Sequencing genes and genomes in R DNA Technology

Genome sequencing in recombinant dna technology

1. BSc Biotechnology 3 nd year V Semester BBT 502- Recombinant DNA technology Ganesh Nawkar 03..10.2023 and 12/10/2023 Lecture14/15: Sequencing genes and genomes

2. Sequencing genes and genomes The ultimate objective of a genome project is the complete DNA sequence for the organism being studied. It is important to understand the genetic and/or physical maps of the genome so that genes and other interesting features can be located within the DNA sequence. Techniques for sequencing DNA are clearly of central importance in this context and we will begin the chapter with a detailed examination of sequencing methodology.

3. Different methods available for gene sequencing Sangar’s enzymatic method- Chain termination method Maxam – Gilbert Method Automated DNA sequencing Next generation sequencing

4. Sangar’s enzymatic method- Chain termination method The chain termination method first devised by Fred Sanger and colleagues in the mid-1970s. Chain termination DNA sequencing is based on the principle that single stranded DNA molecules that differ in length by just a single nucleotide can be separated from one another by polyacrylamide gel electrophoresis.

5. Sangar’s enzymatic method- Chain termination method

6. Sangar’s enzymatic method- Chain termination method Reading the sequence generated by a chain termination experiment

7. Maxam – Gilbert Method The starting material is double-stranded DNA, which is first labeled by attaching a radioactive phosphorus group to the 5’ end of each strand. Dimethylsulfoxide (DMSO) is then added, and the DNA is heated to 90°C. This breaks the base pairing between the strands, enabling them to be separated from one another by gel electrophoresis, the basis for this being that one of the strands probably contains more purine nucleotides than the other and is, therefore, slightly heavier and runs more slowly during the electrophoresis. One strand is purified from the gel and divided into four samples, each of which is treated with one of the cleavage reagents.