Question

3.Briefly describe the concepts of DNA sequencing. How it has evolved over the time? What is the current status of sequencing and pick a method to outline the principles as well as reasons you think it is the best?

4.What do you understand by “omics” revolution? Why do we need different “omics” to understand genes and proteins functions? Pick a specific omics and elaborate how it may help to understand fundamental levels as well as helping us to live a better life

Answer 1

A process that aims to determine the order of nucleotides (adenine, guanine, cytosine, and thymine) in a DNA molecule is termed as DNA sequencing.

Methods used for DNA sequencing have been greatly evolved over time.

• Sanger and the team introduced the concept of DNA sequencing for the first time in 1970. Its working principle was to insert a dideoxythymidine triphosphate (ddTTP) molecule and terminating the growth of growing nucleotide chain. Later, terminated sequences were determined using the radiography technique on Polyacrylamide Gel Electrophoresis (PAGE). Sanger sequencing is also known as Chain termination sequencing.

However, the Sanger sequencing method was expensive and inadequate for the sequencing of the large genome. Thus, sequencing methods were modified with time.

• Later, radiography was replaced with fluorescence for detection as separate fluorescence markers were introduced in each nucleotide which allowed easy detection.
• PAGE was replaced with the use of capillary electrophoresis.
• capillary electrophoresis was further replaced with capillary array electrophoresis

However, this technique depicted low sensitivity for low-level mutant alleles and inefficiency in the analysis of highly polymorphic regions like Major Histocompatibility Complex (MHC). So, the sequencing approach was further evolved.

• Most recently, Next Generation Sequencing (NGS) technologies were introduced that overcame all the limitations showcased by previous methods. It is a set of most recent DNA sequencing methods.

NGS is the best technique for DNA sequencing as it is fast, comparatively cheap, allows multiple sequencing at the same time (highly parallel in nature), allows many reactions to be conducted on 1 chip.

The basic principle involved in the working of NGS methods include-

Multiple DNA clusters are made from a single target DNA molecule. Target -DNA library is generated and loaded on to a flow cell following which, sequencing of DNA clusters is conducted in parallel. Signal originating from each target DNA molecule is then seperated physically.