Tuesday, September 25, 2012

Historical Development of Automated Sequencing Using the Sanger Method

Autnmated sequencing has been a technique utilized since the early 1980s. Although the technology has changed dramatically since its first use, the basic chemistry is still commonly used today. It is based on four basic steps: purification of DNA, amplification using the polymerase chain reaction (PCR), separation by electrophoresis and analysis.
Sanger's Dye Terminator Chemistry
Although several methods were developed during the 1970s, the dye-terminator method invented by Fred Sanger is the accepted method used in automated sequencing. The amplification step in PCR combines a mix of raw DNA bases (dNTPs) and bases that cause termination (ddNTPs). The advantage of using ddNTPs is that a number of DNA products amplified during PCR terminate once the ddNTP is added. This creates a series of products that are different by a single base. The end product is a combined soup that contains products ranging from about 19 bases in length up to hundreds of bases, each different by a single base. On separation media, the total number of products would appear as a ladder. In addition, the ddNTPs are labeled to allow for detection.
Initially Radioactivity Was Used in the Dye-Terminator Method
Each ddNTP that represented one of the four DNA bases also contained a radioactive label. The amplified products were separated on media though electrophoresis. Then the media was removed and photographed to view the base sequence of the sample. However, the problem with this method was that there was no way to detect a difference between the ddNTP labeling a G base versus A, C or T. Therefore, it was necessary to amplify the sample in four separate reactions in which only one base ddNTP was present. One tube would terminate only when the sequence had the G base while the other three tubes labeled either A, C or T.
With this in mind, four separate reactions would be loaded separately on the media. Each base would appear as an incomplete ladder. The researcher conducting sequencing would need to draw a line across four separate lanes on the media in order to determine the sequence of all four bases.
Fluorescent Labels Replaced Radioactivity
The requirement of using four lanes to determine one sequence was soon replaced with fluorescent labels on the ddNTPs. Each ddNTP representing one of the four bases was labeled with a different fluorophore that would be detected as a different color: green for As, blue for Cs, red for Ts and yellow for Gs. The need for four lanes was eliminated. This expanded the capacity to sequence samples by four tiles.
In addition, a system capable of detecting the bases was also developed in the early 1980s, soon after Sanger developed the Dye-Terminator method. Samples were loaded on the same medium initially used for the radioactive method. Then they were fitted into a machine that would run electrophoresis. This provided a second advantage. It was no longer necessary to keep the base ladder on the gel for photographing later. Instead, as each band representing a base reached the end point on the media, the automated machine would photograph the color and send this information to a computer. Once complete, the media was simply discarded appropriately. This allowed more of the amplified products to be determined increasing capacity of the radioactive method an additional two times.
Automated Sequencing Equipment has Evolved
Separation of the radioactive products of DNA amplification was performed using a procedure called electrophoresis. Essentially a reagent called acrylamide was poured between two glass plates where it would polymerize into a gel-like matrix called polyacrylamide. The samples would be loaded into the top of the matrix and electrical current would cause the DNA to migrate through the gel. Small products migrate faster than large products when an electrical current is applied because they incur less resistance.
The same process was used when the first automated sequencers were developed. Over time this technology continued to improve so researchers could determine longer pieces of DNA and load more samples. Overall the technology changed very little until the invention of capillary sequencers. Thin glass capillaries replaced the bulky glass plates. It was no longer necessary to pour gels. Instead, a new polymer was injected automatically each time samples were to be loaded. Even better was the amount of time to run an average sample. Glass plate gel electrophoresis could take more than 12 hours to determine a sequence of 400 or 500 bases. Glass capillaries could do the same job in a little over 2 hours.
Like slab gel (glass plate) electrophoresis, capillary sequencing has continued to develop faster methods of sequencing more samples. The overall output is tremendous when compared to the original automated sequencers.
Today, science has continued to develop better methods for sequencing DNA. Next generation sequencing has the capacity to sequence an entire two megabase genome in a few days. This same job would require even the most high-tech Sanger sequencers months of preparation and processing. This does not mean automated sequencers will be replaced. There is still a great need to sequence shorter pieces of DNA at a substantially lower overall cost.

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