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The field of DNA sequencing is transforming, and it can be challenging to keep up with the rapid evolution among all the platforms and technologies on the market.
FREMONT, CA: DNA sequencing has undergone significant advances since the first Sanger sequencing method that was introduced in 1977. Sanger sequencing randomly added chain-terminating deoxynucleotides during the DNA synthesis to generate DNA chains of lengths differing by one base pair. Today, there is a wide range of enhanced methods with rapid and precise results.
In the late 2000s, the Human Genome Project drove enhancements on the Sanger as mentioned above method that included whole-genome shotgun sequencing and next-generation sequencing processed. Breaking up large sequences of DNA and then sequencing them simultaneously in a huge parallel fashion, data can be generated faster. With advanced computer technology, the sequence could then be reassembled, and whole genomes began to be decoded.
New methods are now available. Single-molecule sequencing (SMS) is an approach that bypasses amplification by PCR, a crucial step in all next-generation technologies. PCR amplification doubles the original template DNA fragment by thousands of times so that optical detection methods can visualize it.
The Helicos Genetic Analysis Platform sequences the individual DNA molecules that are bonded to a flat surface as they are elongated. The technology still needs a pause after each round of extension to read the sequence, but it removes the PCR step needed for next-generation technologies.
The raw error rates on the sequencing of an individual DNA strand are more than 5 percent. But, when the sequencing is carried out in a highly parallel manner, the consensus finished sequence accuracy is over 99 percent. The method can also be used to sequence RNA directly, by alternating a reverse transcriptase for DNA polymerase.
The Single Molecule Real-Time (SMRT)
SMRT sequencing use of a nanometer-scale chamber to isolate a single DNA strand and DNA polymerase. The DNA polymerase molecule is then anchored and flooded with fluorescently labeled nucleotides.
These nucleotides can be detected as they diffuse down to the bound DNA strand and are recognized and incorporated by DNA polymerase. The diffusion and extension method happens in milliseconds, which means that DNA polymerase is added several bases per second. As the fluorescent dye is attached to the nucleotide through a phosphate chain, it disconnects from the nucleotide during the DNA polymerase reaction, freeing itself from the growing DNA chain.
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