New PNA-based Nanopore Sequencing Method May Aid Molecular Diagnostics
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Researchers in the laboratory of Amit Meller at Boston University (BU) have developed a single-molecule detection system that identifies the bases in a DNA strand by analyzing a peptide nucleic acid (PNA)–tagged genomic fragment as it travels through a nanopore.
PNAs are a class of synthetic nucleic acid analogs, consisting of nucelobases bound to a peptide-like backbone in a specifically designed sequence. PNAs can be used to tag a genome sequence when it binds with one of the stands of a double stranded DNA (dsDNA) molecule.
“Nanopore research is growing at an unprecedented rate,” Meller, an associate professor in BU’s biomedical engineering and physics departments, told BioTechniques. “We wanted to take it to the next level to see if we could detect short DNA sequences embedded in a long DNA molecule.”
According to Meller, this purely electrical method vastly improves nanopore sequencing, which is highly regarded for its potential to sequence whole genomes quickly and cheaply. Though the method solves the problem of how to detect single nucleotide bases-a problem that has plagued nanopore technology-Meller said he doesn’t see the method being used for whole-genome sequencing. “While there is something to be said regarding this method for complete genome sequencing, we feel that this method would be better suited for tasks in molecular diagnostics.”
PNAs have previously been used as part of a microfluidic approach to DNA sequencing. Sequence detection through PNA invasion of the DNA strand was problematic because it was primarily achieved through electrophoresis gel assays.
“While highly useful, this process ultimately has a number of limitations specifically in throughput, sample size requirements, and resolution,” said Meller. “By utilizing the inherent single-molecule nature of the nanopore system, we now have the ability to look at individual molecules, one by one, and judge whether or not they contain the PNA tag—and with it, the DNA sequence of interest.”
The single-molecule detection device is a solitary nanopore fabricated in a free-standing silicon nitride membrane using a focused electron beam. When a positive current is applied across the membrane using electrodes, negatively charged PNA-tagged DNA molecules are captured and guided through the nanopore. This provides control over the speed of the strand and enables researchers to identify bases by evaluating changes in ion current. The current is reduced to a value that reflects the displacement of electrolytes from the nanopore by the DNA segment.
“Most single-molecule sensing methods require that the molecules will be ‘located,’ which, needless to say, is not an easy task,” said Meller. “With the nanopore method, we found that long-range electrical fields can be used to focus the DNA molecules from far away into the nanopore. The same electrodes that are used to generate this focusing field also induce ion flow across the pore, giving way to the ion current which is our main detection method.” According to Meller, researchers can draw the DNA toward and through the nanopore by manipulating the electric field, without having to modify either the DNA strand or the pore itself.
“Ultimately, PNA induces DNA structure change,” said Meller. “Therefore, by detecting the PNA tags along the DNA molecule, we are in fact detecting the presence of its pre-defined corresponding sequence; thus, [this method] is a perfect match for the nanopore system.” The PNA signal is direct, so the method is highly quantitative, fast, and does not require large sample sizes. Meller said these characteristics will aid the development of new diagnostics.
Research into DNA mapping technology has focused on developing the best mechanism for sequencing complete genomes, but Meller suggested that research should also explore methods geared toward detection of specific sequences for molecular diagnostics. “We feel that by developing a method [that] searches for key sequences, we can forego the need to sequence blood samples for every sickness [and] instead look for key molecular signatures,” said Meller.
Despite its prescribed use in targeted molecular diagnostics, Meller pointed out that their new technique was still competitive with whole-genome sequencing platforms. “We do believe that our method has the ability to be rather inexpensive when compared to current sequence detection methods,” said Meller. “Given the single-molecule nature of our method, it is quite feasible that we would be able to forego the current practice of target amplification. This would not only reduce errors, but also simplify the sample preparation process, ultimately reducing costs dramatically.”
The research team intends to continue improving the technology. “We are looking as to how far we can push this method,” said Meller. “The goal is to identify a genome simply based on the manner in which it was tagged.”
Funding for the research was provided by the National Institutes of Health and the National Science Foundation. The paper, “Nanopore based sequence specific detection of duplex DNA for genomic profiling,” was published Jan. 20 in Nano Letters, a journal of the American Chemical Society.
PNAs are a class of synthetic nucleic acid analogs, consisting of nucelobases bound to a peptide-like backbone in a specifically designed sequence. PNAs can be used to tag a genome sequence when it binds with one of the stands of a double stranded DNA (dsDNA) molecule.
“Nanopore research is growing at an unprecedented rate,” Meller, an associate professor in BU’s biomedical engineering and physics departments, told BioTechniques. “We wanted to take it to the next level to see if we could detect short DNA sequences embedded in a long DNA molecule.”
According to Meller, this purely electrical method vastly improves nanopore sequencing, which is highly regarded for its potential to sequence whole genomes quickly and cheaply. Though the method solves the problem of how to detect single nucleotide bases-a problem that has plagued nanopore technology-Meller said he doesn’t see the method being used for whole-genome sequencing. “While there is something to be said regarding this method for complete genome sequencing, we feel that this method would be better suited for tasks in molecular diagnostics.”
PNAs have previously been used as part of a microfluidic approach to DNA sequencing. Sequence detection through PNA invasion of the DNA strand was problematic because it was primarily achieved through electrophoresis gel assays.
“While highly useful, this process ultimately has a number of limitations specifically in throughput, sample size requirements, and resolution,” said Meller. “By utilizing the inherent single-molecule nature of the nanopore system, we now have the ability to look at individual molecules, one by one, and judge whether or not they contain the PNA tag—and with it, the DNA sequence of interest.”
The single-molecule detection device is a solitary nanopore fabricated in a free-standing silicon nitride membrane using a focused electron beam. When a positive current is applied across the membrane using electrodes, negatively charged PNA-tagged DNA molecules are captured and guided through the nanopore. This provides control over the speed of the strand and enables researchers to identify bases by evaluating changes in ion current. The current is reduced to a value that reflects the displacement of electrolytes from the nanopore by the DNA segment.
“Most single-molecule sensing methods require that the molecules will be ‘located,’ which, needless to say, is not an easy task,” said Meller. “With the nanopore method, we found that long-range electrical fields can be used to focus the DNA molecules from far away into the nanopore. The same electrodes that are used to generate this focusing field also induce ion flow across the pore, giving way to the ion current which is our main detection method.” According to Meller, researchers can draw the DNA toward and through the nanopore by manipulating the electric field, without having to modify either the DNA strand or the pore itself.
“Ultimately, PNA induces DNA structure change,” said Meller. “Therefore, by detecting the PNA tags along the DNA molecule, we are in fact detecting the presence of its pre-defined corresponding sequence; thus, [this method] is a perfect match for the nanopore system.” The PNA signal is direct, so the method is highly quantitative, fast, and does not require large sample sizes. Meller said these characteristics will aid the development of new diagnostics.
Research into DNA mapping technology has focused on developing the best mechanism for sequencing complete genomes, but Meller suggested that research should also explore methods geared toward detection of specific sequences for molecular diagnostics. “We feel that by developing a method [that] searches for key sequences, we can forego the need to sequence blood samples for every sickness [and] instead look for key molecular signatures,” said Meller.
Despite its prescribed use in targeted molecular diagnostics, Meller pointed out that their new technique was still competitive with whole-genome sequencing platforms. “We do believe that our method has the ability to be rather inexpensive when compared to current sequence detection methods,” said Meller. “Given the single-molecule nature of our method, it is quite feasible that we would be able to forego the current practice of target amplification. This would not only reduce errors, but also simplify the sample preparation process, ultimately reducing costs dramatically.”
The research team intends to continue improving the technology. “We are looking as to how far we can push this method,” said Meller. “The goal is to identify a genome simply based on the manner in which it was tagged.”
Funding for the research was provided by the National Institutes of Health and the National Science Foundation. The paper, “Nanopore based sequence specific detection of duplex DNA for genomic profiling,” was published Jan. 20 in Nano Letters, a journal of the American Chemical Society.
