Genome Sequencing – Chain Termination Method

//Genome Sequencing – Chain Termination Method

Genome Sequencing – Chain Termination Method

DNA sequencing is the process of determining the sequence of nucleotides within a DNA molecule. It includes technology that is used to determine the order of the four bases: adenine, guanine, cytosine and thymine —in a strand of DNA.

Method of Sequencing:

Sanger sequencing

This method is also known as the chain termination method. This method is developed by the British biochemist Fred Sanger and his colleagues in 1977. The DNA of upto 900 bp has been sequenced using this method.

Sanger sequencing was used in the Human Genome Project 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 then entire chromosomes.

This method of genome sequencing is widely used for the sequencing of individual pieces of DNA, such as fragments used in DNA Cloning or generated through polymerase chain reaction (PCR).

Ingredients for Sanger sequencing

Sanger sequencing requires many copies of a target DNA region, hence its ingredients are similar to those needed for DNA replication in an organism, or for polymerase chain reaction (PCR), which copies DNA in vitro. They include:

  • A DNA polymerase enzyme
  • A primer, which is a complementary single-stranded DNA that binds to the template DNA and acts as a “starter” for the polymerase
  • The four DNA nucleotides (dATP, dTTP, dCTP, dGTP)
  • The template DNA to be sequenced

However, a Sanger sequencing reaction also contains a unique ingredient:

  • Dideoxy, or Chain terminating versions of all four nucleotides (ddATP, ddTTP, ddCTP, ddGTP), each labeled with a different color of dye.
  • Dideoxy nucleotides are similar to regular, or deoxy, nucleotides, but with one key difference: they lack a hydroxyl group on the 3’ carbon of the sugar ring. In a regular nucleotide, the 3’ hydroxyl group acts as a “hook,” allowing a new nucleotide to be added to an existing chain.
  • The method is based on the process that involves a dideoxy nucleotide added to the chain, hence there is no hydroxyl available and no further nucleotides can be added. The chain ends with the dideoxy nucleotide, which is marked with a particular color of dye depending on the base (A, T, C or G) that it carries.

Process of Sanger sequencing

  • All the ingredients i.e. the DNA sample to be sequenced, primer, DNA polymerase, and DNA nucleotides (dATP, dTTP, dGTP, and dCTP), four dye-labeled, chain-terminating dideoxy nucleotides are added in a tube.
  • To denature the template DNA (separate the strands), The mixture is first heated, then cooled so that the primer can bind to the single-stranded template. After the primer has bound, the temperature is raised again, which allows DNA polymerase to synthesize new DNA starting from the primer. DNA polymerase will continue adding nucleotides to the chain until it happens to add a dideoxy nucleotide instead of a normal one. At that point, no further nucleotides can be added, so the strand will end with the dideoxy nucleotide.
  • The above process is repeated in a number of cycles. By the time the cycling is complete, it’s virtually guaranteed that a dideoxy nucleotide will have been incorporated at every single position of the target DNA in at least one reaction. The tube will contain fragments of different lengths, ending at each of the nucleotide positions in the original. The ends of the fragments will be labeled with dyes that indicate their final nucleotide.
  • The fragments are run through a long, thin tube containing a gel matrix in a process called capillary gel electrophoresis after the reaction is done. Short fragments move quickly through the pores of the gel, while long fragments move more slowly. As each fragment crosses the “finish line” at the end of the tube, it’s illuminated by a laser, allowing the attached dye to be detected.
  • The smallest fragment (ending just one nucleotide after the primer) crosses the finish line first, followed by the next-smallest fragment (ending two nucleotides after the primer), and so forth. Thus, from the colors of dyes registered one after another on the detector, the sequence of the original piece of DNA can be built up one nucleotide at a time. The data recorded by the detector consist of a series of peaks in fluorescence intensity, as shown in the chromatogram The DNA sequence is read from the peaks in the chromatogram.

Advantages of Whole Genome Sequencing

  • The Sequencing is used to study gene and protein encoded by the gene. Information helps to identify the changes in the gene associated with the disease. Hence it leads to treat disease which is possible based not only on the mutant genes causing a disease, but also other genes in the patient’s genome.
  • Genotyping of  cancer cells and understanding what genes are misregulated helps the reasearcher to design the treatment of the cancer patients as  chemotherapy and potentially expose the patient to less toxic treatment is tailored.
  • The unknown gene which are contributing to a disease can also be identified
  • As DNA carry a genetic information that passes from one generation to another, DNA sequencing is used in evolutionary biology to know how different organisms are related and how they evolved.
  • DNA sequencing plays major role in the field of metagenomics as it involves identification of organisms present in a body of water, dirt, debris filtered from the air, or swab samples, sewage. Knowing which organisms are present in a particular environment is critical to research in microbiology, ecology, epidemiology and other fields. Sequencing enables researchers to determine which types of microbes may be present in microbiome.
  • DNA sequencing is used along with DNA methods for forensic identification and paternity testing. As the DNA patterns in fingerprint, saliva, hair follicles, etc. are uniquely separate in each living organism. Testing DNA is used to detect specific genomes in a DNA strand to produce a unique and individualized pattern. Every living organism ever created has a one of a kind DNA pattern, which can be determined through DNA testing hence DNA testing is highly successful.

Disadvantages of Whole Genome Sequencing

  • A lot of the “information” found in a human genome sequence is unusable at present as the role of most of the genes in the human genome is still unknown or incompletely understood.
  • Interpretation of genomic data is very difficult.
  • An individual’s genome may contain information that they DON’T want to know. For example, a patient has genome sequencing performed to determine the most effective treatment plan for high cholesterol. In the process, researchers discover an unrelated allele that assures a terminal disease with no effective treatment.
  • The volume of information contained in a genome sequence is vast. Policies and security measures to maintain the privacy and safety of this information are still new.
By |2018-05-04T05:29:08+00:00May 3rd, 2018|Molecular Biology|Comments Off on Genome Sequencing – Chain Termination Method

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