NEED OF GENETIC SEQUENCING - Understanding the particular DNA sequence can shed light on a genetic condition and offer hope for the eventual development of treatment. - An alteration in a DNA sequence can lead to an altered or non functional protein and hence to a harmful effect in a plant or animal. - Simple point mutations can cause altered protein shape and function.

1. GENE SEQUENCING
SUBMITTED BY MAHIMA
(1ST YR, M PHARM,
PHARMACOLOGY
Noida Institute of Engineering and Technology
(Pharmacy Institute), Greater Noida
SUBMITTED TO DR. SAUMYA DAS

2. CONTENT
 Introduction
 Need of sequencing
 DNA, DNA sequencing
 Gene, Gene sequencing
 Genome, Genome sequencing, Genomics
 Different sequencing procedure
chemical cleavage method (Maxam–Gilbert Sequencing method)
chemical termination method (sanger sequencing method)
 Refrences

3. INTRODUCTION
DNA – the hereditary material written in four letter code of nucleotides.
Sequencing or reading the genetic code has become of increasing interest to scientists.
RNA sequencing – earliest form of nucleotide sequencing complete genome of bacteriophage
MS2.
Knowledge on genome organization was based on reverse genetics.
NEED OF GENETIC SEQUENCING
 Understanding the particular DNA sequence can shed light on a genetic
condition and offer hope for the eventual development of treatment.
 An alteration in a DNA sequence can lead to an altered or non functional
protein and hence to a harmful effect in a plant or animal.
 Simple point mutations can cause altered protein shape and function.

4.  DNA
• DNA, also known as deoxyribonucleic acid, is the molecule that contains
the genetic information necessary for the development and functioning of
an organism.
• composed of a double helix, which is a structure characterized by two
linked strands winding around one another to resemble a twisted ladder.
• Each strand has a backbone made of alternating sugar (deoxyribose) and
phosphate groups.
• Attached to each sugar is one of four bases: adenine (A), cytosine (C),
guanine (G) or thymine (T) at position 1C of pentose sugar.
• The two strands are connected by chemical(hydrogen) bonds between the
bases: adenine bonds with thymine, and cytosine bonds with guanine.
• The sequence of the bases along DNA’s backbone encodes biological
information, such as the instructions for making a protein or RNA
molecule.

5. Right handed coiling,
Antiparallel to each other
One base pair is above other
with 36 degree difference

6.  DNA SEQUENCING
• It refers to the general laboratory technique for determining the exact
sequence of nucleotides, or bases, in a DNA molecule.
• The sequence of the bases (often referred to by the first letters of their
chemical names: A, T, C, and G) encodes the biological information that cells
use to develop and operate.
 GENE
• The gene is considered the basic unit of inheritance.
• Genes are passed from parents to offspring and contain the information
needed to specify physical and biological traits.
• Most genes code for specific proteins, or segments of proteins, which have
differing functions within the body.
• Humans have approximately 20,000 protein-coding genes.

7.  GENE SEQUENCING
• Process in which individual base nucleotides in an organism’s DNA are
identified.
• Gene sequencing and DNA sequencing are used interchangeably; also known as
nucleotide sequencing or base sequencing.
• However, not all DNA sequences are genes (i.e, coding regions) as there may,
also be promoters, tandem repeats, introns, etc. depending on the organism and
the source of the source of the DNA sample.
 GENOME
• The genome is the entire set of DNA instructions found in a cell.
• In humans, the genome consists of 23 pairs of chromosomes located in the cell’s
nucleus, as well as a small chromosome in the cell’s mitochondria.
• A genome contains all the information needed for an individual to develop and
function.

8.  GENOME SEQUENCING
• A laboratory procedure that determines the order of bases in the genome of
an organism in one process.
• Breaking the whole genome into small pieces, sequencing the pieces and
then reassembling them in proper order to arrive at the sequence of the
whole genome.
GENOMICS
• Sequencing of genomes, determination of the complete set of proteins
encoded by an organism and functioning of genes and metabolic path ways
in an organism.

10. DIFFERENT SEQUENCING PROCEDURE
Widely Used Two DNA Sequencing Methods;
First, a bit of history. In the mid-1970s, two methods were developed to
sequence DNA directly. These were:
1.The Maxam–Gilbert sequencing method
2.The Sanger chain-termination method
Maxam–Gilbert sequencing is a method for chemically sequencing DNA. It
used to be popular but has been superseded by Sanger sequencing and next-
generation sequencing methods because it is slow, is low-throughput, and
uses dangerous chemicals.
This popularity was because scientists could use purified DNA directly,
while the initial Sanger method required DNA cloning for the start of each
read.

11. Chemical cleavage method: Maxam–Gilbert Sequencing method
How Does Maxam–Gilbert Sequencing Work?
Maxam–Gilbert sequencing is also known as chemical sequencing because chemical reactions,
rather than DNA and RNA amplification, are the basis of the method.
It’s quite easy to understand, though, and proceeds in just 4 steps:
1. Preparation of Your Sample
• The DNA used in Maxam-Gilbert sequencing is first denatured into single-stranded chains
and radiolabeled on the 5′ end, usually with 32P.
2. Electrophoresis and Autoradiography
• The next step cleaves the DNA. And this is where the Maxam–Gilbert sequencing gets really
interesting.
• By taking advantage of piperidine and two chemicals that selectively attack purines and
pyrimidines (dimethyl sulfate and hydrazine, respectively), the DNA is cleaved at specific
points.

12. • To be more accurate, using different combinations of these chemicals, you can cleave a DNA
sequence wherever there is a C, wherever there is a C or a T; wherever there is a G, or wherever
there is a G or an A.
• So, if you put your sample into these 4 different reaction tubes, you obtain different fragments
depending on the combination of chemicals!
3. Electrophoresis and Autoradiography
• These reactions are then loaded onto a high-percentage agarose gel to differentiate fragment
sizes. The fragments are visualized via the radioactive tag.
4. Reading the Sequence
• To read the sequence, you begin with the smaller fragments at the bottom of the gel. “Calling”
each base involves interpreting the band pattern relative to the four chemical reactions.
• For example, if a band in the DNA sequence appears in both the G-reaction and the G+A-
reaction lanes, then that nucleotide is a G.
• If a band in the DNA sequence appears only in the G+A-reaction lane, then it is an A. The same
decision process works for the C-reaction and the C+T-reaction lanes. Sequences are confirmed
by running replicate reactions on the same gel and comparing the autoradiographic patterns
between replicates.
• Then the process repeats like you are solving a puzzle.

14. Why Did It Lose Popularity?
Maxam–Gilbert sequencing, although based on simple principles, came with a
whole lot of trouble. Here are a few of its major drawbacks.
 It’s Slow
First, it was time-consuming. And that was supposing that everything went well on
the first try. A lot of steps in the method could cause problems. Examples of
potentially problematic steps include:
1.Radiolabeling.
2.The cleavage reactions.
3.Preparing the gel.
4.Electrophoresis.
5.Developing the X-ray film to visualize results.
Plus, you can’t easily parallelize the method. So, you can only confirm about 200–
300 bases of DNA every few days!

15.  It Uses Dangerous Chemicals
Working with radioactive isotopes and hazardous chemicals.
In particular, 32P is a nasty isotope as they go. It’s a high-energy beta emitter with a
short half-life about 14 days, thus it can put a lot of energy into your tissue and do
so quickly.
And hydrazine is a potent neurotoxin that can cause organ damage.
 It’s Comparatively Information-Poor
Next-generation sequencing methods, such as the Illumina dye method, offer
genome-wide sequencing data. And Sanger sequencing is the incumbent method for
plasmid sequencing—a fundamental service required by almost all biology research
labs.
Partly because of the drawbacks mentioned above, and partly because of the
intrinsic limitations of the technique, Maxam–Gilbert sequencing cannot compete
with these methods.

16. What Is Maxam–Gilbert Sequencing Used for Today?
While the Maxam-Gilbert method is not used as much as it once was, it is
still used in some specialized applications where aspects of the process
(namely the chemical cleavage step) make it useful.
• DNA Footprinting
• Identifying DNA Modifications
• Structural DNA Analysis

17. Chain termination method: sanger sequencing method
Sanger sequencing, also known as the “chain termination method,” was developed
by the English biochemist Frederick Sanger and his colleagues in 1977.
This method is designed for determining cg the sequence of nucleotide bases in a
piece of DNA (commonly less than 1,000 bp in length).
Sanger sequencing with 99.99% base accuracy is considered the “gold standard”
for validating DNA sequences, including those already sequenced through next-
generation sequencing (NGS). Sanger sequencing was used in the Human
Genome Project to determine the sequences of relatively small fragments of
human DNA (900 bp or less). These fragments were used to assemble larger DNA
fragments and, eventually, entire chromosomes.

18. How Does Sanger Sequencing Work?
In Sanger sequencing, a DNA primer complementary to the template
DNA (the DNA to be sequenced) is used to be a starting point for DNA
synthesis. In the presence of the four deoxynucleotide triphosphates
(dNTPs: A, G, C, and T), the polymerase extends the primer by adding
the complementary dNTP to the template DNA strand. To determine
which nucleotide is incorporated into the chain of nucleotides, four
dideoxynucleotide triphosphates (ddNTPs: ddATP, ddGTP, ddCTP, and
ddTTP) labeled with a distinct fluorescent dye are used to terminate the
synthesis reaction. Compared to dNTPs, ddNTPs has an oxygen atom
removed from the ribonucleotide, hence cannot form a link with the next
nucleotide. Following synthesis, the reaction products are loaded into
four lanes of a single gel depending on the diverse chain-terminating
nucleotide and subjected to gel electrophoresis. According to their sizes,
the sequence of the DNA is thus determined. The structure of ddNTP and dNTP.

19. Sanger Sequencing Steps
The Sanger sequencing method consists of 6
(1) The double-stranded DNA (dsDNA) is
single-stranded DNA (ssDNA).
(2) A primer that corresponds to one end of
attached.
(3) Four polymerase solutions with four types
only one type of ddNTP are added.
(4) The DNA synthesis reaction initiates and
extends until a termination nucleotide is
incorporated.
(5) The resulting DNA fragments are
(6) The denatured fragments are separated by
electrophoresis and the sequence is
The Sanger sequencing method in 6 steps

20. Applications of Sanger Sequencing:
Sanger DNA sequencing is widely used for research purposes like
(1) targeting smaller genomic regions in a larger number of
samples,
(2) sequencing of variable regions,
(3) validating results from next-generation sequencing (NGS)
studies,
(4) verifying plasmid sequences, inserts, mutations,
(5) HLA typing,
(6) genotyping of microsatellite markers, and
(7) identifying single disease-causing genetic variants.

21. OTHER DNA SEQUENCING METHODS:
 CYCLE SEQUENCING
Modification of the traditional sanger sequencing method.
Principle are the dame as in sander sequencing as in cycle sequencing thermostable DNA polymerase is used
which can be heated to 95 degree celsius.
HIGH-THROUGHPUT SEQUENCING
Produce thousands or millions of sequences at once.
Commonly called dye-terminator sequencing.
CAPILLARY ELECTROPHORESIS
PYROSEQUENCING
SEQUENCING BY LIGATION
SEQUENCING BY HYBRIDIZATION
Non-enzymatic method that uses a DNA microarray.

22. NEXT GENERATION SEQUENCING METHODS
 Mass spectrophotometric sequences
 Direct visualization of single DNA molecules by atomic force microscopy
(AFM)
 Single molecule sequencing techniques
 Single nucleotide cutting
 Read out of cellular gene expression
 Use of DNA chips or micro arrays
 Nano sequencing

23. SOME COMMERCIAL SEQUENCERS
 rochel45FLXpyrosequencer - pyrosequencing
 Illumina genome analyzer – sequencing by synthesis
 Applied biosystems SOLID sequencer – sequencing by ligation
 Helicos heliscope
 Pacific biosciences SMRT – zeromode waveguide

24. Refrences :
1. Sidote DJ et al. (2008) Structure of the Staphylococcus aureus AgrA LytTR domain bound
to DNA reveals a beta fold with an unusual mode of binding. Structure 16:727–35
2. Gauthier, Michel G. Simulation of polymer translocation through small channels: A
molecular dynamics study and a new Monte Carlo approach. Diss. University of Ottawa
(Canada), 2008.
3. Sikkema‐Raddatz, Birgit, et al. Targeted next‐generation sequencing can replace Sanger
sequencing in clinical diagnostics. Human mutation 34.7 (2013): 1035-1042
4. Crossley BM, Bai J, Glaser A, et al. Guidelines for Sanger sequencing and molecular assay
monitoring. Journal of Veterinary Diagnostic Investigation. 2020 Nov;32(6).
5. Mardis ER. DNA sequencing technologies: 2006–2016. Nature protocols. 2017 Feb;12(2).