News Column

Patent Issued for Methods Using Pores

August 5, 2014

By a News Reporter-Staff News Editor at Life Science Weekly -- From Alexandria, Virginia, NewsRx journalists report that a patent by the inventors Bayley, Hagan (Oxford, GB); Astier, Yann (Oxford, GB); Braha, Orit (Oxford, GB), filed on December 28, 2011, was published online on July 22, 2014 (see also Isis Innovation Limited).

The patent's assignee for patent number 8785211 is Isis Innovation Limited (Oxford, GB).

News editors obtained the following quote from the background information supplied by the inventors: "The current method for sequencing DNA involves a number of costly reagents such as fluorescent ddXTPs, dXTPs, primers and polymerase. This method requires sophisticated equipment, which needs to be operated by a qualified technician. Also, this method is limited to sequences of less than one thousand nucleotides in length.

"Other sequencing methods have been considered in order to reduce cost, simplify the method, and allow sequencing to take place out of the lab. Cycle extension, polymerase reading, exonuclease sequencing, and DNA micro-arrays are methods that have been considered (Braslaysky, I., B. Herbert, et al. (2003), PNAS 100(7): 3960-3964). These methods have been comprehensively reviewed (Marziali, A. and M. Akeson (2001), Ann. Rev. Biomed. Eng. 3: 195-223).

"One potential method of sequencing DNA is based on threading a single strand of DNA through a nanopore and identifying its sequence from the variation in the ionic current flowing through the pore as the strand is threaded (Kasianowicz, J. J., E. Brandin, et al. (1996), Proc. Natl. Acad. Sci. 93: 13770-13773). A second potential approach is exonuclease sequencing (Chan, E. Y. (2005), Mutat. Res. 573: 13-40). This method involves digesting the DNA one nucleotide at a time (Dapprich, J. (1999), Cytomet. 36: 163-168; and Matsuura, S.-I., J. Komatsu, et al. (2001), Nuc. Ac. Res. 29(16): e79) and then identifying each of the released nucleotides. However, these methods require modification of the DNA before digestion or modification of the nucleotides once they have been released from the DNA by exonuclease. The development of exonuclease sequencing is currently being held back by the difficulty in identifying the nucleotides at the single molecular level as they are released by the enzyme. Investigators have tried to identify the nucleotides using fluorescent labeling with limited success.

"Stochastic sensing involves placing a nanometer sized pore in an insulating lipid bilayer membrane and measuring the ionic transport through the pore. When an analyte interacts with a binding site within the pore, a change in the ionic current is detected (Braha, O., B. Walker, et al. (1997), Chem. & Biol. 4: 497-505; and Bayley, H. and P. S. Cremer (2001), Nature 413: 226-230). The extent and duration of the current block resulting from each binding event can reveal the identity of the analyte. The frequency of the binding events can reveal the analyte concentration. Various binding sites can be created within the pore by way of protein mutation, chemical modification, and by use of molecular adaptors and carriers (Gu, L.-Q., O. Braha, et al. (1999), Nature 398: 686-690; and Braha, O., J. Webb, et al. (2005), Chem. Phys. Chem. 6: 889-892)."

As a supplement to the background information on this patent, NewsRx correspondents also obtained the inventors' summary information for this patent: "It has been surprisingly demonstrated that individual nucleotides can be identified at the single molecule level from their current amplitude when they interact with a transmembrane pore. Hence, stochastic sensing may be used to identify individual nucleotides and to sequence nucleic acid sequences via exonuclease sequencing.

"Accordingly, the invention provides a method of identifying an individual nucleotide, comprising: (a) contacting the nucleotide with a transmembrane protein pore so that the nucleotide interacts with the pore; and (b) measuring the current passing through the pore during the interaction and thereby determining the identity of the nucleotide.

"The invention further provides: a method of sequencing a target nucleic acid sequence, comprising: (a) digesting an individual nucleotide from one end of the target sequence using a processive exonuclease; (b) contacting the nucleotide with a transmembrane protein pore so that the nucleotide interacts with the pore; measuring the current passing through the pore during the interaction and thereby determining the identity of the nucleotide; and (d) repeating steps (a) to at the same end of the nucleic acid sequence and thereby determining the sequence of the nucleic acid; and a kit for sequencing a nucleic acid, comprising: a cyclodextrin; and a processive exonuclease.

"The method of sequencing of the invention is a rapid and simple DNA sequencing method at the single molecule level. It is also a cheap method of sequencing DNA because it does not involve the use of expensive reagents, such as fluorophores."

For additional information on this patent, see: Bayley, Hagan; Astier, Yann; Braha, Orit. Methods Using Pores. U.S. Patent Number 8785211, filed December 28, 2011, and published online on July 22, 2014. Patent URL:

Keywords for this news article include: Esterases, Polymerase, DNA Research, Exonucleases, Enzymes and Coenzymes, Isis Innovation Limited.

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Source: Life Science Weekly

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