The field of Molecular Biology has made major bounds and leaps in the quest to understand the code of life. After James Watson and Francis Crick discovered the structure of DNA, the next task was to determine what was the precise order for the genetic code in living cells. Not only would this unlock the structure of the genetic makeup of life, but it could also benefit modern medicine in treating disease. The technique used to analyze DNA became known as DNA Sequencing. But what is the story of this novel concept and how has it shaped the way we look at genetics? I hope this post will help to shed some light on the matter.
DNA sequencing first became a plausible technique once Robert Holley was able to sequence a whole nucleic acid strand from Saccharomyces cerevisiae. Coupled this with the application of 2-D fractionation and short RNA sequences, the messenger material for DNA, could be sequenced to offer a glimpse into the DNA sequence. The next breakthrough occurred when scientists Alan Coulson, Allan Maxam, and Walter Gilbert used a combination of “plus and minus” system, and chemical cleavage, to produce the next genetic breakthrough. The process involved using a primer, or template, to allow DNA polymerase, an enzyme used in DNA synthesis, to add radiolabelled nucleotides to a DNA sequence so that it can be viewed on a gel matrix to determine the sequence. This technique offered the chance to sequence the first genome, bacteriophage ϕX174, a common positive control DNA sequence still used today.
Both Gilbert, and Maxam, also developed an alternative method which cleaved DNA at specific nucleotides, with specific enzymes, to run on a gel to determine the size of the fragment. The major breakthrough for DNA sequencing was the introduction of dNTP’s, which are individual nucleotides, that are incorporated into the DNA strand but can not bond with the next nucleotide in a strand; thus offering a break in the DNA strand that is compared with other dNTP’s (one each for Adenine, Guanine, Cytosine, and Thymine). This method proved revolutionary in comparing the breaks for each DNA strand at specific segments in the DNA strand.
The next major breakthrough came when luminescent nucleotides could be measured in an ATP driven reaction. ATP, which is the energy currency for the cell, acts as an activator for enzymes and can be useful in experiments that generates light. In this case, a pyrophosphate, a form of ATP, is released during the nucleotides being passed through a sensor, and the sensor measures the level of light emittance based upon the nucleotide present. With this technique, gene sequencing could be conducted with greater speed and could benefit in using the DNA present rather than dNTP’s.
After the creation of DNA sequencing, the first human to have their genome completely sequenced was Craig Venter. Venter was President of Celera Genomics which sequenced most of Venter’s DNA to decode its mystery. Surprisingly, Venter’s company was directly competing with government scientists to sequence the entire human genome. Shortly after Venter published his personal genome, James Watson, one of the original scientists who discovered the sequence of DNA, had his DNA sequence completed. Back then, genome sequences cost around $1 million dollars and was costly. Today, whole genomes can be sequenced for as little as $1000 by high-throughput sequencers. This advancement has accelerated scientists ability to analyze genomes with greater efficiency that revolutionized molecular biology.
What does the future hold for gene sequencing? With the costs of sequencing drastically decreased, private citizens could now have their genomes sequenced to be used in personalized medicine. Companies like 23andme offer the chance for people to understand their genomes and offer a glimpse into where their ancestors originated. The future for gene sequencing is looking very bright and I believe we will see new advances that could forever change the nature of molecular biology for generations to come.
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