We will now take a closer look at the Illumina sequencing method, which uses a technique called reversible termination sequencing:


Click to enlarge.

In this video, pay close attention to how bridge amplification works.

  1. Much like in first generation sequencing, the Illumina method requires first denaturing the DNA. However, DNA is adhered to a surface that holds the DNA while it is being sequenced and it is copied through a process called bridge amplification.
  2. Polymerase, regular nucleotides, and modified nucleotides that prevent further elongation of the complementary DNA strand are also added like in the Sanger method (first generation sequencing- review if you forgot what the Sanger method is). However, the modified nucleotide in this case is an RT-base instead of a ddNTP. An important difference in RT-bases from ddNTPs is that the end of an RT-base containing the dye can be cut off so that it becomes a normal nucleotide. In other words, unlike a ddNTP, an RT-base does not create a permanent "dead-end"; after treatment, it becomes a normal nucleotide that can permit further elongation.
3. Next, the extra bases and DNA polymerase are "washed" off. Instead of needing a capillary tube for electrophoresis, cameras can simply record the color of the RT-base added to the end in the "scanning" phase, since each RT-base has its own color: blue for cytosine RT-bases, green for thymine RT-bases, yellow for guanine RT-bases, and red for adenine RT-bases.
4.  The dye attachment at the end of the RT-base is subsequently cut off so that the next base to be sequenced is exposed, and DNA polymerase and bases are added again, going back to step two.
5. This "wash-and-scan" cycle repeats until the entire DNA fragment has been sequenced; it is done simultaneously with millions of DNA fragments at a time and can sequence a complete human genome in less than a week. (31) (38)

 <<<Previous                                                                                           Next>>>


This free website was made using Yola.

No HTML skills required. Build your website in minutes.

Go to www.yola.com and sign up today!

Make a free website with Yola