Sanger Sequencing Video Lecture | Crash Course for CSIR NET Life Sciences

Ans. Sanger sequencing, also known as the chain termination method, is a technique used to determine the nucleotide sequence of DNA. It involves the use of dideoxynucleotides (ddNTPs), which terminate DNA strand elongation when incorporated. The process begins with the amplification of the target DNA segment, which is then mixed with DNA polymerase, normal deoxynucleotides (dNTPs), and a small proportion of ddNTPs. As the DNA strands are synthesized, the incorporation of a ddNTP leads to the termination of the elongating strand. The resulting fragments are then separated by size using capillary electrophoresis, allowing for the reading of the sequence based on the fluorescence emitted by the labeled ddNTPs.

Ans. Sanger sequencing offers several advantages, including high accuracy and reliability for short DNA fragments. It is particularly effective for sequencing individual genes or small genomic regions. The method produces longer reads compared to many next-generation sequencing techniques, which aids in resolving complex regions of the genome. Additionally, Sanger sequencing is well-established, with a robust set of protocols and reagents available, making it a preferred choice for certain applications, especially in clinical diagnostics and validation of next-generation sequencing results.

Ans. Despite its strengths, Sanger sequencing has limitations. It is relatively slow and costly compared to high-throughput next-generation sequencing methods, making it less suitable for large-scale genomic projects. The method is also limited in the length of DNA it can effectively sequence, typically up to about 1,000 base pairs, which can be restrictive for more extensive genomic studies. Furthermore, Sanger sequencing requires a higher amount of starting DNA material, which can be a challenge when working with limited samples.

Ans. Sanger sequencing is widely used in various fields, including medical diagnostics, genetic research, and evolutionary biology. In medicine, it is employed for the detection of genetic mutations associated with diseases, such as cancer and inherited disorders. In research, Sanger sequencing is used to confirm mutations identified by high-throughput sequencing, to sequence plasmids, and to analyze genetic variation within populations. Its precision makes it a valuable tool for validating findings from other sequencing methods.

Ans. Sanger sequencing was first developed in the 1970s by Frederick Sanger and his colleagues. Over the decades, the method has evolved with advancements in technology, such as automated sequencing machines that improve throughput and decrease the time required for sequencing. Innovations like fluorescent labeling and capillary electrophoresis have enhanced the accuracy and efficiency of the method. While next-generation sequencing technologies have largely supplanted Sanger sequencing for large-scale projects, it remains a crucial technique for specific applications, maintaining its relevance in the genomics field.