Over thirty years after the beginning of the Human Genome Project, the human genome has finally been completed. How did they do it?
On April 14, 2003, the International Human Genome Sequencing Consortium announced that the Human Genome Project was complete: they had successfully mapped the first complete human genome. Twenty years later, the Telomere-to-Telomere (T2T) consortium has announced that they have mapped a truly complete sequence of the human genome. Although the Human Genome Project accounted for an impressive 92% of the human genome, their sequence could only be considered “essentially complete.” With the help of new technologies, however, the T2T consortium has been able to fill in the missing gaps and make corrections to the initial human genome sequence.
Challenges
Mapping the complete human genome took over three decades to accomplish— why did it take so long? The sheer amount of DNA that makes up the human genome alone posed a significant challenge. The limits of technology meant that researchers could not simply read the entire human genome at once. Instead, they had to determine sequences of shorter, random pieces of DNA and identify overlapping sections before incrementally piecing the pieces together for a more complete picture of the human genome. Moreover, many sections of the human genome are repetitive with near-identical sequences, making the task infinitely more difficult. At the time of the Human Genome Project, researchers were only able to read about 500 bases at a time. Currently, researchers are able to read up to 100,000 bases at once. The increased perspective researchers part of the T2T consortium got from longer sequences allowed them to more easily detect these repeating sequences and to then fill in missing pieces.
DNA Sequencing
The Human Genome Project utilized Sanger sequencing, also known as the chain-termination method, to sequence the human genome. Sanger sequencing consists of three main steps: chain termination PCR (polymerase chain reaction), size separation by gel electrophoresis, and determination of the DNA sequence. PCR is a method based on DNA replication that allows researchers to copy small segments of DNA in large amounts. In standard PCR, the two strands of the target DNA are separated using high temperature. Primers (sequences which serve as the starting point for DNA synthesis) are then bound to the ends of these separated strands. A polymerase enzyme is attached to the primer and begins to synthesize new complementary strands of DNA by adding dNTPs. Chain termination PCR differs from standard PCR in that chain-elongating inhibitors of DNA polymerase, ddNTPs, are mixed in with the dNTPs. Therefore, when the polymerase enzyme randomly adds a ddNTP instead of a dNTP, chain synthesis is terminated, resulting in DNA strands of varying lengths. In gel electrophoresis, the strands are placed on one end of a gel matrix, an electric current is applied, and the negatively-charged DNA is attracted towards the positive electrode on the opposite side of the gel. Smaller fragments will move faster than bigger fragments, meaning the strands will be arranged from smallest to largest from bottom to top. Finally, the gel is read to determine the DNA sequence. As each terminal ddNTP corresponds to a specific nucleotide in the original sequence, researchers are able to determine the sequence of the original DNA strand. Although Sanger sequencing is time consuming and costly, it is the gold standard for DNA sequencing.
454 sequencing, or pyrosequencing, like Sanger sequencing, begins with dividing the DNA sequence into fragments and using PCR to create copies of each fragment. Fragments of the same type are put into a well and incubated with various substances. One of the four types of nucleotides are added to the wells and then are incorporated onto the single-strand DNA fragments by the polymerase enzyme. The pyrophosphate released from this process is converted into ATP, and following a series of reactions, a light of intensity proportional to the amount of ATP produced is emitted. This process is repeated until the complementary strand is completed. A detector picking up the intensity of light emitted throughout the process determines the number and type of nucleotides added. While pyrosequencing is much more cost-effective and accurate than Sanger sequencing, it can only read short sequences.
Researchers are now working on third-generation sequencing which will allow them to read longer sequences. There are two main types: nanopore sequencers and single molecule, real-time sequencing (SMRT) platforms. Nanopore sequencing utilizes the differences in size and electrical properties of each nucleotide base, measuring the electrical current changes to determine the DNA sequence. The SMRT platform detects fluorescence events that correspond to the addition of specific nucleotides. Each nucleotide is labeled with its own color. Every time the polymerase enzyme adds a nucleotide on a single-stranded DNA, a camera takes a picture. The different colors then allow researchers to determine which base was added. However, third-generation sequencing platforms are still expensive and lack the same accuracy as Sanger sequencing. Nevertheless, these advances in DNA sequencing technologies have allowed for great breakthroughs in science, including the complete sequencing of the human genome.
BIBLIOGRAPHY
Adams, Jill U. “DNA Sequencing Technologies.” Nature News, Nature Publishing Group, https://www.nature.com/scitable/topicpage/dna-sequencing-technologies-690/.
“A Brief Guide to Genomics.” Genome.gov, https://www.genome.gov/about-genomics/fact-sheets/A-Brief-Guide-to-Genomics.
Green, Eric D. “Completing the Human Genome Sequence (Again).” Scientific American, Scientific American, 31 Mar. 2022, https://www.scientificamerican.com/article/completing-the-human-genome-sequence-again/.
Greenwood, Michael. “What Is Pyrosequencing?” News, 31 Oct. 2018, https://www.news-medical.net/life-sciences/What-is-Pyrosequencing.aspx.
Hartley, Gabrielle. “How Scientists Finally Completed the Human Genomic Puzzle.” PBS, Public Broadcasting Service, 1 Apr. 2022, https://www.pbs.org/newshour/science/how-scientists-finally-completed-the-human-genomic-puzzle.
“Sanger Sequencing Steps & Method - Sigmaaldrich.com.” MilliporeSigma, https://www.sigmaaldrich.com/IS/en/technical-documents/protocol/genomics/sequencing/sanger-sequencing.
Sharman, Sarah. “Piecing Together the Genome: The Long and Short of It All.” HudsonAlpha Institute for Biotechnology, 3 Feb. 2021, https://www.hudsonalpha.org/piecing-together-the-genome-the-long-and-short-of-it-all/.
“What Is PCR (Polymerase Chain Reaction)?” Facts, The Public Engagement Team at the Wellcome Genome Campus, 21 July 2021, https://www.yourgenome.org/facts/what-is-pcr-polymerase-chain-reaction.
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