Course: SAT 170 Introductory Biotechnology

Unit 6 Reading and Multimedia Assignment

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“DNA sequencing” by Boundless, CC BY SA 3.0

https://www.boundless.com/biology/biotechnology/biotechnology-reveals-gene-sequence-expression-and-function/dna-sequencing/

Reading Assignment: DNA Sequencing
DNA sequencing includes several technologies that are used for determining the order of the nucleotide bases in a molecule of DNA.

DNA Sequencing

DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA.

Knowledge of DNA sequences has become indispensable for basic biological research. Other research branches utilize DNA sequencing, as well as numerous applied fields such as diagnostic, biotechnology, forensic biology and biological systematics. The advent of DNA sequencing has significantly accelerated biological research and discovery. The rapid speed of sequencing attained with modern DNA sequencing technology has been instrumental in the sequencing of the human genome, the Human Genome Project. Related projects, often by scientific collaboration across continents, have generated the complete DNA sequences of many animal, plant, and microbial genomes.

The first DNA sequences were obtained in the early 1970's by academic researchers using laborious methods based on two-dimensional chromatography. Following the development of dye-based sequencing methods with automated analysis, DNA sequencing has become easier and orders of magnitude faster. Automated DNA sequencing instruments (DNA sequencers) can sequence up to 384 DNA samples in a single run and perform up to 24 runs a day. DNA sequencers carry out capillary electrophoresis for size separation, detection, and recording of dye fluorescence, and data output as fluorescent peak trace chromatograms. The DNA to be sequenced is first amplified by PCR, cleaned up to remove free dNTPs and buffer salts, and resuspended in a buffer solution before being loaded onto the sequencer.

The high demand for low-cost sequencing has driven the development of high-throughput sequencing technologies that parallel the sequencing process, producing thousands or millions of sequences at once. High-throughput sequencing technologies are intended to lower the cost of DNA sequencing beyond what is possible with standard dye-terminator methods. A popular method for sequencing is called the dideoxy method, or Sanger method (named after its inventor, Frederick Sanger, who was awarded the 1980 Nobel prize in chemistry for this achievement and is one of only four people to win the award twice). The dideoxy method gets its name from the critical role played by synthetic nucleotides that lack the -OH at the 3′ carbon atom. A dideoxynucleotide can be added to the growing DNA strand, which causes chain elongation stops because there is no 3′ -OH for the next nucleotide to be attached to Figure 0, Figure 1. For this reason, the dideoxy method is also called the chain termination method.

In recent years, new sequencing technology has been developed that has been referred to as second generation, and now third generation sequencing methods are available. The second generation method is sequencing by synthesis, which involves taking a single strand of the DNA to be sequenced and synthesizing its complementary strand while detecting the activity of DNA polymerase with another chemiluminescent enzyme. Essentially, this method allows sequencing of a single strand of DNA by synthesizing the complementary strand along it, one base pair at a time, and detecting which base was actually added at each step. The template DNA is immobile, and solutions of A, C, G, and T nucleotides are sequentially added and removed from the reaction. Light is produced only when the nucleotide solution complements the first unpaired base of the template. The sequence of solutions that produce chemiluminescent signals allows the determination of the sequence of the template.

One of the strongest pieces of evidence for common descent comes from the study of gene sequences. Comparative sequence analysis examines the relationship between the DNA sequences of different species and has produced several lines of evidence that confirm Darwin's original hypothesis of common descent. If the hypothesis of common descent is true, then species that share a common ancestor will have inherited that ancestor's DNA sequence. They will have inherited mutations unique to that ancestor. More closely-related species will have a greater fraction of identical sequence and will have shared substitutions when compared to more distantly-related species.