Sanger Sequencing Technique

Figure 1.1: A schematic representation of Sanger sequencing. The Sanger method uses dideoxynucleotides that terminate newly synthesized DNA fragments at specific bases either A, T, C or G. Then, the resulting fragments are resolved by electrophoresis through a denaturing polyacrylamide gel in four parallel lanes, and the DNA sequence can be read (Rosenberg and Pascual, 2014).

Exome sequencing is one example of NGS that is useful when the identity of the specific genes involved in the disease has not yet been identified. The technique, which entails sequencing only the 1% of the genome that is exons and thus codes for proteins, is less costly than whole-genome sequencing and is increasingly being used for the diagnosis of rare inherited disorders. A 2013 study on MHS using exome sequencing was able to identify over 100 RYR1 and CACNA1S variants in an asymptomatic population, reclassify several previously classified pathogenic variants as likely non-pathogenic due to high prevalence, as well as alert several individuals with a high likelihood of MHS based on known pathogenic mutations7. The major use of large scale exome sequencing efforts in MHS is that they will allow researchers to identify the pathogenicity of variants identified in RYR1 and CACNA1S genes. In addition, the technique can be used in combination with functional studies to identify new mutations in families for whom traditional methods have not identified a mutation. A limitation of this technology, however, is that many variants of unknown significance (VUS) are frequently identified, making interpretation and large scale clinical application of the results

The genome is the total genetic information carried by an organism. The 10K Genomes Initiative was the plan to sequence all DNA from 10000 species. The Human Genome Project established databases and refined analytical software to make data available on the Internet. Bioinformatics and genomics are two terms that are often hashed up. While bioinformatic is the application of computational methods to the storage and analysis of biological data, genomics is the study of whole sets of genes and their interactions the two are often confused. Next-Generation Sequencing is the different modern sequencing technologies. NCBI genbank is the NCBI database of sequences. Systems biology is used to define gene circuits and protein interaction networks. A

Sanger sequencing is a technique to sequencing DNA and Applied Biosystems were the first to make it commercial. It has been widely used for more than 40 years and is still one of the best even today. "Next-Gen" methods have more recently replaced the original Sanger sequencing technique especially for sequences with more than 1000 data points. For smaller-scale projects, Sanger method is still most prevalent where sequences of 500 up to a 1000 nucleotides are common.

Genomics and genome sequencing branched out from the modern genetics field of biology. In 1865, Gregor Mendel became the father of modern genetics. He was the first person to cross breed plants to see how physical traits were passed on from generation to generation. In 1953, James Watson and Francis Crick discovered the double helix structure of DNA (Timeline). Frederick Sanger developed a method for rapidly decrypting DNA to determine the order of bases in a strand in 1977. In 1990, the Human Genome Project (HGP) was started. It was an endeavor that intended to develop the technology needed to map genomes, as well as to map human, mice, and fruit fly genomes. This project ended in 2003 when the human genome was completely mapped up to 99.9% accuracy (Timeline).

Following the discovery of DNA’s structure, the research possibilities that could be conducted on the DNA that makes up all of life was endless. In 1961, the genetic code for protein synthesis was discovered by Marshall Nirenberg. Also in 1977, Frederick Sanger developed the “rapid DNA sequencing” technique to determine the bases’ order in a DNA strand; this would later be known as the Sanger method. Huntington’s disease was the first genetic disease to be mapped in 1983, and was later isolated in 1993. Also, polymerase chain reaction (PCR), the technology still used today to amplify DNA, was developed in 1983. Lastly, in 1989, the mutation for cystic fibrosis was discovered, and in 1990 scientists found the

The Human Genome Project is one of the most widely discussed topics in genetics today. The United States human genome project began in 1990, when the $3 billion dollar project to map 3 billion DNA base pairs was announced.(4) When the initial funding was provided, it was anticipated that the project would require 15 years to complete and the target date for completion was 2005.(8) Recent technological advances have shortened that time period, and it is now estimated that the program will be complete by 2003.(1) The program now has been expanded to an international effort involving research facilities in France, Germany, Japan, the United Kingdom and the United States. There are also several private companies

The HGP is progressing faster than what was expected, but a race to be the first to complete the human genetic sequence has emerged and become very aggressive. For example, in 1998, the Perkin-Elmer Corporation and Dr. J. Craig Venter, head of the Institute for Genomic Research, announced that they intended to start a new genomics company. The companies' intent was to

Next-generation sequencing (NGS) has lately been adapted to diagnostic prerequisites and a growing number of diagnostic facilities offer an assortment of NGS-based services. However, variable NGS-based methodologies ranging from amplicon-based (targeted) NGS, whole exome (WGS) or whole genome NGS (WES) exist and are suited for different applications. As regards to tumor diagnostics in molecular pathology amplicon-based NGS predominantly is usually applied this is due to the facts that

Due to our profound genomic studies, we have been able to sequence many genomes of various organisms. With the

The Human Genome Project was a scientific program aimed at getting to know all of the complementary sequence of base pairs that make up the human genome, containing approximately 30 thousand genes. The project began in 1990 and was originally planned to last 15 years, but rapid technology advances accelerated the completion date to 2003. (WJEC A2 Biology, 2012) says main aims of the project were to:

In HGP there are two types of sequences, the draft and the high quality. To get the draft sequence, scientist determine the order of base pairs in each chromosomal area at least four to five times to ensure data accuracy and to help with reassembling DNA fragments in their original order. This repeated sequencing is known as genome "depth of coverage." The draft sequence for the human genome was completed on June 26, 2000. To generate high quality sequencing , additional sequencing is needed to close gaps, reduce mistakes and allow only a single error in 10,000 bases. The finished version will provide an estimated eight to nine times the coverage of each chromosome. Thus far, high quality

The original technology used to determine the order of DNA building blocks was known as Sanger sequencing. As useful as it is, this technique is very time consuming and expensive. It has been developed to be more automated and faster, keeping it existent in many laboratories today. Other options are out there, such as Next-Generation sequencing. Although, much faster and more efficient, it’s not nearly as thorough. (Adv. in DNA Seq.)

Furthermore, this essay into how sequencing of Deoxyribonucleic (DNA) has lead to researchers understanding human genome better.

Illumina HiSeq is one of the more accurate forms of DNA sequencing technology, about 99% accurate. The newest version of HiSeq is TruSeq nano and NEBNext Ultra. Sequencing