Sequencing entails the base-by-base determination of nucleotide content within a strand of DNA. There are two widely used methods for DNA sequencing—terminator-based sequencing (also known as “Sanger” sequencing) and sequencing-by-synthesis (associated with a variety of high throughput sequencing methods such as Ion Torrent and pyrosequencing).

Traditional Terminator Sequencing

Terminator sequencing is based on the process of ss DNA replication with DNA Pol. As described previously, DNA Pol synthesizes new strands of DNA by binding to a primer/template complex and adding new nucleotides to the 3’ hydroxyl group of the new strand. The sequence of the new strand depends on (i.e., is complementary to) the sequence of the template strand. In terminator sequencing, a mixture of regular and special nucleotides is added to an in vitro DNA replication reaction. These special nucleotides are called terminators because, unlike regular deoxyribonucle- otides (dNTPs), they lack the 3’ hydroxyl group required for chain elongation. These terminating nucleotides are called di-deoxyribonucleotides, or ddNTPs (e.g., ddGTP, ddCTP, ddTTP, and ddATP). DNA Pol utilizes both dNTPs and ddNTPs for the extension of new DNA strands. When a dNTP is added, the extension reaction continues and the chain grows. When a ddNTP is added, the extension reaction is terminated because no more nucleotides can be attached. DNA Pol falls off the template, having produced a truncated strand, and starts again. After many rounds, new strands—terminating at every possible position within the chain—are produced. For example, new strands may be A*, AG*, AGT*, AGTT*, AGTTC*, AGTTCG*, AGTTCGA*, and so on, where the asterisk represents the inclusion of a ddNTP at the end of the chain. The collection of sequentially truncated strands can be used to reconstruct the sequence of the original template, assuming that the final ddNTP in each truncated chain can be identified as a G, C, A, or T.

The method of final ddNTP detection has changed significantly over time. Originally, radioactive ddNTPs were added to four individual DNA replication reactions. Each reaction was spiked with just one type of ter- minator—ddGTP, ddCTP, ddATP, or ddTTP. Thus, if a truncated chain was detected in the ddGTP-containing reaction mixture, the researcher could deduce that the final nucleotide in that chain was a G (and so on). The products of the four reactions (each containing complex mixtures of truncated strands, but each terminating with the nucleotide matching the ddNTP added to the mixture) were separated based on size, using gel electrophoresis at a single-nucleotide resolution. Bands were detected by autoradiography, as the radioactive ddNTPs expose X-ray film to produce dark bands. The sequence was deduced by reading the fragment sizes in the various lanes.28

Current detection methods include the use of differentially labeled fluorescent ddNTPs, which are simultaneously added to a single DNA replication reaction. For example, ddATPs may emit green, ddCTPs may emit blue, ddGTPs may emit red, and ddTTPs may emit yellow fluorescence. Thus, a green strand ends with an A, and so on. The complex mixture of products is again separated by gel electrophoresis, but this time using a single lane (usually a capillary tube). A laser is mounted at the end of the gel to detect the color of the fragments as they pass by. Peaks of fluorescence are produced to create a chromatogram. The sequence of the newly formed strand, and by deduction the template strand, is determined by the order of the colors emerging from the gel.

Typically, 800 to 1,500 quality nucleotides can be determined in a single-terminator sequencing reaction. If a larger length of DNA sequence is desired, multiple reactions must be performed on overlapping regions of the template. Sequence overlaps are used to piece the sequences from each independent reaction together into one contiguous sequence. In this and similar contexts, individual sequences are called reads while the pieced-together sequence is called a contig. As you might imagine, terminator-based sequencing of extremely long pieces of DNA, such as whole genomes, is time-consuming and expensive. And yet, terminator sequencing is still the preferred method for short sequences, such as single genes and PCR products.

< Prev   CONTENTS   Source   Next >