DNA sequencing

How the sequence of nucleotide bases (As, Ts, Cs, and Gs) in a piece of DNA is determined.
  • DNA sequencing is the process of determining the sequence of nucleotides (As, Ts, Cs, and Gs) in a piece of DNA.
  • In Sanger sequencing, the target DNA is copied many times, making fragments of different lengths. Fluorescent "chain terminator" nucleotides mark the ends of the fragments and allow the sequence to be determined.
  • Next-generation sequencing techniques are new, large-scale approaches that increase the speed and reduce the cost of DNA sequencing.

You may have heard of genomes being sequenced. For instance, the human genome was completed in 2003, after a many-year, international effort. But what does it mean to sequence a genome, or even a small fragment of DNA?

DNA sequencing is the process of determining the sequence of nucleotide bases (As, Ts, Cs, and Gs) in a piece of DNA. Today, with the right equipment and materials, sequencing a short piece of DNA is relatively straightforward.

Sequencing an entire genome (all of an organism's DNA) remains a complex task. It requires breaking the DNA of the genome into many smaller pieces, sequencing the pieces, and assembling the sequences into a single long "consensus." However, thanks to new methods that have been developed over the past two decades, genome sequencing is now much faster and less expensive than it was during the Human Genome Project.

Regions of DNA up to about 900900900900 base pairs in length are routinely sequenced using a method called Sanger sequencing or the chain termination method. Sanger sequencing was developed by the British biochemist Fred Sanger and his colleagues in 1977.

In the Human Genome Project, Sanger sequencing was used to determine the sequences of many relatively small fragments of human DNA. (These fragments weren't necessarily 900900900900 bp or less, but researchers were able to "walk" along each fragment using multiple rounds of Sanger sequencing.) The fragments were aligned based on overlapping portions to assemble the sequences of larger regions of DNA and, eventually, entire chromosomes.

Although genomes are now typically sequenced using other methods that are faster and less expensive, Sanger sequencing is still in wide use for the sequencing of individual pieces of DNA, such as fragments used in DNA cloning or generated through polymerase chain reaction (PCR).

There are a variety of next-generation sequencing techniques that use different technologies. However, most share a common set of features that distinguish them from Sanger sequencing:

  • Highly parallel: many sequencing reactions take place at the same time
  • Micro scale: reactions are tiny and many can be done at once on a chip
  • Fast: because reactions are done in parallel, results are ready much faster
  • Low-cost: sequencing a genome is cheaper than with Sanger sequencing
  • Shorter length: reads typically range from 50505050 -700700700700 nucleotides in length

In the Human Genome Project, Sanger sequencing was used to determine the sequences of many relatively small fragments of human DNA. (These fragments weren't necessarily 900900900900 bp or less, but researchers were able to "walk" along each fragment using multiple rounds of Sanger sequencing.) The fragments were aligned based on overlapping portions to assemble the sequences of larger regions of DNA and, eventually, entire chromosomes.

Although genomes are now typically sequenced using other methods that are faster and less expensive, Sanger sequencing is still in wide use for the sequencing of individual pieces of DNA, such as fragments used in DNA cloning or generated through polymerase chain reaction (PCR).

A single-nucleotide polymorphism, often abbreviated to SNP is a variation in a single nucleotide that occurs at a specific position in the genome, where each variation is present to some appreciable degree within a population (e.g. > 1%).

For example, at a specific base position in the human genome, the C nucleotide may appear in most individuals, but in a minority of individuals, the position is occupied by an A. This means that there is an SNP at this specific position, and the two possible nucleotide variations - C or A - are said to be alleles for this position.

Next-generation sequencing technologies have accelerated the discovery of novel SNPs and other variants, and these discoveries require confirmation with an orthogonal technology. Confidence and speed to results are of utmost importance in the validation of novel variants, and Grace Laboratories provides both the gold-standard Sanger sequencing and fragment analysis technology for individual variant confirmation, and Ion Torrent Next-Generation Sequencing for fast confirmation of hundreds to thousands of variants in a single day.

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