Sanger Sequencing: Methodology, Advantages, and Its Place in the Genomics Era (Comprehensive Guide)

Table of Contents

• What is Sanger Sequencing
• The History of DNA Sequencing
• Principle of Sanger Sequencing
• Steps Involved in Sanger Sequencing
• Advantages of Sanger Sequencing
• Limitations of Sanger Sequencing
• Applications of Sanger Sequencing
• Sanger Sequencing vs. Next-Generation Sequencing

What is Sanger Sequencing

Sanger sequencing is a technique used to determine the sequence of nucleotide bases in DNA by utilizing chain termination with modified nucleotides known as dideoxynucleotide triphosphates (ddNTPs).

Also referred to as the dideoxy sequencing or chain termination method, this approach was first developed by Frederick Sanger and his team in 1977. Renowned for its high accuracy and capacity to generate long reads, Sanger sequencing is considered the gold standard in sequencing, making it invaluable for a variety of research applications.

The History of DNA Sequencing

Sequencing technologies have evolved significantly over the past years but Sanger sequencing continues to be the most widely used technology. It is particularly effective for small sequencing projects. However, for higher-throughput sequencing projects, next-generation sequencing (NGS) technologies such as Illumina, PacBio, and Nanopore have emerged. Although NGS technologies have largely replaced Sanger sequencing due to their ability to sequence larger amounts of DNA more quickly and at a lower cost, Sanger sequencing remains in use today. The development of newer DNA sequencing platforms has led to the automation of Sanger sequencing.

Principle of Sanger Sequencing

The principle of Sanger sequencing is based on the termination of DNA strand elongation by ddNTPs. These modified molecules are chemical analogs of DNA nucleotides that lack the 3’ hydroxyl group necessary for the formation of a phosphodiester bond that elongates the DNA strand. The addition of ddNTPs in the Polymerase Chain Reaction (PCR) reaction terminates DNA elongation.

During the sequencing process, labeled ddNTPs, deoxyribonucleotide triphosphates (dNTPs), and template DNA are mixed in a PCR reaction. When ddNTPs are added, they terminate the DNA chain, producing fragments of different lengths. These fragments are separated by electrophoresis. The fluorescent labels on the ddNTPs indicate which base terminated each fragment and are used to determine the DNA sequence.

Steps Involved in Sanger Sequencing

Sanger Sequencing involves the following 4 steps.

1. DNA Template Preparation

• The initial step in Sanger sequencing is preparing the DNA template. The DNA of interest must first be extracted from its source. There are several methods for DNA extraction, including chemical extraction, column-based extraction, and magnetic bead-based extraction. Once extracted, identical single-stranded molecules are prepared for sequencing.

2. Chain Termination PCR

• The next step involves amplifying the target DNA using Polymerase Chain Reaction (PCR). This process begins with initial denaturation, followed by multiple cycles of denaturation, annealing, and extension, and concludes with a final hold at 4°C. The PCR reaction mixture contains template DNA, primers, all four deoxyribonucleotide triphosphates (dNTPs), and DNA polymerase.
• In addition to dNTPs, a small amount of the four dideoxynucleotide triphosphates (ddNTPs) is also added. Each ddNTP is labeled with a distinct fluorescent marker. Since ddNTPs lack the 3’ hydroxyl group required for further elongation, their incorporation leads to chain termination.
• Because ddNTPs are present in smaller quantities compared to dNTPs, chain termination occurs at various points, resulting in a mixture of newly synthesized DNA strands of differing lengths, each terminating at a ddNTP.
• In the traditional sequencing method, the PCR reaction is carried out in four separate tubes, each containing one type of labeled ddNTP.
• In automated Sanger sequencing, all four ddNTPs are included in a single reaction, with each ddNTP labeled with a unique fluorescent marker.
• Following the PCR reaction, sequencing clean-up is performed to remove unincorporated ddNTPs and other contaminants before electrophoresis.

3. Separation of DNA Fragments

• Electrophoresis is used to separate the DNA fragments based on their lengths. This can be achieved using either polyacrylamide gel or a capillary gel system.
• In the traditional method, DNA fragments are separated through polyacrylamide gel electrophoresis, which distinguishes the fragments by size. The fragments from each PCR reaction are run in separate lanes to identify the corresponding ddNTP.
• Capillary electrophoresis (CE) uses glass capillaries filled with a gel polymer, with each sequencing reaction being run in a single capillary.

4. Detection and Analysis

• Once the DNA fragments are separated, they pass through a fluorescence detector.
• The detector identifies the fluorescent label attached to the ddNTP at the end of each DNA fragment. Each fluorescent signal corresponds to the nucleotide that terminated the fragment.
• The emitted signals from the nucleotides are captured, generating a chromatograph that displays the fluorescent peaks of each labeled fragment, representing the DNA sequence.
• The DNA sequence is then determined by reading the order of the fluorescent labels associated with the terminated DNA fragments.

Advantages of Sanger Sequencing

• Sanger sequencing is widely regarded as the gold standard for numerous research and clinical applications due to its exceptional accuracy. This high level of precision makes it particularly valuable for validating sequences obtained from other methods, such as next-generation sequencing (NGS) technologies.
• The technology behind Sanger sequencing is well-established and involves a straightforward process, resulting in consistent and reproducible outcomes.
• This method is ideal for small-scale projects or those focused on short DNA regions, as it generates relatively long read lengths.
• Additionally, data analysis from Sanger sequencing is simpler and does not require the complex bioinformatics tools and expertise often needed for interpreting NGS data.
• The quality of data produced by Sanger sequencing is high, with clear and easily interpretable chromatograms.

Limitations of Sanger Sequencing

• Sanger sequencing is limited to sequencing short DNA fragments and has a low throughput, as it can only process one fragment at a time. This makes it slow and costly for sequencing large genomes.
• Due to its complexity and expense, Sanger sequencing is not well-suited for long DNA sequences or large-scale sequencing projects.
• Compared to next-generation sequencing (NGS) workflows, Sanger sequencing involves more manual steps and is a time-consuming process, with longer preparation and handling times.
• Its low sensitivity makes it challenging to detect rare mutations and study complex mixtures.
• Additionally, Sanger sequencing requires high-quality and pure DNA samples, as degraded or contaminated DNA can result in unreliable outcomes.

Applications of Sanger Sequencing

• Sanger sequencing is widely used in medical diagnostics to identify genes associated with various diseases. Its ability to target specific regions of the genome makes it a valuable tool in clinical diagnostics and genetic testing.
• In species identification, Sanger sequencing helps identify new species by comparing their gene sequences with those of known species. It is also instrumental in studying the evolutionary history of many species.
• In forensic science, Sanger sequencing plays a crucial role in personal identification and DNA fingerprinting, aiding in the analysis of DNA evidence in criminal cases.
• In agriculture, Sanger sequencing is used to identify different breeds of crops and livestock, as well as in the conservation of these species.
• Sanger sequencing also has applications in emerging technologies such as single-cell sequencing and synthetic biology.

Sanger Sequencing vs. Next-Generation Sequencing