Therefore, new dNTPs have to be added to the reaction one at a time in a certain time interval, which is 65 s. As the added nucleotide is known, the sequence of the template can be determined.
Moreover, pyrosequencing is a widely applicable technique with high accuracy, parallel processing, and easily automated. Also, it avoids the usage of labeled primers, labeled nucleotides, and gel electrophoresis. Additionally, it is suitable for both confirmatory sequencing and de novo sequencing.
Further, the main important feature of pyrosequencing is its sequencing depth, which allows the detection of variants with high sensitivity. However, the main drawback of the technique is its suitability to sequence up to several hundreds of bases. Sanger sequencing refers to a method of DNA sequencing by the selective incorporation of chain-terminating dideoxynucleotides , while pyrosequencing refers to a method of DNA sequencing based on the sequencing-by-synthesis principle.
Sanger sequencing is the first generation sequencing approach, while pyrosequencing is next-generation sequencing chemistry, which is a second-generation sequencing approach. Above all, the main difference between Sanger sequencing and pyrosequencing is that Sanger sequencing uses the dideoxy chain termination method, while pyrosequencing is based on the sequencing-by-synthesis principle. In Sanger sequencing, the identification of nucleotides is by capillary electrophoresis after the amplification of the whole DNA fragment while in pyrosequencing, the identification of nucleotides is done with the release of pyrophosphate during the synthesis.
Furthermore, Sanger sequencing involves the detection of fluorescent light, while pyrosequencing involves the detection of visible light at nm. Additionally, Sanger sequencing can read up to to base pairs while pyrosequencing can read up to base pairs. Sanger sequencing is a complex process with many steps, while pyrosequencing is a less complex process with fewer steps. Also, another difference between Sanger sequencing and pyrosequencing is that Sanger sequencing has a lower sensitivity, while pyrosequencing has a higher sensitivity.
Sanger sequencing is the first generation sequencing approach, which is the conventional method of sequencing. However, it uses the dideoxy chain termination method followed by capillary electrophoresis. On the other hand, pyrosequencing is the first alternative to Sanger sequencing, and it is a type of next-generation sequencing.
Further, it has a higher sensitivity and fewer steps to cover. Generally, it uses the sequencing-by-synthesis method, which determines nucleotides during the synthesis of DNA fragment as it is. Therefore, the main difference between Sanger sequencing and pyrosequencing is the method of sequencing and their benefits. Hence, once the ddNTP is attached, chain elongation ceases and terminates from that point. Capillary gel electrophoresis is used to organize these short DNA strands by their sizes on a gel as shown in Figure Figure 1: Capillary gel electrophoresis of synthesized short DNA.
For in vitro replication of DNA, few requirements should be provided. Deoxynucleotides are not totally replaced by the respective ddNTPs. Four separate tube products are run on a gel in four separate wells. Then by reading the gel, the sequence can be constructed as shown in Figure Sanger sequencing is an important technique that helps in many areas of molecular biology.
Human genome project was successfully completed with the aid of Sanger sequencing-based methods. Sanger sequencing is also useful in target DNA sequencing, cancer and genetic disease research, gene expression analysis, human identification, pathogen detection, microbial sequencing etc. Therefore, new advanced sequencing techniques were developed with time to overcome these problems.
However, Sanger sequencing is still in use due to its highly accurate results up to approximately base pair length fragments. This technique relies on the detection of pyrophosphate release upon the nucleotide incorporation. The process starts with the primer binding with the single-stranded DNA template and DNA polymerase starts the incorporation of nucleotides complementary to it.
When the nucleotides join together nucleic acid polymerization , it releases pyrophosphate two phosphate groups bound together groups and energy. Each nucleotide addition releases equimolar quantity of pyrophosphate. The generated ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin, producing visible light in amounts that are proportional to the amount of ATPs.
Light is detected by a photon detection device or by photomultiplier and creates a pyrogram. Since the addition of nucleotide is known according to the incorporation and detection of light, the sequence of the template can be determined. Pyrogram is used for generating the nucleotide sequence of the sample DNA as shown in Figure Pyrosequencing is very important in single nucleotide polymorphism analysis and sequencing of short stretches of DNA.
The high accuracy, flexibility, ease of automation and parallel processing are the advantages of pyrosequencing over Sanger sequencing techniques. Sanger sequencing and Pyrosequencing are two DNA sequencing methods used in molecular biology. Sanger sequencing constructs the order of the nucleotides in sequence by terminating the chain elongation while the pyrosequencing constructs the precise order of the nucleotides in sequence by incorporation of nucleotides and detecting the release of pyrophosphates.
Therefore, the main difference between Sanger sequencing and Pyrosequencing is that Sanger sequencing works on sequencing by chain termination while pyrosequencing works on sequencing by synthesis. Reference: 1. Fakruddin, Md, and Abhijit Chowdhury.
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