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SU6.5 2022 notes
Genetics (GTS 251)
University of Pretoria
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Lecture 6 introduces the concept of DNA sequencing, see Pierce pages 590
DNA sequencing is a powerful molecular method for analyzing DNA, as it allows you to quickly determine the sequence of bases in a DNA molecule.
Analogous to some of the other technologies we have explored thus far, scientist exploit and manipulate a system already present in nature. The dideoxy or Sanger method of DNA sequencing is based on replication.
The fragment to be sequenced is used as a template to make new DNA molecules. In the process, replication is sometimes (but not always) terminated when a specific abnormal base is encountered. These special dideoxyribonucleoside triphosphate (ddNTP), used only in sequencing and not present in cellular DNA replication, lack a 3′-OH.
To set up a Sanger sequencing reaction, you require a ssDNA template. You split the solution into four tubes, and to each you add a primer, the four normal dNTPs and a DNA polymerase – all the components required to synthesize a new daughter strand. You also add one of the ddNTPs (either ddATP, or ddCTP, or ddGTP, or ddTTP) to each tube. One component should be labelled, to allowed visualization of the new daughter strands.
Inside the tube DNA replication proceeds from the primer, synthesizing a complementary daughter strand using the normal dNTPs. This happens in parallel for multiple different copies of the template. Should a ddNTP be encountered and incorporated, replication is terminated in that specific strand. Remember, a tube contains only one kind of ddNTP. In e. the tube containing ddATP, a set of fragments of different lengths will be produced that were all terminated opposite a T in the template.
Here is an illustration of the sequencing process. You start with a ssDNA template, the one used here has the sequence 3’ CTAAGCTCGACT 5’. You add a primer, which anneals to its complementary region, four normal dNTPs and DNA polymerase. As in a PCR, the primers used in sequencing are DNA (not RNA), and form part of the new strand. The components are sufficient to form a new strand.
The mixture is then split into four tubes, each receiving on of the special modified ddNTPs, or terminator nucleotides.
If we consider the events in the tube containing ddATP – a new strand is synthesized, complementary to the template, with sequence 5’ GATTCGAGCTGA. Occasionally, when a T is read on the template strand, instead of a normal dATP, the modified ddATP will be incorporated. This will result in termination of DNA synthesis in that specific strand. In this example there are three Ts in the template, so there are three different daughter strands terminating in ddA possible. They have lengths of 2, 7 and 12 bp respectively.
In the next tube, containing ddCTP, the same process happens except that termination here is at the two positions where there is a G in the template, and a ddCTP is incorporated. Here two possible daughter strands can form with lengths of 5 and 9 bp.
The equivalent processes occur in the ddGTP and ddTTP tubes.
The content of each of the four tubes is then loaded into adjacent wells on an acrylamide gel, and separated by electrophoresis. You cannot use agarose for this, acrylamide allows separation of fragments that differ from each other in size by only 1 bp.
Consider the content of the ddATP tube, it has three different newly synthesized daughter molecules, 2, 7 and 12 bp in length. Upon electrophoresis they will separate based on size, with the smallest molecule migrating the furthest distance, and results in 3 bands at positions 2, 7 and 12. The equivalent process takes place in the other 3 lanes.
The DNA sequence is then read from the gel, starting at the bottom.
At position 1 there is band in the ddGTP lane, it is read as G. Position 2 contains an A, 3 is T, 4 is T etc. As you move up the gel, you will read the full sequence of the new DNA strand, i. 5’ GATTCGAGCTGA 3’. This is the complement of the original template strand sequence.
Review this process carefully, if you get confused remember to also view the animations.
In automated sequencing the four ddNTPs are labelled with four different fluorescent dyes, and the reactions placed in the same well for electrophoresis. As the different sized fragments separate, their fluorescent signals are captured via a laser beam and detector, and saved onto a computer. The electropherogram then shows peaks in four different colours, from which the DNA sequence can be read.
Here is an example question for you to try. Note that in this case the DNA template sequence is provided 5’ to 3’, the other way around as the previous textbook example, with the primer site at the 3’ end of the template. Once the primer has annealed, the new strand will be formed 5’ to 3’ right to left relative to the template.
Next-generation sequencing technologies have made sequencing hundreds of times faster, and much cheaper, than traditional Sanger sequencing. Millions of DNA fragments can be sequenced simultaneously. The first sequencing of the human genome, completed in 2003, took more than ten years. Now it can be done within a day or two.
Illumina sequencing is based on the dideoxy chain termination principles.
In the third generation sequencing technologies the sequence of a single DNA molecule is determined.
You need to be aware that these technologies exist, but we will not require that you understand any of these methods’ details for this year.
SU6.5 2022 notes
Course: Genetics (GTS 251)
University: University of Pretoria
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