CS262: COMPUTATIONAL GENOMICS LECTURE 11 SCRIBE: ANJALEE SUJANANI 1 LECTURE 11: CONTEXT FREE GRAMMARS AND RNA STRUCTURE Lecturer: Serafim Batzoglou 14 February 21, 2006 1 New Sequencing Technologies Molecular invasion probes were discussed in the previous lecture as a new technology for sequencing. Outlined below are a few more new technologies that may be used to sequence or re-sequence a genome and which have the potential to dramatically alter the way we do genomics. 1.1 Single Molecular Array for Genotyping: Solexa Solexa is a company based in Cambridge, which uses a single molecular array for genotyping. The goal of this technology is to re-sequence human genomes. Firstly, genomic DNA is extracted from sample cells taken from an individual and in a single tube reaction the DNA is processed into single-stranded oligonucleotide fragments: These are then prepared for attachment to Solexa’s Single Molecule Arrays using proprietary primer and anchor molecules:CS262: COMPUTATIONAL GENOMICS LECTURE 11 SCRIBE: ANJALEE SUJANANI 2 The single stranded DNAs may now be extended to double stranded ones in a number of cycles or phases. At each cycle, a fluorescently labeled nucleotides and a polymerase enzyme is added to the Single Molecule Array. Complementary nucleotides base-pair to the first base of each oligonucleotide fragment and are added to the primer by the enzyme. Remaining free nucleotides are removed. Hence each single stranded DNA molecule is extended according to what the complementary base is. Laser light of a specific wavelength for each base excites the label on the incorporated nucleotides, which fluoresce. This fluorescence is detected by a Charge-coupled Device (CCD) camera that rapidly scans the entire array to identify which particular nucleotide was incorporated on each fragment. The identity of the incorporated nucleotide reveals the identity of the base in the sample sequence to which it is paired – for example, Cytosine (C) in the diagram. Fluorescence is then removed, so that the reaction can happen again, and the DNA strands be extended by one base.CS262: COMPUTATIONAL GENOMICS LECTURE 11 SCRIBE: ANJALEE SUJANANI 3 According to Solexa, this cycle of incorporation, detection and identification can typically be repeated approximately 25 times; hence it is possible to determine the first 25 bases in each oligonucleotide fragment. This reaction can happen in parallel for hundreds of millions of oligonucleotide fragments, so it may be possible to sequence a whole human genome on one chip or on a few chips, with very little cost. Assuming that 24 out of 25 letters give a unique position, each of the hundreds of millions of sequences can then be aligned and compared to a reference original human genome sequence (such as from the human genome project) using Solexa’s proprietary bioinformatics system. A single letter of difference between the current human genome being sequenced and the reference sequence can be detected and known or unknown single nucleotide polymorphisms (SNPs) and other genetic variations can be determined. 1.2 Nanopore Sequencing In Nanopore sequencing a single stranded DNA or RNA piece is electrophoretically driven through a nano-scale pore in such a way that the molecule traverses the pore in a linear sequence. The reaction is very rhythmic and happens for only one nucleotide per given time frame. As the DNA passes, it partially obstructs the nanopore, there by changing its electrical properties. Thus it is possible to obtain a reading of which particular nucleotide passed since the properties of the nanopore change differently depending on which nucleotide is translocating.CS262: COMPUTATIONAL GENOMICS LECTURE 11 SCRIBE: ANJALEE SUJANANI 4 The underlying goal is that DNA may be read in a very similar way to CD ROMs or DVD ROMs, however in practice this has not yet been worked to such good effect. Currently this technology does not seem to be making a lot of progress; however this could change with some technological advances in the near future. 1.3 Pyrosequencing This is a promising new technology which has been commercialized and marketed by Biotage AB. 1.3.1 Outline The technique adds one nucleotide into a mixture of four enzymes (DNA polymerase, ATP sulfurylase, luciferase and apyrase) and uses the bioluminescence to monitor the incorporation of nucleotides into the DNA. As shown in the diagram, the following is carried out: Polymerase - One of the four nucleotides dNTP (dATP, dCTP, dGTP, dTTP) is added to the reaction mixture, which will incorporate this nucleotide if it is complementary to base in the currently extended strand of DNA. If the nucleotide is incorporated, the reaction will result in the release of inorganic pyrophosphate (PPi).CS262: COMPUTATIONAL GENOMICS LECTURE 11 SCRIBE: ANJALEE SUJANANI 5 ATP sulfurylase - The PPi is converted by the enzyme ATP sulfurylase into ATP (which is basically energy). Luciferase - The luciferase enzyme catalyzes a reaction using ATP to produce light. The amount of light is proportional to the amount of ATP and so is also proportional to the amount of incorporated nucleotides via the PPi. This light is detected by a CCD camera. Apyrase - With addition of Apyrese, the remaining dNTP and ATP are degraded to (d)ADP and then (d)AMP and then the reaction can start again with the addition of a different nucleotide to the reaction mixture. 1.3.2 Pyrosequencing on a Chip Pyrosequencing may be done on a chip composed of many small wells. Each well contains several copies of one nucleotide strand. During the reaction, the number of incorporations can be seen by how much light each particular well emits. This technology is the basis of the 454-sequencing machine which is in production and is being sold for $500,000. The machine is fairly quite powerful already and costs about $5,000 to sequence simultaneously 250,000 reads. This means that one can take a microbial DNA and sequence it to fairly high coverage. One cycle of the machine takes about 4 hours currently, so
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