ION TORRENT™ SEQUENCING THE FUTURE OF DNA SEQUENCING Another widely used strategy for rapid DNA sequencing is called the ion torrent method. Here, a genome is fragmented, and the individual fragments are attached to microscopic beads. Using PCR, each DNA fragment is then amplifed so that copies of it eventually coat the bead to which it was initially attached. This process produces a library of billions of individual beads, each covered with identical copies of a particular DNA fragment. Like eggs in a carton, the beads are placed into individual wells on an array that can hold a billion beads in a square inch. Beginning with a primer, DNA synthesis is then initiated on each bead. A hydrogen ion (H+) is released (along with pyrophosphate) each time a nucleotide is incorporated into a growing DNA chain (see Figure 5–3), and the ion torrent method is based on this simple fact. Each of the four nucleotides is washed in, one at a time, over the array of beads; when a nucleotide is incorporated in the DNA of a given bead, the release of an H+ ion changes the pH, which is registered by a semiconductor chip placed beneath the array of wells. In this way, the DNA sequence on a given bead can be read from the pattern of pH changes observed as nucleotides are washed over them. Like a high-resolution sensor in a digital camera, the ion torrent semiconductor chip can register enormous amounts of information and can thus keep track of billions of parallel sequencing reactions. Using this technology it is currently possible, using a single chip, to determine the nucleotide sequences of several human genomes in just a few hours. PPP PP 5˜DNA template A OH OH H+ pH change detected volts 1 µm voltage-sensing chip well bead coated with DNA template A DNA sequencing by the ion torrent method. Beads, each coated with a DNA molecule that has been amplifed many times, are placed in wells along with primers and DNA polymerase. As nucleotides are sequentially washed over the beads, those incorporated by the polymerase cause a pH change. In the example shown, an A is incorporated; thus, the template must have a T in this position. As the four nucleotides are sequentially washed over the beads, the sequence of the DNA on each bead can be “read” by the pattern of pH fuctuations. Billions of beads are monitored at once by a voltage-sensitive semiconductor chip placed below the array of beads. Even newer, potentially faster, methods of sequencing DNA are being developed. Some of these “third-generation” technologies bypass the DNA amplifcation steps altogether and determine the sequence of single molecules of DNA. In one technique, a DNA molecule is pushed through a tiny channel, like a thread through the eye of a needle. As the DNA molecule moves through the pore, it generates electrical currents that depend on its sequence of nucleotides; the pattern of currents can then be used to deduce the nucleotide sequence. Other methods visualize single DNA molecules using electron or atomic force microscopy; the nucleotide sequence is read from the small differences in the “appearance” of the DNA as it is scanned. Finally, another method is based on immobilizing a single DNA polymerase molecule (with a template) and measuring the “dwell” time of each of the four nucleotides, which are labeled with different removable fuorescent dyes. Nucleotides that reside longer on the polymerase (before their dye is removed) are those incorporated by the polymerase. Although the two methods we have described in detail (Illumina and ion torrent) are now used extensively, it is likely that faster and cheaper methods will continue to be developed. 2001 2003 2005 2007 2009 2011 2013 2015 100,000,000 10,000,000 1,000,000 100,000 10,000 1000 100 cost in dollars years Shown here are the costs of sequencing a human genome, which was $100 million in 2001 and about a thousand dollars by the end of 2014. (Data from the National Human Genome Research Initiative.) 481 (A) reading direction for sequence of top DNA strand frames –3 reading direction for sequence of bottom DNA strand 500 base pairs contain a stop codon about every 20 amino acids; protein-coding regions will, in contrast, usually contain much longer stretches without stop codons (Figure 8–40). Known as open reading frames (ORFs), these usually signify bona fide protein-coding genes. This assignment is often “double-checked” by comparing the ORF amino acid sequence to the many databases of documented proteins from other species. If a match is found, even as imperfect one, it is very likely that the ORF will code for a functional protein (see Figure 8–23).