Sanger Sequencing
– Sanger sequencing is the gold standard in DNA sequencing.
– The presentation was prepared by Mohsen Al-Saleh on 17/02/2025.
Outlines:
– The presentation will cover the introduction and background of Sanger sequencing.
– It will also delve into the principles and key components of this method.
– Finally, it will discuss the applications, advantages, limitations, and conclusion.
DNA Sequencing:
– DNA sequencing determines the exact sequence of nucleotides in a DNA molecule.
– Methods include Maxam – Gilbert sequencing (chemical degradation method), Sanger sequencing (dideoxy chain-termination method), and High-throughput sequencing technologies (NGS).
What is Sanger sequencing?
– Sanger sequencing is a method used to determine the nucleotide sequence of DNA and relies on chain-terminating dideoxynucleotides (ddNTPs).
– It was developed by Frederick Sanger in 1977 and played a key role in the Human Genome Project (1990).
– Sanger sequencing offers high accuracy and remains a benchmark for validating genetic data.
Key Principles:
– ddNTPs lack a 3’-OH group, which halts DNA synthesis during chain termination.
– Key components include a DNA template, primer, DNA polymerase, dNTPs, and fluorescently labeled ddNTPs.
– Fluorescent tags on ddNTPs are used for detection, with each base having a unique color, and capillary electrophoresis is used for separation of DNA fragments by size.
Procedure Overview:
– The procedure includes isolating target DNA for template preparation.
– Then performing linear amplification with ddNTPs and dNTPs during cycle sequencing.
– Fragments are separated by size in a capillary tube via electrophoresis, a laser excites fluorescent tags for detection, and software generates a chromatogram.
Applications, Pros/Cons, and Conclusion:
– Applications include genetic disease diagnosis, forensic analysis, microbial identification, and mutation detection.
– Advantages include high accuracy (~99.99%) and reliability for short reads (up to 1,000 bp).
– Limitations include low throughput and being costly/time-consuming for large genomes, but it is still vital for small-scale projects and validating NGS results.
Sanger Sequencing vs. NGS:
– Sanger sequencing has an accuracy of 99.9%, while NGS has an accuracy of 99-99.9%.
– Sanger sequencing is more cost-effective for fewer than 20 samples, while NGS is more cost-effective for more than 20 samples.
– Sanger sequencing has lower sensitivity (15-20%) compared to NGS (1%).