Polynucleotide Sequencing Method

Abstract

The subject invention pertains to a method for determining the sequence of a polynucleotide comprising the steps of (i) contacting a polynucleotide processive enzyme immobilised in a fixed position, with a target polynucleotide under conditions sufficient to induce enzyme activity; (ii) detecting an effect consequent on the interaction of the enzyme and polynucleotide, wherein the effect is detected by measurement of a non-linear optical signal or a linear signal coupled to a non-linear signal.

Description

FIELD OF THE INVENTION[0001]This invention relates to a method for determining the sequence of a polynucleotide.BACKGROUND OF THE INVENTION[0002]The ability to determine the sequence of a polynucleotide is of great scientific importance, as shown by the Human Genome Project in mapping the three billion bases of DNA encoded in the human genome.[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 the nascent polynucleotide chain in a polymerase reaction. Upon incorporation, the dideoxy derivatives terminate the polymerase reaction and the products are then separated by gel electrophoresis and analysed to reveal the position at which the particular dideoxy derivative was incorporated into the chain.[0004]Alt...

AI Technical Summary

Benefits of technology

[0012]Numerous advantages are achieved with the present invention. Sequencing can be carried out with small amounts of polynucleotide, with the capability of sequencing single polynucleotide molecules, thereby eliminating the need for amplification prior to initiation of sequencing. Long sequence read lengths can be obtained and secondary structure considerations minimised. Obtaining long read lengths eliminates the need for extensive fragment reassembly using computation. Further, as the invention is not dependent upon the need for fluorescently-labelled nucleotides or any measurement of fluorescence, the limitation of read length at the single molecule level as a function of photobleaching or other unpredictable fluorescence effects, is circumvented. The present invention also permits long polynucleotide fragments to be read sequentially by the same enzyme system. This has the benefit of allowing a single enzyme system to be used which can be regenerated and re-used allowing many different polynucleotide templates to be sequenced. Finally, the utilisation of Second or Third Harmonic Generation offers advantages due to the lack of photodamage and photobleaching. This is due to the fact that no photochemistry occurs, even in the focal plane because the signal, stimulated by non-resonant radiation, does not involve an excited state with a finite lifetime.

Problems solved by technology

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

However, these techniques have the disadvantage of increasing background interference from the fluorophores.

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

The most serious limitation of polynucleotide sequencing systems built around fluorescent dyes, however, is the problem of photobleaching.

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