DNA sequencing is a technique used to determine the nucleotide sequence of a particular DNA fragment. Sanger sequencing and next-generation sequencing are two types of sequencing methods. Fluorescent markers are used to identify each nucleotide in the sequence. PCR is used for the incorporation of the fluorescent markers into the DNA fragment. PCR (polymerase chain reaction) is a technique used in the laboratory to produce millions of copies of a particular DNA fragment. The analysis of the PCR fragments in the gel allows the determination of the nucleotide sequence of the DNA fragment.
Key Areas Covered
1. What is Sequencing
– Definition, Types of Sequencing – Next-Generation Sequencing, Sanger Sequencing
2. Why is PCR Used in the Process of DNA Sequencing
– Incorporation of Fluorescent Dyes During PCR
Key Terms: ddNTPs, dNTPs, DNA Sequencing, Fluorescent Dyes, Next-Generation Sequencing, PCR, Sanger Sequencing
What is Sequencing
Sequencing is a laboratory technique used to determine the nucleotide sequence of a DNA molecule. Sanger sequencing and next-generation sequencing are the two main methods of DNA sequencing. Both DNA sequencing methods are involved in the incorporation of fluorescent makers to the DNA strand by PCR for the determination of the nucleotide sequence of a particular DNA strand.
The first method of sequencing, which is known as Sanger sequencing, is first developed by Fredric Sanger in 1975. Consequently, it is known as Sanger sequencing. Sanger sequencing is involved in the selective incorporation of chain-terminating dideoxynucleotides (ddNTPS) by DNA polymerase during in vitro DNA synthesis. Therefore, it is also known as chain-termination method. Regular deoxynucleotides (dNTPs) are used for the elongation of DNA strand. ddNTPs are also added to the reaction mixture for the termination of the chain growth. The four types of ddNTPs are added to four separate PCR mixtures. Therefore, four separate PCR reactions are carried out by adding ddATP, ddGTP, ddCTP, and ddTTP. For each reaction mixture, a single type of added ddNTP (if ddATP is added), the growth of different amplicons are terminated at each (A) nucleotide in the DNA fragment. Then the four reactions are separated by gel electrophoresis. The emitting fluorescence is detected by a fluorometer. Sanger sequencing is widely-used for the determination of the sequence of the fragments used in DNA cloning and the fragments amplified by PCR. The general procedure of Sanger sequencing is shown in figure 1.
Next-generation sequencing is the collective name for the most recent DNA sequencing technologies. Several sequencing reactions are performed in microscale on a chip at once in next-generation sequencing. Both sequencing methods use labeled nucleotides with fluorescence that are incorporated into the amplicon during PCR, allowing the determination of the nucleotide sequence. Chain terminating addition of fluorescent markers is also involved in the next-generation sequencing. However, the main difference between Sanger sequencing and next-generation sequencing is the use of capillary electrophoresis for the separation of differently-labeled amplicons in next-generation sequencing. Capillary electrophoresis is an analytical separation method by which the molecules are separated based on their electrophoretic mobility.
Why is PCR Used in the Process of DNA Sequencing
During sequencing, fluorescent markers should be incorporated into the DNA strand for the determination of the nucleotide sequence. This incorporation takes place during PCR. Generally, the four types of dNTPs are incorporated into the newly-synthesizing DNA strand during PCR. This phenomenon is used in DNA sequencing to incorporate fluorescent-labeled dideoxynucleotides (ddNTPs) into the amplicon while determining the DNA sequence.
Generally, a mixture of regular four bases (dNTPs; dATP, dGTP, dCTP, dTTP) is added to the PCR reaction mixture during DNA sequencing.
In addition, one of the four dideoxynucleotides (ddNTPs; ddATP, ddGTP, ddCTP, and ddTTP) are added as components of the PCR reaction in a low concentration. Finally, four PCR reactions have to be carried out to determine the complete sequence.
The ddNTPs lack 3’-OH group to which the incoming nucleotide is added by DNA polymerase. Hence, the incorporation of the ddNTP terminates the chain growth. Thus, in each of the four PCR reactions, the chain termination occurs at a particular base. These ddNTPs are also incorporated with different fluorescent dyes (The ddATP is labeled with green dye; the ddGTP is labeled with yellow dye; the ddCTP is labeled with blue, and the ddTTP is labeled with red dye). The incorporation of the fluorescent dyes and the chain termination take place during PCR. The amplicons are run on a gel, and the gel is scanned for the fluorescence by a fluorometer in the automated sequencer for the determination of the nucleotide sequence.
DNA sequencing is a laboratory technique used to determine the nucleotide sequence of a particular DNA fragment. Sanger sequencing and next-generation sequencing incorporate different fluorescent dyes into the DNA fragment for the determination of the nucleotide sequence during a PCR.