After the DNA fragments have been flanked by adaptor sequences, individual DNA fragments must be physically separated in some way. They are then amplified independently to give separated clusters of monoclonal DNA that will provide the DNA templates for sequencing. (It is important that the amplified DNA within each cluster is composed of one type of DNA; if not, the recorded sequencing signals would be unreadable.) Two amplification methods have been particularly popular, as listed below.
• Emulsion PCR. This amplification method was pioneered in the Roche/454 sequencing approach, and variant methods have been used for the ABI SOLiD and Life Sciences/Ion Torrent DNA sequencing platforms. The object is to separate individual DNA fragments in individual tiny water droplets so that each fragment in the starting DNA can be amplified separately. This is done by mixing an aqueous phase containing a library of DNA fragments (plus primers and reagents for PCR amplification) with oil to create an emulsion: tiny water droplets become suspended in the oil and can entrap individual DNA fragments (Figure 1).
• Bridge amplification. Originally developed by the Solexa company, this amplification method is used by the Illumina sequencing platforms. It involves a type of two-dimensional PCR amplification of well-separated DNA fragments that are covalently bound to the surface of a glass slide within a flow cell (Figure 2). A related but different method of amplification known as Wildfire has more recently been adopted for use with ABI SOLiD sequencing, as described below.
Fig1. Principle of emulsion PCR. (A) Generating tiny water-droplet microreactors. DNA fragments (1, 2, 3, 4, and so on) prepared from the DNA source are ligated to double-stranded oligonucleotide adaptors so that each fragment has at one end an adaptor containing the same sequence as the sequencing primer (labeled S here) and at the other end a different adaptor (labeled R here). Multiple copies of the sequencing primer are tethered by a short linker sequence at the 5′ end to the surface of tiny beads (about 28 μm in diameter); each bead has very many copies of the S primer, but for the sake of clarity, only eight S primer copies are shown here. The adaptor linked DNA fragments are mixed with the primer-linked beads plus reverse-strand primer and a heat-stable DNA polymerase, and the resulting aqueous reaction mix is inserted into the emulsion oil, and thoroughly mixed. Tiny water droplets are formed. By ensuring a low concentration of starting DNA, most bead-containing water droplets will have zero or one DNA template molecule. Productive droplets contain a single DNA fragment with all the necessary components for DNA amplification: a bead with bonded sequencing primers, free reverse primers, and DNA polymerase molecules (not shown), constituting a “microreactor.” (B) DNA amplification in microreactors. After the DNA fragment (number 1 in this example) is denatured and cooled, the reverse strand can hybridize to a complementary sequencing primer (S) protruding from the bead, allowing DNA synthesis of a forward strand from the primer bound to the bead. Further denaturation and primer annealing allows a reverse DNA strand to bind to a sequencing primer attached to the bead, and also allows a reverse-strand primer to bind to the forward strand previously synthesized. After further cycles, clonal amplification results so that each bead will have a cluster of many copies of a single type of DNA fragment covalently bound to it.
Fig2. Bridge (cluster) amplification. (A) The surface of an Illumina flow cell is carpeted with two types of single-stranded oligonucleotide (red and blue bars) that are tethered to the surface at their 5′ ends by a short, flexible linker. The two different types of fixed oligonucleotides represent the two types of adaptor sequence fixed at the ends of the test DNA fragments (1, 2, 3, 4, and so on). Test DNA fragments that have been made single-stranded can bind at one end to a tethered oligonucleotide with a complementary sequence. The DNA templates are present at a low concentration to ensure wide spacing between individual bound DNA fragments. (B) Individual DNA fragments are amplified by cycles consisting of: (i) DNA synthesis using the bound test DNA fragment as a template; (ii) denaturation leading to exit of the original test DNA fragment; and (iii) bridging (where the tethered DNA bends so that the adaptor sequence at the free end hybridizes to a neighboring complementary adaptor-specific oligonucleotide fixed on the surface). (C) The end result is a series of physically separate DNA clusters, each containing multiple copies of just one type of DNA fragment (monoclonal DNA clusters). For clarity, this example shows four clusters, but in practice there will be many millions of DNA clusters.
