Volume 6, Issue 7, July – 2021 International Journal of Innovative Science and Research Technology

ISSN No:-2456-2165

DNA Synthesis and Sequence

1: 2:
VIVEK SHUKLA MANOJ KUMAR
(Btech/Mtech Dual Degree 3rd Year Student atKIIT School (MSc Botany 1st year Student at Seth MotiLal College,
of Biotechnology(KSBT), KIIT University, Rajasthan)
Bhubaneswar, Odisha)

Abstract:- DNA(Deoxyribonucleic acid) is a chemical  DNA molecule comprises of two poly nucleotide strands.
molecule which consists of all the necessary information • The bases are occupied the interior of the helix, the
that a living human being must possess. These sugar–phosphate chains are wrapped about its periphery,
instructions are then passed on from one generation to thus reducing the repulsions among charged phosphate
another. Our main objective via this article is to explain groups
how DNA can be obtained by many different techniques • DNA comprises of two long chains of simple units
and methods of analysis. DNA synthesis techniques are which are called nucleotides, with backbones made of
becoming a major part of modern day biology and plays base, sugars and phosphate groups.
a vital role in the field of synthetic biology. Through this • Each nucleotide is always attached via hydrogen bond to
review paper we will try to explore the various form specific complementary base pairing.
techniques that are widely used in the synthesis of DNA. • The aromatic bases have a vander Waals thickness of
We have also discussed the applications of DNA to gene 3.4Å and are always partially piled upon each other.
expression. We will also provide a brief introduction and • Sugar & Base is together called as nucleoside
discuss about DNA replication and what are processes • Phosphate, sugar & Base are collectively known as
responsible for DNA replication. nucleotide
• Adenine and Guanine are together known in a group as
I. INTRODUCTION – Purines
• Cytosine & Thymine are together known in a group as -
• Genes consists of DNA and re responsible for the source Pyrimidines.
of information to makemolecules called proteins.
• BASE PAIRING : Adenine pairs with Thymine with a
• In1868, Friedrich Miescher secluded a phosphorus-
double bond(A =T) in case of DNA whereas it pairs with
involving material. He discovered that the material
Uracil in case of RNA(A=U), Guanine pairs with
consisted of DNA, and a basic portion(protein). Kossel
Cytosinewith a triple bond(G≡C)
showed that DNA contains four nitrogenous bases A, C,
G, and T. • Major difference between DNA and RNA is the sugar,
• Levene later showed that DNA comprises of a huge with the 2-deoxyribose in DNA being substituted by the
quantity of reoccurring units called nucleotides; which different pentose sugar ribose in RNA.
consisted of a sugar, a phosphate, and a base. • DNA is a negatively charged molecule due to the
• He also incorrectly suggested that DNA comprises of a presence of phosphate group.
chain of four-nucleotide units, consisting of all the four
bases—―Adenine, Guanine, Cytosine, and Thymine”— DNA as the Genetic Material :-
in a fixed sequence(tetra nucleotide theory). The genetic material has four criteria.
1. Information: comprises of information essential to
construct an entire organism.(codingregions/genes)
2. Transmission: It is carried from parents to offspring
during the process of reproduction(vertical
transmission).
3. Replication: It must be copied(haploid to diploid).
4. Variation: The genetic material must differ in ways that
are responsible for the the known phenotypic differences
among all species(recombination).

Chargaff (late 1940s) –DNA molecules have distinctive

basecompositions:-
1. The base formation of DNA is generally different
among all genus.
2. DNA specimen extracted from different tissues of the
same species have similarcomposition.
3. The base composition of DNA in a given species
remains unaltered with an organism’s age, nutritional
state, or changing environment.
Figure-1: DETAILED ARRANGEMENT 4. Among all genus:

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Volume 6, Issue 7, July – 2021 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
• The total purine residues = the total pyrimidine (i)less expensive(ii)accurate
residues
 Chargaff base pairing rule: [Purines = Pyrimidines] Sanger methods:
(1) The double-stranded DNA (dsDNA) is reduced to two
 Applies to ds DNA, important for maintaining a
single-stranded DNA(ssDNA).
stable DNA structure.
(2) A primer that harmonizes to one terminal end of the
sequence is joined.
II. DNA SEQUENCING
(3) Four polymerase solutions consist of four types of
dNTPs however only onetype of dNTP is added.
It is a method of establishing the nucleic acid
(4) The DNA synthesis reaction starts and the length
pattern– that is the arrangement of nucleotides in DNA. It
elongates unless any stopnucleotide is added casually
consists of any technique that can be taken into
(5) The consequential DNA portions are reduced into
consideration in order to predict the arrangement of the four
ssDNA.
bases: adenine, guanine, cytosine, and thymine.
(6) The reduced portions are discarded via the process of gel
electrophoresis andthe sequence is analysed.
Different technologies:-
 Sanger sequencing.- is performed during low-
throughput, targeted, or short-read.
 Capillary electrophoresis. Capillary electrophoresis
(CE) instruments are efficient in performing both Sanger
sequencing and fragment analysis.
 Next-generation sequencing(NGS), they are vast-scale
techniques that enhance the velocity and lower the
expenditure.

DNA sequencing can establish a error-free diagnosis

influencing the medical administration of symptoms, or
supply treatment options.

Figure-3:- SANGER SEQUENCING METHODS AND

STEPS

Sanger sequencing provides high-value sequence for

approximately long stretches of DNA (up to about 900 base
pairs). It is mostly done to sequence separate pieces of
DNA, such as bacterial plasmids or DNA transcribed in
Polymerase Chain Reaction.

Capillary electrophoresis and fragment analysis:-

Capillary electrophoresis (CE) is an alternative to
conventional slab gel electrophoresis for the removal of
DNA portions. The bulk of DNA needed for separation is in
Figure-2:- STRUCTURE OF DNA & COMPLENTARY the nanogram range. Single-base resolution can be obtained
BASES on portions up to several hundred base pairs.

(i)SANGER SEQUENCING:-. Capillary electrophoresis is a method that removes

This method is established for predicting the pattern of ions based on their electrophoretic mobility with the
nucleotide bases in a portion of DNA.It was used in the application of an applied voltage. The electrophoretic
Human Genome Project to predict the pattern of small mobility is dependent upon the charge of the molecule, the
portions of human DNA. These fragments and constituted viscosity, and the atom's radius Capillary electrophoresis
larger DNA portions and, ultimately, entire chromosomes. (CE) is the most frequently used for separating and
The development of NGS technologies has exaggerated detecting short tandem repeat (STR) alleles in forensic DNA
genomics research.. Sanger sequencing remains widely used laboratories spread worldwide.
in the sequencing field as it offers several prominent
advantages:

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Volume 6, Issue 7, July – 2021 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
Phosphoramidite-based synthesis of oligonucleotides.
This synthesis process is the most commonly used for the
synthesis of DNA oligonucleotides for gene synthesis.

The main difference between protein synthesis and

DNA replication is that the protein synthesis is the
formation of a functional protein molecule based on the
information in the genes whereas DNA replication is the
production of an exact replica of an existing DNA molecule.

The most important event occurring in S phase is the

replication of DNA. The aim of this process is to produce
double the amount of DNA, providing the basis for the
chromosome sets of the daughter cells

The raw materials for DNA synthesis are the

Figure-4:- CAPILLARY ELECTROPHORESIS nucleotides:-
 deoxyadenosine triphosphate (dATP),
ii) Next-generation sequencing(NGS):-  deoxythymidine triphosphate (dTTP),
Next generation sequencing (NGS), explains a DNA
 deoxycytidine triphosphate (dCTP), and
sequencing technology which has modernised research in
genomics. The basic next-generation sequencing processes  deoxyguanosine triphosphate (dGTP)—collectively
comprises of breaking up DNA or RNA in numerous referred to as deoxyribonucleoside
portions, inclusion of connectors, and reorganising. It is  triphosphates (dNTPs) or deoxyribonucleotides
mostly identical to capillary electrophoresis.

Methods of Next-Generation Sequencing:-

1. Reversible Terminator Sequencing .
2. Single-Molecule Real-Time Sequencing
3. Ion Torrent

The new methods came to be known as next-

generation sequencing as they could formulate collateral
methods to develop high quantity of sequence from
numerous samples at very fast rate.

Advantages of NGS include:

Higher sensitivity to track and identify low-frequency
variants. Quick replication time for high sample volumes.
High genome analysis in details.

III. DNA SYNTHESIS

DNA synthesis is a biological process by which a Figure-5:- DNA REPLICATION DIAGRAM

DNA molecule is formed. In the cell, each of the two strands
of the DNA molecule acts as a template for the synthesis of IV. DNA REPLICATION
a complementary strand. DNA biosynthesis happens when
a cell divides, in a process called replication. It comprises DNA replication is the biological process of forming
of separation of the DNA double helix and thereafter two similar replicas of DNA from one original DNA
synthesis of complementary DNA strand, using the parent molecule. DNA replication happens in all living organisms
DNA chain as a template. The other strand (lagging strand) acting as the mostnecessary part for biological legacy..
also has to be produced in segments (Okasaki fragments)
The purpose of DNA replication is to produce two
Oligos are synthesized from building blocks which identical copies of a DNA molecule. This is essential for cell
replicate natural bases. The process has been automated division during growth or repair of damaged tissues. DNA
since the late 1970s and can be used to form desired genetic replication ensures that each new cell receives its own copy
sequences as well as for other uses in medicine and of the DNA.
molecular biology.

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Volume 6, Issue 7, July – 2021 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
DNA replication is semiconservative:-
Semiconservative replication supports copied DNA to
remain loyal to naïve template. The type of RNA molecule
which forms ribosomes.

Replication occurs in three major steps:

 The beginning of the double helix and removal of the
DNA strands,
 The reading of the template strand, and
 The gathering of the new DNA segment. During
separation, the two strands of the DNA double helix
uncoil at a definite location called the origin.

Three models were proposed for DNA replication:- Figure-6:- VARIOUS MODELS OF DNA REPLICATION

The semi conservative model is correct

Figure-7:-ROLE OF THE ACTIVITY OF THE VARIOUS ENZYMES INOVLVED IN DNA

STAGES IN DNA REPLICATION:-

 Step 1: Replication Fork Formation. Before DNA can be
replicated, the double stranded molecule must be
―unwinded‖ into two single strands.
 Step 2: Primer Binding. The leading strand is the
simplest to replicate.
 Step 3: Elongation.
 Step 4: Termination.

Figure-8:- DIRECTION OF MOVEMENT OF FORK

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Volume 6, Issue 7, July – 2021 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
ELONGATION:-
During elongation, RNA polymerase "walks" along
one strand of DNA, known as the template strand, in the 3'
to 5' direction. For each nucleotide in the template, RNA
polymerase adds a matching (complementary) RNA
nucleotide to the 3' end of the RNAstrand.

DNA is interpreted with the help of DNA polymerase

only in the 3′ to 5′ direction

Figure-9:- REPLICATION FORK BUBBLE

Primer Binding:-

Primers are portable pieces of RNA, ribonucleic acid, about

five to fifteen nucleotides long. Primase forms a short piece
of RNA that is complementary to the template DNA strand
Figure-11:- DNA ELONGATION
and forms hydrogen bonds with it. This gives DNA
polymerase the starting point it needs to initiate synthesis.
TERMINATION:-
Termination of DNA replication initiates as soon as
Okazaki fragments are small arrangements of DNA
the two replication forks coincide with the stretched DNA,
nucleotides formed abruptly and then joined together by the
whereafter the forks intersect until all intervening DNA is
enzyme DNA ligase to form the lagging strand during DNA
unwound; any remaininggaps are filled and ligated
replication. The Okazaki fragments are vital for DNA
synthesis because there is no 3' to 5' strand of DNA for the
The RTP is one of only two well-described proteins
polymerase to use as a perpetual template.
known to be involved in arresting DNA replication forks,
the other being a protein known as tus (termination
A primer is essential to start DNA synthesis by providing a
utilisation substance) from E.coli.
3' end to add nucleotides to. This is usually a combination of
Primase, a short RNA primer, and DNA Polymerase alpha,
Termination happens because the two replication
a short DNA primer..
forks coincide at the identical level of DNA, the replicating
forks broadcast until all mediating DNA is unwound; any
left over gaps are filled and joined.

Figure-10:- PRIMER BINDING

Figure-12:- REPLICATION OF TELOMERE ENDS BY
TELOMERASE ENZYME

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ISSN No:-2456-2165
Eukaryotic Chromosomes are rod shaped Results in PCR reaction is mostly visualized via gel
implementing they have terminal endings. These endings electrophoresis. Gel electrophoresis process involves
give rise to an issue for DNA replication. The DNA at the separating DNA portions conforming to their proportions.
very end of the chromosome cannot be completely copied in Applications includes various research laboratories,
each round of replication, resulting in a moderate, cautious forensics, genetic testing, diagnostics, and amplification of
tallowing of the chromosome. genes.

RNA polymerase continues transcribing unless it For proper endpoint with fast and productable results,
receives signals to terminate. the five key steps to consider:
Termination occurs when the ribosome reaches a  DNA isolation.
stop codon (UAA, UAG, and UGA). Since there are no  Primer designing
tRNA molecules that can recognize these codons, the  Enzyme selection techniques
ribosome recognizes that translation is complete. The new  Thermal cycling.
protein is then released, and the translation complex comes  Amplicon analysis and designing
apart.
V. CONCLUSION
Transcription termination occurs in a reaction
integrated to RNA 3′-end processing. Most eukaryotic DNA Synthesis and sequencing is a technique which is
mRNA precursors are splitted in a site-specific manner in used to figure out the bases in a DNA molecule. It can also
the 3′-UTR, followed by polyadenylation of the upstream be used to study the genome structure. Through this paper
cleavage product. Numerous proteins assist these reactions. we have tried to explain the various DNA sequencing
methods and also the various applications of DNA synthesis
THE CENTRAL DOGMA:- and sequencing. We have also explained many
terminologies regarding the DNA sequencing and synthesis
It is pathway of the formation DNA to RNA(in this methods. Through this review paper we have also discussed
case mRNA) to Proteins. Polymerase Chain Reaction and its applications in brief.
1: Replication Fork Formation.2: Primer Binding. New methods in understanding DNA sequencing is the key
3: Elongation. to the future aspects and applications in the field of
4: Termination. molecular biology.
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