第17章バイオテクノロジーとゲノミクス

Although there have been significant advances in the medical sciences in recent years, doctors are still confounded by some diseases, and they are using whole-genome sequencing to discover the root of the problem. Whole-genome sequencing is a process that determines an entire genome’s DNA sequence. Whole-genome sequencing is a brute-force approach to problem solving when there is a genetic basis at the core of a disease. Several laboratories now provide services to sequence, analyze, and interpret entire genomes.

For example, whole-exome sequencing is a lower-cost alternative to whole genome sequencing. In exome sequencing, the doctor sequences only the DNA’s coding, exon-producing regions. In 2010, doctors used whole-exome sequencing to save a young boy whose intestines had multiple mysterious abscesses. The child had several colon operations with no relief. Finally, they performed whole-exome sequencing, which revealed a defect in a pathway that controls apoptosis (programmed cell death). The doctors used a bone-marrow transplant to overcome this genetic disorder, leading to a cure for the boy. He was the first person to receive successful treatment based on a whole-exome sequencing diagnosis. Today, human genome sequencing is more readily available and results are available within two days for about $1000.

The basic sequencing technique used in all modern day sequencing projects is the chain termination method (also known as the dideoxy method), which Fred Sanger developed in the 1970s. The chain termination method involves DNA replication of a single-stranded template by using a primer and a regular deoxynucleotide (dNTP), which is a monomer, or a single DNA unit. The primer and dNTP mix with a small proportion of fluorescently labeled dideoxynucleotides (ddNTPs). The ddNTPs are monomers that are missing a hydroxyl group (–OH) at the site at which another nucleotide usually attaches to form a chain (Figure 17.13). Scientists label each ddNTP with a different color of fluorophore. Every time a ddNTP incorporates in the growing complementary strand, it terminates the DNA replication process, which results in multiple short strands of replicated DNA that each terminate at a different point during replication. When gel electrophoresis processes the reaction mixture after separating into single strands, the multiple newly replicated DNA strands form a ladder because of the differing sizes. Because the ddNTPs are fluorescently labeled, each band on the gel reflects the DNA strand’s size and the ddNTP that terminated the reaction. The different colors of the fluorophore-labeled ddNTPs help identify the ddNTP incorporated at that position. Reading the gel on the basis of each band’s color on the ladder produces the template strand’s sequence (Figure 17.14).

In shotgun sequencing method, several DNA fragment copies cut randomly into many smaller pieces (somewhat like what happens to a round shot cartridge when fired from a shotgun). All of the segments sequence using the chain-sequencing method. Then, with sequence computer assistance, scientists can analyze the fragments to see where their sequences overlap. By matching overlapping sequences at each fragment’s end, scientists can reform the entire DNA sequence. A larger sequence that is assembled from overlapping shorter sequences is called a contig. As an analogy, consider that someone has four copies of a landscape photograph that you have never seen before and know nothing about how it should appear. The person then rips up each photograph with their hands, so that different size pieces are present from each copy. The person then mixes all of the pieces together and asks you to reconstruct the photograph. In one of the smaller pieces you see a mountain. In a larger piece, you see that the same mountain is behind a lake. A third fragment shows only the lake, but it reveals that there is a cabin on the shore of the lake. Therefore, from looking at the overlapping information in these three fragments, you know that the picture contains a mountain behind a lake that has a cabin on its shore. This is the principle behind reconstructing entire DNA sequences using shotgun sequencing.

Originally, shotgun sequencing only analyzed one end of each fragment for overlaps. This was sufficient for sequencing small genomes. However, the desire to sequence larger genomes, such as that of a human, led to developing double-barrel shotgun sequencing, or pairwise-end sequencing. In pairwise-end sequencing, scientists analyze each fragment’s end for overlap. Pairwise-end sequencing is, therefore, more cumbersome than shotgun sequencing, but it is easier to reconstruct the sequence because there is more available information.