Sequencing is the process involved in the determination of a nucleotide sequence of a particular DNA fragment. During sequencing, the DNA fragment is terminally labeled with fluorescence-labeled nucleotides by PCR. This process uses four types of fluorescence-labeled nucleotides, and they are dideoxynucleotides (ddNTPs). ddNTPs lack a 3′ OH group to which the phosphate group of the incoming nucleotide is attached. Therefore, when a ddNTP is added to the growing chain, there will be no further addition of nucleotides at the 3′ end of the chain. That means the addition of a ddNTP into the growing chain terminates the chain growth. Since ddNTPs are added to the PCR mixture in low concentrations, each growing chain is terminated at different levels. The emitting fluorescence is detected to determine the nucleotide sequence of the DNA fragment at the end of the PCR.
Key Areas Covered
Key Terms: Dideoxynucleotides (ddNTPs), Fluorescent Marker, Gel Electrophoresis, Next-Generation Sequencing, Nucleotide Sequence, PCR, Sanger Sequencing
What is DNA Sequencing
DNA sequencing is a laboratory technique used in the determination of nucleotide sequence of a particular DNA molecule. It uses fluorescence-labeled nucleotides, which are incorporated during PCR. There are two main methods of sequencing based on the techniques used in the detection of fluorescence: Sanger sequencing and next-generation sequencing.
Sanger sequencing, developed by Fredric Sanger in 1975, is the first developed sequencing method. It is also known as chain-termination method since it is involved in the selective incorporation of chain-terminating ddNTPs during in vitro DNA synthesis. In Sanger sequencing, amplicons are separated by gel electrophoresis. Sanger sequencing is widely-used for the determination of the sequence of the DNA fragments used in cloning and the fragments amplified by PCR. A determined DNA sequence is shown in figure 1.
Most recent DNA sequencing technologies are collectively-known as next-generation sequencing. It is also a chain termination method. Next-generation sequencing uses capillary electrophoresis for the separation of amplicons with various lengths created by chain termination method. Next-generation sequencing is used in the determination of a large number of nucleotides per run such as in genome sequencing.
How Does DNA Sequencing Work
During DNA sequencing, fluorescence-labeled nucleotides are added to a particular DNA fragment by PCR. For the elongation of the DNA strand, regular deoxynucleotides (dNTPs) are used. However, ddNTPs are added to the reaction mixture, which is fluorescence-labeled. Since ddNTPs do not have a 3′ OH group in the deoxyribose sugar molecule, further chain growth may not occur, terminating the chain growth. Sugar-phosphate backbone of DNA is formed by the formation of phosphodiester bonds between the 3′ OH group of the deoxyribose sugar and phosphate group of the incoming nucleotide. However, ddNTPs are added in low concentrations; therefore, they do not terminate the chain growth at once.
Four types of ddNTPs are added to four separate PCR mixtures. Four separate PCR reactions are carried out by adding ddATP, ddGTP, ddCTP, and ddTTP. Therefore, in each reaction mixture, the chain growth is terminated at A, G, C, and T nucleotides, respectively. As an example, in the reaction mixture with added ddATP, the growth of different amplicons are terminated at each A nucleotide in the DNA fragment. Determination of DNA sequence by Sanger sequencing is shown in figure 2.
Each of the four types of nucleotides are labeled by separate, fluorescence color; the ddATP is labeled with green dye; the ddGTP is labeled with yellow dye; the ddCTP is labeled with blue; the ddTTP is labeled with red dye. Hence, the amplicons of the four PCR reactions are labeled in separate colors.
After the amplification of the interested DNA fragment, the amplicons are separated either by gel electrophoresis or capillary electrophoresis. The nucleotide sequence of the DNA fragment can be determined by the detection of the emitting fluorescence. The nucleotide sequence of 750-1,000 base pairs long fragments can be easily determined per run by Sanger sequencing. However, the determination of the nucleotide sequence of a whole genome remains challengeable due to a large number of nucleotides in genomes. However, next-generation sequencing techniques such as 454 sequencing, around 20 million base pairs can be read per single run.
DNA sequencing is a molecular biology technique used in the determination of nucleotide sequence of DNA fragments. During sequencing, fluorescence-labeled nucleotides are added to the DNA fragments by PCR. By detecting the emitting fluorescence, the nucleotide sequence can be determined.
1. “DNA Sequencing.” Khan Academy, Available here.
1. “DNA sequence” By Sjef – Own work, Public Domain) via Commons Wikimedia
2. “Didesoxy-Methode” By Christoph Goemans (modifiziert) – Dr. Norman Mauder, auf Basis einer Datei von Christoph Goemans (CC BY-SA 3.0) via Commons Wikimedia