DNA sequencing

DNA sequencing is the process that is used to map out the sequence of the nucleotides. These nucleotides contain strands of DNA. In 1970s, using methods on two-dimensional chromatography, the academic researchers found the first DNA sequences. However, dye-based sequencing methods made DNA sequencing easier and faster. The DNA sequencing is a new technology. It is in use since the invention of the microscope. DNA sequencing is the important process in which scientists unravel genetics. DNA sequencing reactions are the PCR reactions for replicating DNA.  This reaction includes the template DNA, an enzyme, free nucleotides and a 'primer'. The primer is a small piece of single-stranded DNA, which is 20-30 nt long. This single stranded DNA can hybridize to one strand of the template DNA.

The DNA sequencing reaction is started by heating the DNA strands. This heating would separate the DNA strands. Then the primer sticks to the particular location and these DNA polymerase starts elongating the primer. When completed, it gives a new strand of DNA. The method of DNA sequencing transforms the DNA of a specific organism into a format that can be used and easily understood by researchers and scientists. With the DNA sequencing, fields like forensic biology and biotechnology have gained advantage. With DNA sequencing, scientists understand genes and their role. In Forensic biology, DNA sequences identify the unique organism. In Agriculture, DNA sequencing has allowed the scientists to make insect and pests resistant.

Sequencing Methods

The DNA sequencing includes various methods. Let us now discuss a few methods of DNA sequencing:

·         Maxam-Gilbert Sequencing

The Maxam-Gilbert technique of DNA sequencing is a very common method. It depends on the cleaving of nucleotides by chemical. This method is more efficient with small nucleotide polymers. As the name suggests, it was developed by Maxam-Gilbert in 1976-1977. This is also called as the Sanger method as it was published by Sanger and Coulson. But in Sanger’s first method, each read should be cloned for the processing of single-stranded DNA. This method used directly the purified DNA. This method of DNA sequencing was unsafe because of the extensive use of toxic chemicals.

·         Chain Termination methods

Another simple way to perform chain sequencing is by manipulating the chemistry of the molecule. Here, processing of one DNA with normal nucleotides in absence of the hydroxyl group is done. This is good for the polymerase that adds to the next stage. This method is also known as Sanger method as it was discovered by Fredrick Sanger. This chain reaction has a DNA polymerase, DNA template, normal dexoynucleotide (dNTP) and changed deoxynucleotides (ddNTP). The strand synthesis is done four times separately, with ddNTP. This forms the bond between two nucleotides. Thus four discrete families of polynucleotides are obtained. To denature the DNA, polyacrylamide gel electrophoresis is used. This gives the synthesized strands from the given template. 60 degree high voltage is used to heat the gel.  To determine the strands, Autoradiography is used as they are radio labeled.

·         Dye-terminators

The most common method of the DNA sequencing is the Dye-terminator sequencing. This involves labeling of the chain terminating ddNTPs. This also allows the sequencing in single reaction. In this process of Dye-terminator sequencing, each ddNTP is named with a fluorescent dyes. These fluorescent dyes emit lights in different wavelengths. According to the convenience and speed, automated sequencing prefers dye terminator sequencing. However there are many disadvantages related to this type of DNA sequencing method. The main disadvantage is that anomalies or variances in the inclusion of the dye-labeled chain terminators. This is because they fragments the DNA and results in abnormal readings. These readings are made in the electronic DNA sequence trace chromatography after the capillary electrophoresis.