MAWLANA BHASHANI SCIENCE AND TECHNOLOGY UNIVERSITY

SANTOSH,TANGAIL -1902

Department of: BIOTECHNOLOGY & GENETIC ENGINEERING

Assignment NO: 01

Course Code: BGE-2101

Course Title: Molecular Biology 2

Name of Assignment: DNA Sequencing (What, History, Steps, Application,

Limitation)

Date of performance: 18.08.2020 Date of Submission: 23.08.2020

From, To,
Name: Group-05 Roksana Khanam
Student ID: BG-19033, BG-19034, Lecturer,
BG-19036, BG-19037, Department of Biotechnology & Genetic
BG-19038, BG-19040. Engineering,
nd
Year-2 Mawlana Bhashani Science &
semester- 1st Technology University
Session: 2018-19
 DNA SEQUENCING
What is DNA Sequencing?
A laboratory technique, to know the correct DNA sequence
(an order of nucleotides) by the sequential chemical reaction is known as DNA
Sequencing.
History

Steps
There are Two main types of DNA Sequencing.
1. Sanger method- the older, classical chain termination methods.
2. Next-Generation Sequencing methods.
Steps of Sanger method
i. The double standard DNA is denatured into two single standard
DNA(ssDNA)
ii. A primer is attached.
iii. The fragments are added to four polymerase solutions. Each solution
contains four types of bases but one type of termination nucleotide.
iv. DNA synthesizes & extended until a termination nucleotide is added.
 v. The resulting DNA fragments are denatured into ssDNA
vi. Those fragments are separated by gel electrophoresis & the sequence is
read.

NGS’s method
There are Three basic steps
a) Library preparation
b) Sequencing
c) Data analysis.
Library Preparation
This step prepares DNA sample to be compatible with a sequencer.
Fragmented DNA are added with adapter to both ends. These adapters contain
complementary sequence. During adapter ligation “barcodes” are added to each
library.
Sequencing
Libraries are loaded onto a flow cell & placed on the sequencer. The clusters
of DNA fragments are amplified resulting in millions of copies of single stranded DNA.
Then chemically modified nucleotides (contain fluorescent tag & a reversible
terminator) bind to the DNA template strand. The fluorescent signal indicates which
nucleotide has been added & the terminator is cleaved as the next base can bind.
After reading the forward DNA stand, the reads are washed away.

Data analysis
The instrument software identifies nucleotides & find variation, mutation.

Application
 In medical science
- Used in the identification of genes responsible for hereditary disorders &
mutations.
 In forensic science
- Used for parental verification, criminal investigation.
 In Agriculture industries
- Identification of GMO species & any of minor variations in the plant
genome.
 In constructing map
- Whole chromosomal maps;
- Restriction digestion maps;
- Genome maps.
  Identification of open reading & non-open reading frames, protein coding DNA
sequence.
 Used in exon, intron, repeat, tandem repeat indentification.
 Used in gene manipulation, gene editing.
 In microbial identification & study of the new bacterial species.
 On oncology & cancer studies, evolutionary study.

Limitations
 Sanger methods can only sequence short pieces of DNA- about 300 to 1000
basepairs & the quality degrades after 700 to 900 bases
 Tandem repeats, repetitive DNA, fragmented genes, duplicated regions cannot
be studied properly.
 Requires sophisticated bioinformatics systems, equipment, fast data
processing, large data storage.
 In some cases it is very costly.