Name: Muhammad Asif Khan
Class: BS V
Subject: Microbial Genetics
Assignment Topic: DNA sequencing and Gel Electrophoresis
Teacher: Dr Naima Atiq
DNA Sequencing
The technique of determining the precise arrangement of the nucleotides adenine (A), thymine (T),
cytosine (C), and guanine (G) in a DNA molecule is known as DNA sequencing.
Scientists can now investigate and comprehend the genetic composition of organisms thanks to this
approach, which has become a basic tool in molecular biology and genetic research.
British biologist Frederick Sanger created one of the first and most significant DNA sequencing
techniques in 1977. It provided scientists with a dependable, repeatable technique to decode DNA and
was called the Sanger method. This method was very important in groundbreaking projects like the
Human Genome Project. There are now newer, faster sequencing methods, but the Sanger method is
still widely used, especially when working with smaller DNA samples or making sure that the results
from modern high-throughput methods are correct.
Overview of the Sanger Method
The Sanger method, also known as chain termination sequencing, is based on creating complementary
DNA strands to a target DNA sequence. Specially altered building pieces known as dideoxynucleotide
triphosphates (ddNTPs) are inserted at random during the course of DNA synthesis. The DNA strand
stops expanding once these ddNTPs are added because they lack the 3'-OH group required to continue
the chain.
As a result, a collection of different-length DNA fragments containing ddNTPs at the end are produced.
Scientists can then piece together the original DNA sequence by sorting these fragments by size and
determining which ddNTP is at the end of each one.
With small to medium-length DNA strands, this technique performs exceptionally well and provides
exceptional precision.
Key Components and Reagents
1. DNA template
This particular DNA segment requires sequencing. A plasmid vector is frequently used to
introduce it into bacterial cells. Numerous copies of the DNA fragment are created by these
cells' replication.
2. Primers
These are brief, single-stranded DNA segments that serve as the building blocks for DNA
synthesis by attaching to the template strand.
3. Polymerase of DNA
an enzyme that, during replication, adds nucleotides to the expanding DNA strand.
4. Triphosphates of deoxynucleotides (dNTPs)
the common components of DNA, such as dATP, dTTP, dCTP, and dGTP.
5. Dideoxynucleotide Triphosphates (ddNTPs)
These are unique nucleotides that terminate chains. They halt the growth of a DNA strand
when they are introduced. A distinct fluorescent hue that represents one of the four bases (A,
T, C, or G) is applied to each ddNTP.
Step-by-Step Procedure
1. DNA Fragment Preparation
First, a plasmid vector containing the DNA to be sequenced is created.
After introducing this vector into bacterial cells, which multiply and create many copies of the
DNA.
The plasmid DNA is removed and purified so that it may be used in the sequencing reaction.
2. Reaction Setup
The purified DNA is separated into many wells or reaction tubes.
Every reaction includes:
The DNA template
An adhesive primer for the template
DNA polymerase
A combination of the four common dNTPs
A reduced quantity of ddNTPs with fluorescent labels.
3. Thermal Cycling
Denaturation:
The process of heating double-stranded DNA to 96°C splits it into single strands.
Annealing (about 50°C):
Primers attach to the single-stranded DNA's complementary sequences.
Extension:
DNA polymerase begins to add nucleotides at around 60°C. The strand stops
developing when a ddNTP is inserted, leaving behind fragments of different lengths.
4. Fragment Separation
Capillary gel electrophoresis is used to separate the mixture of DNA fragments based on
size.
A distinct size-based separation is made possible by the rapid passage of smaller pieces
through the gel.
5. Sequence Detection
The fluorescent labels on the ddNTPs are stimulated as the pieces move through a laser
detector.
Each of them releases a distinct color signal that is associated with one of the following DNA
bases: A, T, C, or G.
A DNA sequence, which is frequently shown as an electropherogram, is created by recording
and translating these signals.
Conclusion
A ground-breaking development in genetics and molecular biology was the Sanger sequencing
technique. Scientists were able to "read" DNA sequences with remarkable accuracy because to it.
Sanger sequencing is still a vital technique for large-scale sequencing, even if other methods like Next-
Generation Sequencing (NGS) are becoming more popular. This is especially true for applications that
require high precision or for confirming results. Gaining knowledge and comprehension of this
technique lays a strong basis for comprehending the significance of DNA sequencing in contemporary
science.
Gel Electrophoresis
Introduction
A lab technique called gel electrophoresis is used to separate DNA, RNA, or protein combinations
according to their charge and size. An electric current is applied while a sample is submerged in a gel
matrix. When a current is supplied, negatively charged molecules such as DNA and RNA gravitate
toward the positive electrode. Smaller molecules pass through the gel more quickly, allowing
researchers to separate molecules by size.
Principle
The basic idea behind gel electrophoresis is that when an electric field is applied, charged molecules
move. Positively charged molecules flow toward the negative side of the gel, whereas negatively
charged molecules—such as DNA or RNA—migrate toward the positive end. Their size, charge, and
shape are among of the variables that affect how quickly they move.
Procedure
1. Gel preparation
The gel, which is typically composed of polyacrylamide or agarose, is poured into a mold
after being dissolved in a buffer solution. It is ready for use as soon as it solidifies.
2. Getting the Sample Loaded
After being combined with a loading dye, the DNA, RNA, or protein samples are cautiously
pipetted into the gel's wells.
3. Applying the Gel
An electric current is applied to the gel while it is in an electrophoresis chamber. The
molecules start to move across the gel. Compared to larger ones, smaller ones travel farther
and move more quickly.
4. Gel Staining
To see the bands under UV light, the gel is stained with a dye once the run is over; ethidium
bromide is frequently used for DNA.
5. Analysis
To ascertain the size of the molecules in the sample, the separated bands are viewed under
ultraviolet light, and their locations are contrasted with a molecular weight marker.
Applications
A flexible method with numerous biological and biotechnological uses is gel electrophoresis:
1. Analysis of DNA Fragments
Locating and examining particular DNA alterations or pieces.
2. Verification of PCR Products
Verifying the successful amplification of a gene or DNA sequence.
3. Separation of Proteins
Proteins are sorted according to size and charge for additional examination.
4. The use of genetic fingerprints
Used to find genetic patterns in paternity testing and forensic science.
Conclusion
In the biological sciences, gel electrophoresis is a crucial and popular method. It is an essential
technique for research, diagnostics, and biotechnology because it allows scientists to separate,
examine, and analyze complicated mixtures of biomolecules. Gel electrophoresis is a vital component
of contemporary molecular biology, whether it is used for gene identification, PCR result verification,
or protein comparison.
Pictures
DNA Sequencing
Overview of the Sanger Method
Gel Electrophoresis
Refrences
https://www.khanacademy.org/science/ap-biology/gene-expression-and-
regulation/biotechnology/a/gel-electrophoresis
Principles of Genetics(Book)
https://www.britannica.com/science/gel-electrophoresis
The Gene: An Intimate History(Book)
https://biology.unt.edu/~jajohnson/DNA_sequencing_process
Genome: The Autobiography of a Species In 23 Chapters
https://www.sigmaaldrich.com/US/en/technical-
documents/protocol/genomics/sequencing/sanger-
sequencing?srsltid=AfmBOop03sEI9lLLCNWww5ONOE8s4XLCDXNtI531_L-IrYSbl2f3ueT
