First-Generation Sequencing: Modern Sanger Sequencing Method and key for Students

Overview of Sequencing

All living beings have DNA or RNA as hereditary material. RNAs are found in various viruses, such as Human immunodeficiency virus (HIV), Hepatitis C virus (HCV), SARSCoV2, etc., as genetic material. The genetic material either DNA or RNA is made of nucleotides like proteins are made of amino acids. A total of 4 nucleotides make the genetic material (DNA/RNA) of any organism, these are Adenine (A), Guanine (G), Cytosine(C), and Thiamine(T) in DNA, and Uracil(U) replace thymine in RNA.

DNA is a double-stand molecule and these stands are complementary to each other mean, that A will pair with T and G with pair with C and vice versa. This complementary nature of DNA makes sequencing possible. The order of four nucleotides in DNA or RNA of an organism is called sequence and determination of this order is called DNA/RNA sequencing. In 1965 the first sequencing of alanine tRNAs was carried out by Nobel laureate Robert Holley. 

In 1972 Walter Fiers's encoding of the gene of bacteriophage MS2 coat protein made its DNA to be sequenced. The sequencing of tRNA and bacteriophage coat protein gene was the milestone for the beginning of Sequencing and it paved the way for the first-generation sequencing.

Introduction of First-Generation Sequencing

The first generation of sequencing was born in the early 1970s when Allan M. Maxam and Walter Gilbert developed the chemical method of sequencing, and just after that, Frederick Sanger developed dideoxy sequencing.

To determine the complete genome sequence, these steps are required in both techniques: 

• To break down the genome into small fragments of DNA.
• Each DNA fragment shall be sequenced on its own. 
• Alignment of overlapping sequence fragments to complete genome sequencing

The Modern Sanger Sequencing Method

Frederick Sanger developed the method of chain termination known as dideoxy sequencing, which is now commonly known as Sanger sequencing. Since then, Sanger sequencing has become more and more used as a first-generation sequencing method due to various advantages over chemical sequencing.

Principle of Sanger Sequencing: This method is based on the use of Dideoxy nucleotides (ddNTPs) with deoxy nucleotides (dNTPs) in the DNA PCR reaction. The Dideoxy nucleotide lacks the 3’OH group which results in chain termination where this nucleotide is added because polymerase in PCR requires this 3’ OH for chain elongation in PCR chain reaction. 

From its invention till now a lot of innovation has been done to improve Sanger Sequencing previously Sanger sequencing used radiolabelled ddNTPs and autoradiography for visualization, nowadays in place of radiolabelled ddNTPs, fluorescently labelled ddNTPs and fluorescent detector sequencing machine are used. Another important innovation that is done in Sanger sequences which makes it fully automated is the introduction of Capillary electrophoresis.

These are the steps involved in Modern Sanger Sequencing:

1. PCR Amplification: The whole genome (DNA/RNA) is isolated from the samples. PCR amplification amplifies the target region from the whole genome with the help of target primers.
2. PCR Clean Up: After PCR amplification of the target region from the whole DNA/RNA sample, the unused primers and unincorporated dNTPs from the PCR reaction tube would be needed to be cleaned up because these can hinder the sequencing results. The PCR clean-up step purifies the DNA fragments and different technologies are used nowadays such as the spin columns method, beads purification, or enzymatic method. 
3. Dye-Terminator Sequencing: In modern Sanger sequencing, only a single master tube mix is prepared, unlike classical Sanger sequencing in which DNA fragments need to be sequence divided into 4 tubes. The Single master mix tube contains dNTPs (A, C, G, and T), Polymerase, Primer, and all four fluorescent labelled ddNTPs (in very low concentration as compared to dNTPs).During the PCR reaction, when ddNTP is added to the chain, the chain reaction is stopped and this results in the synthesis of varied-length DNA fragments. At this step, the single sample is sequenced with Forward and Reverse primers for full-length resolution of sequence. 
4. Sequencing Clean up:  This step is to clean any sequencing terminator and salts before it goes to the next step. 
5. Capillary Electrophoresis: With the help of electro kinetic injections, varied-size DNA fragments are entered in sub millimetre diameter capillaries from the sequencing plate. In this capillary, DNA fragments are separated based on electrophoretic mobility under the influence of an electric field. At the end of the capillary, the fluorescent detector is present, the smallest fragment with fluorescent ddNTP gets excited and releases fluorescent get detected. All 4 ddNTP are labeled with fluorescent having a different emitted wavelength.
6. Result Analysis: In modern Sanger sequencing, fluorescence is used to identify each terminal ddNTP. Because each of the four ddNTPs is labeled with a different fluorescent label, the emitted light can be directly linked to the identity of the terminal ddNTP.

The output is called a chromatogram and shows the fluorescence peak of each nucleotide along the length of the template DNA.

Clinical Applications of Sanger Sequencing

Although up to 1000bp long DNA sequence is commonly sequenced by Sanger sequencing, and the accuracy is still not matched by any sequencing technology, so it is still widely used in clinical settings for various applications: 

• To confirm the variants or fill up the gap nucleotides reported in next-generation sequencing (NGS).
• Genome editing
• Sequence confirmation for mRNA therapeutic and vaccine manufacturing.
• SARS-CoV-2 Research
• It provides high throughput from a small sample/target region to confirm the clinically significant gene variant 
• Detection of cancer mutations in tumour FFPE samples. 
• Genotyping of viruses and bacteria and detection of drug resistance. 

Importance of Sequencing for Para-medical and Biotechnology Education Students

• To pursue careers in health professions such as Medical Lab technologists, biotechnologists, Molecular biologists, and medical researchers benefit significantly from understanding and mastering the skill of sequencing. 
• Sequencing plays a crucial role in the genetic diagnostic and therapeutic processes, contributing to the overall efficiency and effectiveness of patient care. Students who want to pursue a career in diagnostics should have an understanding of sequencing techniques. 
• Sequencing also plays a vital role in Cancer Genetics, so after the paramedical or Biotechnology courses want to pursue a career in a Cancer hospital as a diagnostic specialist or researcher, acquiring sequencing skills can go a long way. 
• Para-medical professionals involved in research may use Sanger sequencing to explore genetic variations, study the impact of specific genes on health, and contribute to the development of new therapeutic approaches. Understanding sequencing techniques is crucial for conducting meaningful research in medical genetics and molecular medicine.

At Ganesh Paramedical College

Students of paramedical and biotechnology courses at Ganesh Paramedical College will get the theoretical and practical knowledge at a well-equipped molecular lab and skills to enter in health industry.

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