• To carry on with the theme of DNA, I thought today we would talk about a process downstream from PCR – Sequencing! Sequencing is the process of determining the order of the four base pairs in a DNA strand. There are three primary methods of DNA sequencing: Sanger sequencing, Next-generation sequencing, and third generation sequencing methods.


    Sanger sequencing was the first major breakthrough in reliable DNA sequencing. Developed in 1977 by Frederick Sanger, Sanger sequencing relies on a method called chain termination. Sanger sequencing shares some similarities to PCR. Like PCR, Sanger sequencing requires A DNA template, a primer, and DNA polymerase. However unlike PCR, Sanger sequencing uses only one primer which is complimentary to the forward strand of the template DNA. Therefore, the DNA polymerase will only amplify the main strand – since DNA strands are complimentary we only need to know the sequence of one strand, so this is not a problem. Another difference is that all amplification is directly from the template DNA, so you have linear amplification instead of the exponential amplification you get with PCR. The chain termination that occurs in Sanger sequencing is what allows us to determine the sequence. In addition to deoxynulceotides (dNTPs), dideoxynucleotides (ddNTPs) are added to the reaction. ddNTPs lack the 3’-hydroxyl (3’-OH) that is required to form a bond between two nucleotides, so anywhere a ddNTP is incorporated into the growing strand, elongation will stop and the DNA chain will terminate. Sometimes the ddNTP includes a label for easier detection, either radioactive or fluorescent, which helps with automation of the process. To carry out Sanger sequencing, you must perform four separate reactions to amplify the DNA. Each reaction contains all four dNTPs, the DNA polymerase, and only one type of ddNTP. So in a reaction where only ddATP was used, you will get a mixture of DNA fragments of various length and all the DNA fragments produced will end in adenosine. Once you have done this with all four ddNTPs, you separate the fragments by electrophoresis. Electrophoresis will separate the fragments based on size, giving you a range of products ordered by size and terminated in an A, T, G, or C – so you can read the sequence. Here is a video from Thermo Fisher Scientific about how this process can be automated by using a method called capillary electrophoresis and fluorescently tagged ddNTPs for detection of the DNA fragments. Sanger sequencing allows you to read up to 1,000 bases at a time. Although newer sequencing methods have emerged which are better at dealing with longer sequences, Sanger sequencing is still used today. It is often used for small sequencing projects and is also the gold-standard when comparing newer sequencing methods.


    Next-generation sequencing, abbreviated NGS and developed in the 2000s, allows sequencing of much longer sequences – even an entire genome – in one sequencing run. To do this though, NGS doesn’t actually sequence the entire piece in one reaction; it sequences many small pieces simultaneously on microfluidic devices or beads on chips, then puts the information together. The first step in NGS is to break up the target DNA into small fragments – creating what is called a sequencing library. The sequence is broken up randomly, with overlaps between fragments. Small sequences call adaptors are added to the ends of the DNA fragments. The adaptors generally help the DNA move through the device and also act as primers for DNA amplification. The library is amplified to improve signal and processed in different ways depending on the NGS method being used. Each fragment in the library is then amplified again and read simultaneously – the reading process depends on the method. One company called Illumina does this by using fluorescently tagged nucleotides and detecting them with a camera as they are incorporated into the growing strand. Another method, called Ion torrent, detects changes in pH as the nucleotides are incorporated into the growing strand. The process of sequencing as the strand is being polymerized is called sequence by synthesis. Each signal that is detected is called a base call. A computer program interprets the base calls and then assembles the entire sequence based on overlap. NGS is generally faster and cheaper than Sanger sequencing and has made sequencing much more accessible.


    The newest methods, termed third generation sequencing, read the DNA at a molecular level instead of by synthesis. Because third generation sequencing doesn’t rely on amplification of the DNA, they allow for much longer strands of DNA to be read at one time – further improving the speed and lowering the cost. One company doing this, Oxford Nanopore Technology uses bioengineered proteins called nanopores.



    The nanopores are embedded in a polymer membrane with high electrical resistance and current is generated across the nanopore. When molecules like DNA pass through the nanopore, the current flow is disrupted. The current disruption is characteristic of the molecule passing through; e.g. when a guanine on a DNA strand passes through, the disruption will be different than when an adenine on a DNA strand passing through. The change in current is used to make base calls in real time as the DNA moves through the pore. Sample processing is more efficient than NGS; fragmentation of the target DNA can be done to improve efficiency, but is not always required. Adaptors need to be added here as well, they are tags which help an enzyme guide the DNA into the nanopore– see how that works here. This technology has been incorporated into portable microfluidic devices that can be run with a USB port or cell phone! They are also significantly less expensive. While NGS sequencers can cost tens to hundreds of thousands of dollars, the Oxford Nanopore MinION sequencer starts at $1000. The MinION was recently used in combination with MiniPCR’s thermocycler to carry out the first ever start to finish identification of microbes onboard the International Space Station! I hope to start using this technology in TSoG programs in 2018!!



    Sincerely,

    Ms. Dark

  • To carry on with the theme of DNA, I thought today we would talk about a process downstream from PCR – Sequencing! Sequencing is the process of determining the order of the four base pairs in a DNA strand. There are three primary methods of DNA sequencing: Sanger sequencing, Next-generation sequencing, and third generation sequencing methods.

    Sanger sequencing was the first major breakthrough in reliable DNA sequencing. Developed in 1977 by Frederick Sanger, Sanger sequencing relies on a method called chain termination. Sanger sequencing shares some similarities to PCR. Like PCR, Sanger sequencing requires A DNA template, a primer, and DNA polymerase. However unlike PCR, Sanger sequencing uses only one primer which is complimentary to the forward strand of the template DNA. Therefore, the DNA polymerase will only amplify the main strand – since DNA strands are complimentary we only need to know the sequence of one strand, so this is not a problem. Another difference is that all amplification is directly from the template DNA, so you have linear amplification instead of the exponential amplification you get with PCR. The chain termination that occurs in Sanger sequencing is what allows us to determine the sequence. In addition to deoxynulceotides (dNTPs), dideoxynucleotides (ddNTPs) are added to the reaction. ddNTPs lack the 3’-hydroxyl (3’-OH) that is required to form a bond between two nucleotides, so anywhere a ddNTP is incorporated into the growing strand, elongation will stop and the DNA chain will terminate. Sometimes the ddNTP includes a label for easier detection, either radioactive or fluorescent, which helps with automation of the process. To carry out Sanger sequencing, you must perform four separate reactions to amplify the DNA. Each reaction contains all four dNTPs, the DNA polymerase, and only one type of ddNTP. So in a reaction where only ddATP was used, you will get a mixture of DNA fragments of various length and all the DNA fragments produced will end in adenosine. Once you have done this with all four ddNTPs, you separate the fragments by electrophoresis. Electrophoresis will separate the fragments based on size, giving you a range of products ordered by size and terminated in an A, T, G, or C – so you can read the sequence. (Here)[https://www.youtube.com/watch?v=e2G5zx-OJIw] is a video from Thermo Fisher Scientific about how this process can be automated by using a method called capillary electrophoresis and fluorescently tagged ddNTPs for detection of the DNA fragments. Sanger sequencing allows you to read up to 1,000 bases at a time. Although newer sequencing methods have emerged which are better at dealing with longer sequences, Sanger sequencing is still used today. It is often used for small sequencing projects and is also the gold-standard when comparing newer sequencing methods.

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