How Do Fluorescent Markers Help Determine a Nucleotide Sequence

DNA sequencing is a technique that helps to determine the nucleotide sequence of a particular DNA molecule. The two methods of sequencing are Sanger sequencing and next-generation sequencing. Both types of sequencing methods are fully-automated up to date. Any DNA strand is composed of the four nucleotides: adenine (A), guanine (G), cytosine (C), and thymine (T). The nucleotides in the DNA fragment are labeled with four separate, fluorescent markers in both types of sequencing methods. The fluorescent markers or fluorophores are molecules capable of absorbing light and emitting it at a well-defined wavelength. The fluorescent markers are incorporated into the DNA strand by PCR. Then the sequence of the nucleotides is determined by automated techniques.

Key Areas Covered

1. What is Sequencing
     – Definition, Sanger Sequencing, Next-Generation Sequencing
2. How Do Fluorescent Markers Help Determine a Nucleotide Sequence
     – Sequencing Procedure

Key Terms: Dideoxynucleotides (ddNTPs), Fluorescent Marker, Gel Electrophoresis, Next-Generation Sequencing, Nucleotide Sequence, PCR, Sanger Sequencing 

What is Sequencing

Sequencing is a laboratory technique used to determine the nucleotide sequence of a DNA molecule. Two main types of DNA sequencing methods can be identified as Sanger sequencing and next-generation sequencing. Both Sanger sequencing and next-generation sequencing use labeled nucleotides with fluorescence for the determination of the nucleotide sequence.

Sanger Sequencing

Sanger sequencing is the first developed method of DNA sequencing. The method of sequencing was first developed by Fredric Sanger in 1975. Consequently, it is known as Sanger sequencing. The method of Sanger sequencing is also known as chain-termination method as it is involved in the selective incorporation of chain-terminating dideoxynucleotides (ddNTPS) by DNA polymerase during in vitro DNA synthesis. The elongation of the DNA strand is achieved by the regular deoxynucleotides (dNTPs). However, ddNTPs are added to the reaction mixture to terminate the chain growth. These ddNTPs are fluorescent-labelled. 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. In each reaction mixture, the chain growth is terminated at each 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. Then, these four reactions are separated by gel electrophoresis, and a fluorometer is used to scan for the separate fluorescence. 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 determined nucleotide sequences are shown in figure 1.

Figure 1: DNA Sequences

Next-Generation Sequencing

The most recent DNA sequencing technologies are collectively-known as next-generation sequencing. The sequencing reactions are performed in microscale on a chip at once. Therefore, several sequencing reactions are performed in parallel. In next-generation sequencing, capillary electrophoresis is used in addition to gel electrophoresis for the separation of amlicons with various lengths created by chain termination method. Capillary electrophoresis is an analytical separation method by which molecules are separated based on their electrophoretic mobility.

How Do Fluorescent Makers Help Determine a Nucleotide Sequence

During sequencing, the DNA to be sequenced serves as the template strand for DNA synthesis by PCR. A DNA primer is used for the initiation of DNA synthesis by DNA polymerase. A mixture of regular, four bases (dNTPs; dATP, dGTP, dCTP, dTTP) and a low level of one of the four dideoxynucleotides (ddNTPs; ddATP, ddGTP, ddCTP, and ddTTP) are added as components of the PCR reaction. Hence, four, individual PCR reactions are performed by the addition of each of the four ddNTPs. Dideoxynucleotides possess two special characteristics:

1. They lack 3’-OH group to which the incoming nucleotide is added by DNA polymerase. Hence, the incorporation of the ddNTP terminates the chain growth.
2. They are labelled with different fluorescent dyes: the ddATP is labelled with a green dye, the ddGTP is labelled with a yellow dye, the ddCTP is labelled with blue, and the ddTTP is labelled with red dye.

However, the chain terminating ddNTPs are added in low concentrations; they do not terminate the whole PCR process at once. But, when one of the four ddNTPs are incorporated into the growing chain, that particular chain growth is terminated. Therefore, at the end of each four PCR reactions, a series of amplicons (the resulting DNA fragments by PCR) are produced, which are terminated at each nucleotide of the target DNA fragment. These amplicons can be run in a gel. The fluorescent dyes that pass at a defined point of the electrophoretic gel can be scanned by a fluorometer in order to determine the nucleotide sequence in the automated DNA sequencers. The fluorescent-labeled nucleotide sequence obtained in DNA sequencing is shown in figure 2.

Figure 2: Fluorescent-Labeled Nucleotide Sequence

By combining each of the nucleotides in the series, the nucleotide sequence of the initial DNA fragment can be determined. The nucleotide sequence of a fragment with 750-1,000 base pairs can be easily determined per run by Sanger sequencing. However, the sequencing of a whole genome still remains challengeable due to the presence of a large number of nucleotides. 454 sequencing is a type of next-generation sequencing by which 20 million base pairs can be read per single run.

Conclusion

Sequencing is a technique used in the determination of nucleotide sequence of a particular DNA fragment. Sanger sequencing and next-generation sequencing are the two main sequencing technologies. Both technologies use fluorescent markers for the determination of the nucleotide sequence. Each of the four chain-terminating dideoxynucleotides is labeled with four different fluorescent dyes, and they are used in four separate PCR reactions to obtain the sequence.

Reference:

1. Adams, Jill U. “DNA Sequencing Technologies.” Nature News, Nature Publishing Group, Available here.
2. Carr, Steven M. Fluorescent sequencing, Available here.
3. “Sequencing DNA – Automated Sequencing With Fluorescent Dyes.” JRank Articles, Available here.

Image Courtesy:

1. “Alineando secuencias (2)” by Shaury Nash (CC BY-SA 2.0) via Flickr
2. “Radioactive Fluorescent Seq” By Abizar at English Wikipedia (CC BY-SA 3.0) via Commons Wikimedia