ILLUMINA® SEQUENCING Several second-generation sequencing methods are now in wide use, and we will discuss two of the most common. Both rely on the construction of libraries of DNA fragments that represent—in toto—the DNA of the genome. Instead of using bacterial cells to generate these libraries, as we saw in Figure 8–30), they are made using PCR amplifcation of billions of DNA fragments, each attached to a solid support. The amplifcation is carried out so that the PCR-generated copies, instead of foating away in solution, remain bound in proximity to the original DNA fragment. This process generates clusters of DNA fragments, where each cluster contains about 1000 identical copies of a small bit of the genome. These clusters—a billion of which can ft in a single slide or plate—are then sequenced at the same time; that is, in parallel. One method, known as Illumina sequencing, is based on the dideoxy method described above, but it incorporates several innovations. Here, each nucleotide is attached to a removable fuorescent molecule (a different color for each of the four bases) as well as a special chain-terminating chemical adduct: instead of a 3’-OH group, as in conventional dideoxy sequencing, the nucleotides carry a chemical group that blocks elongation by DNA polymerase but which can be removed chemically. Sequencing is then carried out as follows: the four fuorescently labeled nucleotides along with DNA polymerase are added to billions of DNA clusters immobilized on a slide. Only the appropriate nucleotide (that is complementary to the next nucleotide in the template) is covalently incorporated at each cluster; the unincorporated nucleotides are washed away, and a high-resolution digital camera takes an image that registers which of the four nucleotides was added to the chain at each cluster. The fuorescent label and the 3˜-OH blocking group are then removed enzymatically, washed away, and the process is repeated many times. In this way, billions of sequencing reactions are carried out simultaneously. By keeping track of the color changes occuring at each cluster, the DNA sequence represented by each spot can be read. Although each individual sequence read is relatively short (approximately 200 nucleotides), the billions that are carried out simultaneously can produce several human genomes worth of sequence in about a day. Principle behind Illumina sequencing. This reaction is carried out stepwise, on billions of DNA clusters at once. The method relies on a color digital camera that rapidly scans all the DNA clusters after each round of modifed nucleotide incorporation. The DNA sequence of each cluster is then determined by the sequence of color changes it undergoes as the elongation reaction proceeds stepwise. Each round of modifed nucleotide incorporation, image acquisition, and removal of the 3˜block and the fuorescent group takes less than an hour. Each cluster on the slide contains many copies of different, random bits of a genome; in preparing the clusters, a DNA sequence (specifed by the experimenter) is joined to each copy in every cluster, and a primer complementary to this sequence is used to begin the elongation reaction by DNA polymerase. 100 µm A slide showing individual clusters of PCR-generated DNA molecules. Each cluster carries about 1000 identical DNA molecules; the four colors are produced by incorporation of C, G, A, or T, each of which has a different color fuorophore. The image has been taken just after a fuorescent nucleotide has been incorporated into each growing DNA chain. (From Illumina Sequencing Overview, 2013.) PPP PP fuor 3˜ block 5˜3˜ block incorporation into growing chain by DNA polymerase P 5˜free 3˜ end P DNA template OH fuor removed block removed next cycle no fuor photo taken 480