What is the basic process of sequencing dna by chain termination method?

Chain termination sequencing (Sanger sequencing) is a commonly used DNA sequencing method, and the following is its basic flow:

Preparation of DNA template: firstly, it is necessary to obtain DNA samples to be sequenced and purify them. DNA fragments are usually amplified by PCR or bacterial culture to obtain enough templates. Then, DNA becomes single-stranded by thermal denaturation.

Synthesis of labeled primers: At the end of DNA template, DNA was synthesized by DNA polymerase and binary nucleotide (ddNTPs). Among them, ddNTPs is an anoxic derivative of common dinucleotide (dNTPs), which is similar to dNTPs, but its 3'-OH terminal has no hydroxyl group, so the extension of DNA chain will be terminated.

Reaction segmentation: the synthetic DNA fragments were separated by gel electrophoresis. DNA fragments are separated according to their length, and there are color differences according to the types of terminal bases (ddtp, ddCTP, ddGTP, ddTTP).

Gel electrophoresis: the reaction product is injected into polyacrylamide gel, and then the DNA fragment is moved along the gel by applying an electric field. Due to the different pore sizes in the gel, shorter DNA fragments move faster, while longer DNA fragments move slower.

Analysis of electrophoresis results: The distribution of DNA fragments in the gel was observed by appropriate detection methods, such as fluorescent labeling or radioactive labeling. Different bases will produce different colors or radiation intensities, so the DNA sequence can be determined.

Data analysis: the sequence and length of DNA fragments can be determined by analyzing the band pattern on the gel electrophoresis image. According to the relative position and color of the bands, the base sequence of DNA can be inferred.

Chain termination sequencing is a method to determine DNA sequence by using the characteristics of terminated bases during DNA chain replication and combining gel electrophoresis technology.