a conformational change in an enzyme that interacts with and processes along the polynucleotide. The detection of a conformational change may be carried out by measuring changes in a fluorophore bound to the enzyme.

Description

FIELD OF THE INVENTION [0001] This invention relates to polynucleotide sequence determinations. BACKGROUND OF THE INVENTION [0002] The ability to determine the sequence of a polynucleotide is of great scientific importance. For example, the Human Genome Project is an ambitious international effort to map and sequence the three billion bases of DNA encoded in the human genome. When complete, the resulting sequence database will be a tool of unparalleled power for biomedical research. The major obstacle to the successful completion of this project concerns the technology used in the sequencing process. [0003] The principle method in general use for large-scale DNA sequencing is the chain termination method. This method was first developed by Sanger and Coulson (Sanger et al., Proc. Natl. Acad. Sci. USA, 1977; 74: 5463-5467), and relies on the use of dideoxy derivatives of the four nucleoside triphosphates which are incorporated into a nascent polynucleotide chain in a polymerase react...

AI Technical Summary

Benefits of technology

[0018] The present invention offers several advantages over conventional sequencing technology. Once a polymerase enzyme begins its round of polynucleotide elongation, it tends to polymerase several thousand nucleotides before falling off from the strand. Additionally, certain specific polymerase systems are able to anchor or tether themselves to the template polynucleotide via a ‘sliding clamp’ (e.g. Polymerase III) which encircles the template molecule or via a molecular hook (e.g. T7:thireodoxin complex) which partially encircles the template.

[0019] The invention may also enable tens of kilobases (kb) or more to be sequenced in one go, at a rate of hundreds of base pairs per second. This is a result of sequencing on a single fragment of DNA. An advantage of sequencing a single fragment of DNA is that sequencing rates are determined by the enzyme system utilised and not upon indirect, summated reactions, and are therefore correspondingly higher. Just as important as the high rate is the ability to sequence large fragments of DNA. This will significantly reduce the amount of subcloning and the number of overlapping sequences required to assemble megabase segments of sequencing information. An additional advantage of the single fragment approach is the elimination of problems associated with the disposal of hazardous wastes, such as acrylamide, which plague current sequencing efforts.

Problems solved by technology

When complete, the resulting sequence database will be a tool of unparalleled power for biomedical research.

The major obstacle to the successful completion of this project concerns the technology used in the sequencing process.

Although this method is widely used and produces reliable results, it is recognised that it is slow, labour-intensive and expensive.

However, limitations of this method are recognised; the most serious for the SPR technique being that, as the size of the copy strand grows, the absolute size of the signal also grows due to the movement of the strand out of the evanescent wave field, making it harder to detect increments.

The fluorescence measuring techniques have the disadvantage of increasing background interference from the fluorophores incorporated on the growing nascent polynucleotide chain.

This severely restricts the use of the method for sequencing large polynucleotides.

However, limitations of this method are recognised; the most serious for the exonuclease technique being the fact that the labelled nucleotides severely affect the processivity of the exonuclease enzyme.

Other limitations of this method include ‘sticking’ of the nucleotide(s) to the biotin bead used to immobilise the polynucleotide fragment, thus resulting in the nucleotide flow becoming out of phase; inefficiency and length limitation of the initial enzymatic labelling process; and the excitation ‘cross-over’ between the four different dye molecules resulting in a greatly increased error rate.

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