This document provides in-depth knowledge on sequencing methods used in genetics field. they cover various types of sequencing that fall under traditional and next generation sequencing majorly.
Genome projects-
Cloned and sequenced the entire genome of an organism.
Characterizing whole genomes is fundamental to understand entire body of genetic information
underlying the physiology and development of living organisms, and to the discovery of new genes such
as those having roles in human genetic disease.
DNA sequencing-
To determine order of bases in section of DNA or entire genome. Starting point for analysing gene
structure and its expression. Or detect mutations and polymorphisms.
New strategies based on automation as presently no sequencing machine to read the entire genome at
once. Now automated sequences differ in accuracy, speed, cost and length of DNA obtained.
1) Sanger sequencing (dideoxy sequencing)-
Used for short DNA sequences, goal to use it to sequence entire genomes. Accurate expensive and slow.
● ddNTP is modified nucleotide called dideoxynucleotide triphosphate, has no 2’ and 3’-OH.
Therefore it cannot form phosphodiester bond in synthesis and terminates the chain.
● First DNA amplified by PCR or plasmid insert.
● Denatured to form two separate strands and primer created that binds to exactly one location of
DNA strand.
● Nucleotides present are dNTP of all four bases and small amount of ddNTP of a specific base.
Polymerase begins synthesis and stops at any point when ddNTP is incorporated. Eg- when
ddATP is added in small amount, then every time T is present in template there will be dideoxy
fragments. Different array of fragments with different ddNTP added to the different wells of the
electrophoresis-
● Either primers or ddNTP are radioactively labelled to visualise or fluorescently labelled with
different color emitters for each type of ddNTP and detector in the end distinguishes the color.
, Each peak is different sized fragments and colors for the type of ddNTP terminating them.
2) Whole Genome Shotgun sequencing (WGS)-
Determines sequences of many genomic DNA fragments in parallel.
a) Randomly shredding genome to billions DNA fragments
b) Overlapping sequence reads- Finding the consensus sequence in all fragments until all fragments
are linked. Many sequence reads obtained of each base pair in the genome to ensure accuracy.
c) Final genome sequence is now assembled in form of contigs. These genome sequences serve as
a standard, but do not represent the entire species, there are many variations when obtained
from parent leading to differences b/w individuals.
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