The principles and steps of Sanger sequencing

Sanger sequencing, also known as dideoxy chain end synthesis termination method, is based on the natural process of DNA replication. By cleverly introducing chain terminators, the DNA chain is randomly terminated during the synthesis process, thereby generating a series of DNA fragments of different lengths. After these fragments are separated by electrophoresis, they form specific band patterns. It is through the position and order of these bands that scientists can decipher the precise sequence of DNA.

Principles and steps of Sanger sequencing

1. Principle The basic principle of Sanger sequencing is based on the process of DNA polymerase synthesizing a new DNA strand on a DNA template. In this process, by adding specially designed binary deoxynucleotide triphosphates (ddNTPs), DNA polymerase stops synthesis when this special nucleotide is encountered in the new strand. Since ddNTPs do not contain hydroxyl groups at the 5' and 3' positions, they cannot form a phosphodiester bond with the next nucleotide, thereby terminating the extension of the DNA chain. By determining where these stops occur, the DNA sequence can be determined.

2. Step 1. DNA template preparation: Separate the DNA of the sequence to be tested into single strands, and add a short primer of known sequence to the 3' end of the single strand to connect it to the single stranded DNA. 2. Reaction system settings: Add DNA polymerase, four deoxynucleotides (dNTPs: dATP, dGTP, dCTP, dTTP) and low concentrations of ddNTPs to the reaction system. ddNTPs serve as chain terminators, and each ddNTPs corresponds to the termination of A, G, C and T respectively. 3. Chain extension and termination: Under the guidance of primers, DNA polymerase adds dNTPs in a base-pairing manner to extend the chain. When encountering ddNTPs, chain elongation stops, forming a series of DNA fragments of varying lengths. 4. Electrophoretic separation: The reaction products are separated by electrophoresis, and DNA fragments of different lengths are arranged in order of size. 5. Fluorescence detection: Put the electrophoretically separated DNA fragments into an automatic sequencer, and the instrument records the fluorescence signal generated by each ddNTP according to the length of the DNA fragments. 6. Sequence reconstruction: Use computer software to reconstruct the original DNA sequence based on the peaks and order of the fluorescence signal.

3. Features 1. Long read length: The read length of Sanger sequencing is usually between 800 and 1000 bp. Compared with other sequencing methods, it can read longer DNA sequences at one time. 2. High accuracy: Because Sanger sequencing is based on the synthesis process of DNA polymerase, it has high base reading accuracy. 3. Intuitive results: Sequencing results can be interpreted directly through electrophoresis patterns and fluorescence signals, which is intuitive and easy to understand.

4. Application of Sanger sequencing played a key role in the Human Genome Project and is still widely used to obtain highly accurate and reliable sequencing data. Its application fields include but are not limited to: 1. Gene research: used to determine the precise sequence of genes and study the structure and function of genes. 2. Disease diagnosis: Certain genetic diseases can be diagnosed by sequencing the sequence of specific genes. 3. Drug research and development: Understanding the sequence information of drug targets is helpful for drug design and development. 4. Forensic medicine: It is widely used in DNA fingerprint analysis, paternity testing and other fields.

5. Limitations Although Sanger sequencing has many advantages, there are also some limitations: 1. Low throughput: only one or a small number of DNA fragments can be sequenced at a time, which is low-efficiency for large-scale sequencing projects. 2. Time-consuming: The sequencing process takes a long time, including steps such as DNA extraction, library construction, sequencing, and data analysis. 3. High cost: Due to the complexity of the sequencing process and the required reagents, the cost of Sanger sequencing is relatively high.

Sanger sequencing, as a classic DNA sequencing method, is widely used in fields such as genetic research, disease diagnosis, drug research and development, and forensic medicine. However, with the development of high-throughput sequencing technology, Sanger sequencing has been gradually replaced in some aspects.

Related reading recommendations

COVID-19 testing with Opentrons

3 ways automation can lead to better biological laboratory results

Adapter magnet support article

User Manual: Opentrons Calibration Module

How to clean the glass tips of the fully automatic pipetting workstation

The principle and working steps of enzymatic hydrolysis in a fully automatic pipetting workstation