Understanding Sequencing Methodology

Nucleic acids are the “building blocks” of life; they determine all inherited characteristics in life and direct the process of protein synthesis. These important biomolecules create, encode and store information in all cells.

Understanding Sequencing Methodology

Nucleic acids are the “building blocks” of life; they determine all inherited characteristics in life and direct the process of protein synthesis. These important biomolecules create, encode and store information in all cells.

DNA, RNA, mRNA, tRNA are types of nucleic acids and these are made up of a chain of nucleotides stretching from as few as three to millions.

Each nucleotide in DNA contains one of four possible nitrogenous bases adenine (A), guanine (G), cytosine (C), and thymine (T). RNA nucleotides have the same adenine, guanine and cytosine bases but instead of thymine they have uracil (U).

Nucleic acid sequencing is the method for determining the exact order nucleotides present in a piece or strand of DNA or RNA.

The use of this exceedingly important methodology has increased exponentially as the ability to sequence DNA and RNA has become more and more accessible to both researchers and clinicians, alike. Most of this improved access is due to the dramatic reduction in the cost associated with the sequencing methodology. The ability to sequence the DNA of both normal and abnormal cells has revolutionized our basic understanding of disease in both people and pets.

The first generation sequencing technology was developed in 1977 by Fred Sanger and was known as Sanger Sequencing.

This methodology was used during the Human Genome Project completed in 2003 and is still widely used today.

Sanger sequencing is based on the activity of a polymerase enzyme that binds to the single strand of DNA and fills in one nucleotide base at a time. As nucleotides (A, T, G and C) are added, it creates a double-stranded DNA from a single-stranded DNA template. Sanger methodology uses special nucleotides, dideoxynucleotides (ddNTPs), that stops the DNA elongation process at very specific sites so that the size of the DNA strand can be calculated. Each of the 4 chain-terminating nucleotides are labeled with a different color dye. Light signals are analyzed by a computer to determine the sequence of the nucleotides and thus the DNA.

Using Sanger sequencing, Kerstin Lindblad-Toh and her team at the Broad Institute of MIT and Havard and Agencourt Bioscience Corp successfully assembled the genome of a Boxer in 2004.

The high demand of DNA sequencing after the Human Genome Project has driven the development of second-generation sequencing methodology.

The second generation technology also known as Next-Generation Sequencing (NGS) utilizes different technologies that enable rapid and high-throughput sequencing of DNA. There are a number of different NGS platforms, but all of them have one thing in common, they sequence millions of small fragments of DNA in parallel. NGS has largely supplanted first generation sequencing.

FidoCure® uses this newer technology to sequence the DNA of dog tumors.

Read more about this
1000 Genomes Project, Consortium, Durbin RM, Abecasis GR, Altshuler DL, Auton A, Brooks LD, Durbin RM, Gibbs RA, Hurles ME, McVean GA (2010-10-28). A map of human genome variation from a population-scale sequencing. Nature. 2010 Oct 28; 467:1061-1073.

Grada A, Weinbrecht K. Next-generation sequencing: methodology and application. J Invest Dermato. 2013 Aug; 133(8):e11.

Meldrum C, Doyle MA, Tothill RW, Next-generation sequencing for cancer diagnostic: a practical perpective. Clin Biochem Rev. 2011 Nov; 32(4):177-195.

Shigemizu D, Fujimoto A, Akiyama S, Abe T, Nakano K, Boroevich KA, Yamamoto Y, Furuta M, Kubo M, Nakagawa H, Tsunoda T. A practical method to detect SNVs and indels from whole genome and exome sequencing data. Sci Rep. 2013; 3: 2161.

Dr. Gerry Post

Dr. Gerry Post has been practicing veterinary medicine for over 25 years specializing in veterinary oncology. He currently serves as Chief Veterinary Officer at the One Health Company.

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