The chain termination method, also called Sanger sequencing, is a method of DNA sequencing. This technique determines the nucleotide sequence of DNA. This concept was introduced by Frederick Sanger in 1977. The technique has been fundamental in genetics and molecular biology.
About Chain Termination Method
The chain termination method of dna sequencing notes briefly explains the technique, key components, methodology, applications, advantages, and limitations. The technique incorporates dideoxynucleotide triphosphates (ddNTPs) into the DNA strand during replication.
Procedure For Chain Termination Method Of DNA Sequencing
- Template Preparation – The process begins with the preparation of a DNA template, which involves the isolation and purification of the DNA for sequencing.
- Primer Binding – The next step involves primer annealing. It involves a primer, which is a short single-stranded DNA oligonucleotide. The primer is mixed with the template DNA to bind or anneal to the complementary sequence on the template.
- Reaction Setup – Involves the preparation of four reaction mixtures of sequencing. Each preparation contains a primer, template DNA, DNA polymerase, and dNTPs (deoxynucleotide triphosphates).
- DNA Synthesis – Synthesis of new DNA strands happens with DNA polymerase. The process involves extending the primer and adding dNTPs complementary to the template strand.
- Chain Termination – From time to time, a ddNTP gets incorporated instead of a dNTP. This is so, as ddNTPs lack the 3′-OH group crucial for forming a phosphodiester bond with the next nucleotide. As a result, DNA synthesis stops, leading to the termination of a fragment.
- Fragment Separation – DNA fragments are separated using Polyacrylamide Gel Electrophoresis (PAGE). The fragments get separated as per their size. Those with a smaller size move and travel faster in comparison to the larger ones.
- Detection Of The Sequence – In this process, the DNA sequences are detected using laser scanners. It involves the use of fluorescent labels.
- Data Analysis and Reading Of Sequence – This step determines the DNA sequence through pattern analysis of fragment length. The process of sequencing can be carried out manually or automatically.
Applications of Chain Termination Method
The Chain Termination Method, or Sanger Sequencing, has many uses in various genetics fields. These fields include molecular biology, medicine, and forensic research. Some key applications of this technique of DNA sequencing are as follows:
- Genetic Research – This includes gene discovery and identification, achieved through gene analysis. The research involves identifying genetic mutations and variations for understanding genetic disorders and predispositions. The study also helps establish evolutionary relationships among organisms.
- Medical Diagnostics – For diagnosis, the genetic mutations or variations that increase the risk of genetic disorders are detected. Some common types of genetic conditions revealed include cystic fibrosis, muscular dystrophy, and hemophilia. This includes prenatal testing and newborn screening, which helps assess the risk of genetic disorders. Early diagnosis will enable timely treatment and disease management in the fetus or newborn.
- Microbial Genomics – DNA sequencing by chain termination method facilitates pathogen identification. The genomes of various pathogens under the categories of viruses and bacteria are identifiable with the sequencing technique. Genome sequencing and genetic analysis also help with epidemiology, involving the tracing of the source of an outbreak of infectious diseases.
- Cancer Genomics – Analysis of genes through sequencing helps identify gene mutations linked to certain types of cancer and tumors. Determination of cancer cells and studying the mutations associated with them guide personalized treatment plans.
- Biotechnology – DNA sequencing finds its use in genetic engineering and quality control. The method aids in the verification of the sequences of recombinant DNA constructs for genetic engineering. Besides, it also ensures the accuracy and integrity of genetically modified organisms (GMOs) for research purposes and commercial applications.
- Pharmacogenomics – DNA sequencing through chain termination helps assess the drug response of an individual. Certain genetic mutations can influence their response to specific medications. The technique helps identify those mutations.
Advantages Of Chain Termination Method
- Forensic Science – DNA sequencing has a crucial role in events like crime scene evidence or paternity testing. The technique also helps identify the remains of unknown humans in the case of disaster victim identification. Moreover, it is beneficial for producing legal evidence in criminal investigations.
- High Accuracy – This technique ensures high accuracy and reliability in determining DNA sequences. Plus, it has an error rate as low as 0.001%.
- Simplicity – The ease of operation ensures simplicity and robustness of the technique. It includes clear protocols with minimal computational requirements.
- Versatility – The chain termination method has a range of different applications, making it suitable for use in various fields.
- Ease Of Interpretation – The outcome of sequencing is relatively straightforward to interpret. Also, for analysis of the data, there are well-established standards and guidelines.
- Affordable for Small-Scale Projects – Chain Termination for DNA sequencing is comparatively cost-effective for small-scale projects.
Role Of Chain Termination Method In Genetic Testing
Sanger sequencing, or the chain termination method, has an important function in genetic testing due to its high accuracy and reliability. This method is directly linked to various applications in genetic testing in the following ways:
- Diagnostic Evaluation of Genetic Disorders – Identification of genetic mutations or variants leading to genetic disorders.
- Prenatal And Newborn Screening – Assessing the possibility of risk of a genetic disorder due to the passing of mutated genes in the fetus or newborn.
- Cancer Genomics – Identifying somatic variations in cancer cells and predicting the risk of certain types of cancers.
- Pharmacogenomics – Genetic testing can help determine individual responses to certain types of medications or drugs.
- Forensic Applications – Supports identification of human remains in criminal cases.
- Research and Development – It contributes to the advancement of technologies of genetic analysis.
Conclusion
The Chain Termination Method for DNA Sequencing or Sanger Sequencing is highly accurate and precise. It also offers long read lengths, simplicity, and ease of interpretation. This makes the technique an indispensable tool in many areas of genetic research and diagnostics.
The next-generation sequencing technologies have expanded the possibilities for large-scale genomics. However, the Chain Termination Method continues to be the method of choice for many specific applications. This technique is preferred particularly for its high precision and reliability.
