GENE SEQUENCING AND METHOD OF DNA Sequencing

A laboratory method that is used to determine the entire genetic makeup of a specific organism or cell type.
Establishing the sequence of DNA is key to understanding the function of genes and other parts of the gene.
Genes are the basic unit of inheritance, composed of DNA, which itself is composed of nucleotides that code for proteins.

1. S.K.C.G (Autonomous) college Gene sequencing Represented By MANAS Ranjan sahoo m.sc. 2 nd year Life science department Roll.no – Life-23-026

2. Gene :- The functional and physical unit of heredity passed from parent to offspring. Genes are pieces of DNA, and most genes contain the information for making a specific protein. Genes are found  in chromosomes, which are located in the nucleus of cells

3. Gene Sequencing :- A laboratory method that is used to determine the entire genetic makeup of a specific organism or cell type. Establishing the sequence of DNA is key to understanding the function of genes and other parts of the gene. Genes are the basic unit of inheritance, composed of DNA, which itself is composed of nucleotides that code for proteins.

4. Methods of DNA Sequencing :- First-Generation DNA Sequencing Method :- 1. Maxam-Gilbert (chemical degradation) 2. Sanger (chain termination) Second-Generation Sequencing Method :- Roche 454 Method Illumina Method Solid Method Ion Torrent Method

5. Third-Generation DNA Sequencing :- Third-generation DNA sequencing methods are divided into two techniques :- Pacific Bioscience Method Oxford Nanopore Technology Method

6. Maxam-Gilbert (chemical degradation) The Maxam–Gilbert method is a method based on chemical degradation introduced by Maxam and Gilbert in the late 1970s. In the Maxam–Gilbert procedure, the guanine base is cut using dimethyl sulfate, the cytosine base is cut using hydrazine and sodium chloride, the guanine and adenine bases are cut together using formic acid, and the cytosine and thymine bases are cut together using hydrazine

7. The basic principle of Maxam–Gilbert sequencing method

8. Advantages:- High accuracy: The Maxam-Gilbert method can provide high accuracy in determining the DNA sequence, as the chemical cleavage is precise and predictable. Can detect modifications: The Maxam-Gilbert method can detect modifications to the DNA, such as methylated or damaged bases. Applicable to different DNA types: The Maxam-Gilbert method can be used to sequence different types of DNA, including single-stranded DNA and RNA. Can sequence through difficult regions: The Maxam-Gilbert method can sequence through difficult regions of DNA, such as regions with high GC content or secondary structures.

9. Disadvantages:- Complexity and cost: The Maxam-Gilbert method is a relatively complex and costly method of DNA sequencing, which has limited its use in modern DNA sequencing technologies. Limited read length: The read length of the Maxam-Gilbert method is limited to around 50-500 nucleotides, depending on the specific conditions used. This is much shorter than the read lengths that can be achieved with modern sequencing technologies. Time-consuming: The Maxam-Gilbert method is a time-consuming process, as it involves several steps

10. Sanger (chain termination) methods :- The Sanger method, which was introduced by F. Sanger in 1977, is a method based on chain elongation termination, which is used in DNA sequencing with the help of polymerase and special nucleotides. The elongation of the chain is terminated by synthetic ddNTPs (dideoxynucleoside triphosphate), which are the monomers of the DNA chain added to the reaction. The only difference between these ddNTPs and dNTPs (deoxynucleotide triphosphate) is that they lack a hydroxyl group on the deoxyribose sugar's third carbon.

11. Lack of hydroxyl causes polymerization to stop, and thus, chain elongation is stopped because it cannot establish a bond with the 5' phosphate of the following dNTP

12. Schematic representation of the Sanger method

13. Advantages:- Sanger sequencing is considered the gold standard method for many research and clinical applications .  It provides highly accurate results. This accuracy is particularly useful for validating sequences obtained through other methods, such as NGS technologies. Sanger sequencing is suitable for small-scale projects or those including short DNA regions.  Data analysis from Sanger sequencing does not require complex bioinformatics tools and expertise often necessary for NGS data interpretation.  The quality of the data obtained from Sanger sequencing is high with clear and easily interpretable chromatograms.

14. Disadvantages:- Sanger sequencing can only sequence short fragments of DNA. It has a limited throughput. It can sequence only one fragment at a time, making it slow and expensive for large genomes. Sanger sequencing is not suitable for long DNA sequences or large-scale sequencing projects due to its complexity and cost.  It involves manual steps compared to NGS workflows and is a time-consuming method. The preparation and handling time for Sanger sequencing can be longer. Sanger sequencing requires relatively high-quality and pure DNA samples as degraded or contaminated DNA can lead to unreliable results.

15. References:- https://microbenotes.com/sanger-sequencing/#advantages-of-sanger-sequencing https://www.eajm.org/Content/files/sayilar/224/8.pdf https://sciencevivid.com/maxam-gilbert-sequencing-introduction-steps-advantages-applications/ https://www.slideshare.net/slideshow/gene-sequencing-215490507/215490507 Pareek, C.S., Smoczynski , R. & Tretyn , A. Sequencing technologies and genome sequencing.  J Appl Genetics   52 , 413–435 (2011).

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