A team of UCSC researchers has devised a method of analysing DNA molecules that can rapidly discriminate between nearly identical DNA strands.
The technique may someday find applications in clinical settings to test patients for certain genetic traits. But for now, the ability simply to distinguish individual DNA molecules in a mixed solution is a notable achievement, said David Deamer, professor of chemistry and biochemistry.
The instrument used to perform the analysis, called a nanopore detector, is built around a membrane containing a tiny pore just big enough for a single strand of DNA to pass through. A voltage applied across the membrane generates an ionic current and pulls the negatively charged DNA molecules through the pore. A characteristic decrease in the current occurs when a DNA molecule temporarily blocks the opening.
Deamer’s lab has been working on this prototype nanopore detector for several years. The group’s latest results come from experiments with a variety of synthetic DNA molecules. Using machine-learning techniques, a computer program was ‘trained’ to recognise the signals generated by different DNA molecules. The detector was able to analyse a mixed sample and indicate the proportions of each type of molecule present in the sample.
The pore in this nanopore detector is actually a kind of toxin, known as the alpha-hemolysin ion channel, produced by Staphylococcus bacteria to punch holes in cell membranes. Because of the toxin’s role in staph infections, it has been studied extensively and its structure is known in great detail.
‘We know the environment of the ion channel very well, which helps us to understand how DNA molecules interact with it,’ said Wenonah Vercoutere, a graduate student in Deamer’s lab.
The mouth of the pore in the alpha-hemolysin ion channel is about 2.5 nanometers wide. Double-stranded DNA can enter the mouth of the pore, but the channel then narrows to less than 2 nanometers, so that only single-stranded DNA can pass all the way through. Double-stranded DNA gets stuck in the pore until the strands separate.
A typical DNA molecule–in human chromosomes, for example–consists of two strands that wrap around each other in a double helix resembling a twisted ladder. Each strand is a string of repeating units called nucleotides, and each nucleotide contains one of four ‘bases’ (abbreviated A, T, C, and G). The rungs of the ladder consist of complementary pairs of bases, one from each strand, which form weak bonds (A pairing with T, and C with G).
The DNA molecules used in the nanopore detector experiments had a ‘hairpin’ structure, consisting of a single strand of DNA folded back on itself to form a double-stranded segment (the ‘stem’) with a single-stranded loop at one end.
‘We used hairpins as a model for double-stranded DNA because they are easy to synthesise, we can control their size and the sequence of bases, and we can make single-nucleotide changes to see if we can detect those differences,’ Vercoutere said.
The detector can, in fact, differentiate between two hairpin molecules that differ by only a single base-pair in the stem, or even by just one base in the single-stranded loop. Winters-Hilt said the sensitivity of the instrument makes it a perfect match for the sophisticated computational tools he used to analyse the signals. ‘Because of the inherent sensitivity of the data, there is a real advantage to using these cutting-edge methods for signal analysis,’ he said.
Deamer first conceived of using an ion channel to analyze DNA molecules while driving back from a scientific meeting in 1989. He eventually developed a prototype with collaborators at Harvard University and the National Institute of Standards and Technology. Deamer thought it might be possible to use this system to perform rapid sequencing of DNA by detecting and identifying each base in a strand as it passes through the pore. One obstacle to this approach is that single-stranded DNA flies through the pore too fast to allow detection of individual bases. The team is now working to resolve that problem.
Another potential application might be in the detection of single nucleotide polymorphisms (SNPs), common variations in DNA sequences that account for most of the genetic differences between individuals. Many SNPs (pronounced ‘snips’) are clinically significant, and an easy method of detecting them in patients’ DNA would be extremely valuable.
‘But the best application may be entirely unforeseen by any of us, as is commonly the case when basic science is later put to use,’ Akeson said.