Complete DNA Structures
“Whole genome sequencing is on the way to becoming a common research tool,” says David Bentley, Ph.D., chief scientist at Solexa(www.solexa.com). “There are many other applications beyond SNP identification. We are focusing on changes in DNA sequences that occur in drug-resistant pathogens and on subtle somatic DNA mutations leading to cancer. In the majority of cases, cancer arises due to aberrations in multiple genes. With a virtually unlimited DNA sequencing capacity, we can reveal the changes in the entire genome.”
The company aims to lower DNA sequencing costs by orders of magnitude and it projects that as early as next year a complete human genome could be sequenced for $100,000. Solexa’s technology is based on clonal expansion of DNA fragments captured on a solid support. Each DNA strand carries forward and reverse adaptors and forms a loop when both ends bind to the immobilized, complementary primers. The loops become the substrate to the DNA polymerase, thus amplifying and forming a 1,000-copy cluster. A 100-micrometer square of the flow cell may contain as many as 1,000 clusters.
All four proprietary fluorescent terminator nucleotides are added at the same time. High efficiency of incorporation is achieved by using a DNA polymerase specifically evolved for this purpose. The fluorophores and 3´ termination are cleaved, and the reaction is repeated. The technology has been validated in projects as large as successful resequencing of the human X chromosome. According to the company, its sequencing results average Q18 in quality over 35 bases on both BAC and human genomic DNA.
“Bacteria often incorporate new DNA sequences that are not found in reference DNA,” continues Dr. Bentley. “To decode these new loci, we developed the method for sequencing paired ends of the same fragment. The fragment is circularized to include an adaptor in between the two ends. A universal sequence and the intervening adaptor are used to initiate two separate sequencing reads. The two sequence datasets are mapped to the original template in order to extract pairing information.” In mid-2006 the company shipped first-generation instruments to its early access partners, which include the Broad Institute in Cambridge and the Genome Sequencing Center (GSC) at the Washington University School of Medicine in St. Louis.
In July, Applied Biosystems (ABI; www.appliedbiosystems.com) entered the realm of high-throughput sequencing with the acquisition of Agencourt Personal Genomics (APG). “We have carefully evaluated over 40 types of high-throughput sequencing technologies, from those in embryonic stages to those that are well developed,” says Andy Watson, Ph.D., senior director of market development in the genetic analysis department of the molecular and cell biology division.
“APG’s technology is complementary to current ABI platforms and applicable to many experiments. We chose cancer gene resequencing, high-throughput gene expression, and resequencing of bacterial genomes as our first target applications.”
APG’s sequencing chemistry, Sequencing by Oligonucleotide Ligation and Detection (SOLiD™), has a unique, inherently built ability to interrogate two bases at the same time, Dr. Watson reports, resulting in extremely accurate readings. The SOLiD method utilizes random 8-mer primers with a fifth position containing A, G, T, or C. Primers are labeled with one of the four fluorescent dyes. The color of a fluorescent dye indicates the base in the fifth position. A random primer is ligated to the template only when the labeled nucleotide complements the fifth nucleotide on the template, counting from the end of the previously ligated primer.
After visualizing the color, the fluorescent tag is removed by cleaving the primer between the fifth and sixth positions. The process is repeated and every fifth position is recorded. Next, the system is reset to generate the recording for every n-1, n-2, n-3, and n-4 positions.
“Sequencing by synthesis starts generating noise after 25 nucleotides. Our technology demonstrated feasibility up to 50 bases,” adds Dr. Watson.
“We have also developed two-step encoding with known nucleotides in every fourth and fifth position of the primer. This is a unique method offering outstanding base-calling accuracy for SNP detection and is being used successfully in our cancer-resequencing work with Victor Velculescu, M.D., Ph.D., at John Hopkins University.” The company maintains that APG would not be in competition with ABI’s line of microcapillary sequencers based on the Sanger method. “Our primary competition is really microarrays,” states Dr. Watson.
Visigen Biotechnologies (www.visigenbio.com) utilizes a fluorescently tagged polymerase and color-coded fluorescent nucleotides to detect the addition of a particular base to a growing DNA chain, thus determining DNA sequence. The polymerase is modified with a donor fluorophore and immobilized on a glass slide. When an acceptor-labeled nucleotide is incorporated into the growing polymer, energy transfers from the polymerase to the nucleotide, stimulating emission of a base-specific signal. Because nucleotides are modified with the acceptor fluorophore on ¡-phosphate, the fluorescent moiety is released in a pyrophosphase complex.
“Our technology results in native nucleotides incorporated into the growing DNA strand and immediate detection of sequence information,” says Susan Hardin, Ph.D., CEO of Visigen. “Moreover, we are observing and detecting the DNA synthesis in real time. We expect to achieve the sequencing rate of 1 megabase per second.”
To detect the emission of an individual nucleotide on each individual strand, the company developed an extremely sensitive detection system. Visigen demonstrated proof-of-principle by detecting interactions between the polymerase and 30 sequential acceptor-labeled nucleotides. The company plans to enter the market with custom sequencing services. “We believe, however, that our system will become a solution for any DNA-sequencing project,” adds Dr. Hardin. Although Visigen technology is still in development, it has attracted attention from ABI, which completed an equity investment and entered into a scientific collaboration agreement with the company last year.