Unit 3 Basic concept of nucleic acid and recombinant DNA technology

Nucleotides
Nucleotides are the building blocks of nucleic acids. They are composed of three components: a nitrogenous base
(adenine, thymine, cytosine, or guanine), a sugar molecule (ribose or deoxyribose), and a phosphate group.
Nucleotides are linked together through phosphodiester bonds to form nucleic acids, such as DNA and RNA.
The nucleotides present in DNA and RNA are:
DNA Nucleotides:
Deoxyribose Sugar: DNA nucleotides contain a deoxyribose sugar molecule, which is a five-carbon sugar with
one oxygen atom missing compared to a ribose sugar.
Nitrogenous Bases: DNA nucleotides contain four different nitrogenous bases:
Adenine (A): It pairs with thymine (T) in DNA.
Thymine (T): It pairs with adenine (A) in DNA.
Cytosine (C): It pairs with guanine (G) in DNA.
Guanine (G): It pairs with cytosine (C) in DNA.
Phosphate Group: DNA nucleotides also have a phosphate group attached to the 5' carbon of the sugar molecule.
The phosphate group is responsible for linking the nucleotides together through phosphodiester bonds to form the
DNA backbone.
RNA Nucleotides:
Ribose Sugar: RNA nucleotides contain a ribose sugar molecule, which is a five-carbon sugar with one more
oxygen atom compared to deoxyribose sugar.
Nitrogenous Bases: RNA nucleotides also have four different nitrogenous bases:
Adenine (A): It pairs with uracil (U) in RNA.
Uracil (U): It pairs with adenine (A) in RNA instead of thymine.
Cytosine (C): It pairs with guanine (G) in RNA, like DNA.
Guanine (G): It pairs with cytosine (C) in RNA, like DNA.
Phosphate Group: RNA nucleotides have a phosphate group attached to the 5' carbon of the sugar molecule, just
like DNA nucleotides.
Nucleosides
Nucleosides are like nucleotides but lack the phosphate group. They consist of a nitrogenous base and a sugar
molecule (ribose or deoxyribose) bonded together. Nucleosides are involved in various biological processes,
including the synthesis of nucleic acids. DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are both
nucleic acids composed of nucleotides, but they differ in their nucleotide composition.
Nucleic acids
Nucleic acids are complex macromolecules that store and transmit genetic information in living organisms. There
are two types of nucleic acids: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA carries the genetic
instructions necessary for the development, functioning, and reproduction of all known organisms. RNA is
involved in protein synthesis and other cellular processes.

Gene
A gene is a segment of DNA or RNA that contains the instructions for building one or more functional molecules,
usually a protein. Genes are the basic units of heredity and determine the traits and characteristics of an organism
by controlling the synthesis of specific proteins through the process of transcription and translation. Each gene
has a specific location on a chromosome and is composed of a sequence of nucleotides that encodes the
information needed for protein production.

Genome
A genome refers to the complete set of genetic material present in an organism. It includes all the genes, as well
as non-coding DNA sequences. The genome contains the instructions necessary for an organism's growth,
development, and functioning. In humans, the genome is comprised of the DNA contained within the 23 pairs of
chromosomes. It can vary in size and complexity across different organisms.

Chromosome
Chromosomes: Chromosomes are structures within cells that contain DNA and carry genes. They are thread-like
structures composed of DNA tightly coiled around proteins called histones. Chromosomes are visible during cell
division when they condense and become easily observable under a microscope. Humans have 46 chromosomes
(23 pairs) in most cells, except for sex cells (sperm and egg cells), which have 23 individual chromosomes.
Chromosomes are essential for the organization, replication, and transmission of genetic information during cell
division and reproduction.

Difference between DNA and RNA

RNA (ribonucleic acid) and DNA (deoxyribonucleic acid) are two types of nucleic acids that play crucial roles in
the storage and transmission of genetic information. While they share some similarities, they also have several
distinct characteristics. The differentiation between RNA and DNA is
1. Structure
• DNA: DNA is a double-stranded helix composed of two antiparallel strands, each consisting of a
sugar-phosphate backbone and nucleotide bases. The bases in DNA are adenine (A), thymine (T),
cytosine (C), and guanine (G).
 • RNA: RNA is typically single-stranded, although it can fold back on itself to form complex
secondary structures. It also has a sugar-phosphate backbone, but the sugar in RNA is ribose
instead of deoxyribose found in DNA. The bases in RNA are adenine (A), uracil (U), cytosine (C),
and guanine (G). RNA lacks thymine (T) and instead has uracil (U) as one of its bases.
2. Function
• DNA: DNA is the genetic material that carries the instructions necessary for the development,
growth, functioning, and reproduction of living organisms. It serves as a long-term storage
molecule for genetic information.
• RNA: RNA acts as an intermediary between DNA and protein synthesis. It plays multiple roles,
including messenger RNA (mRNA) that carries the genetic instructions from DNA to the
ribosomes, transfer RNA (tRNA) that helps in the translation of mRNA into proteins, and
ribosomal RNA (rRNA) that forms the structural and catalytic components of ribosomes.
3. Stability
• DNA: DNA is more stable and less prone to degradation. It has a relatively long lifespan and is
well-suited for the storage and preservation of genetic information.
• RNA: RNA is generally less stable and more susceptible to degradation. It has a shorter lifespan
and is involved in more transient processes within the cell.
4. Copying and Replication:
• DNA: DNA undergoes a process called DNA replication, where the two strands of DNA separate,
and each strand serves as a template for the synthesis of a new complementary strand. This process
ensures the faithful transmission of genetic information from one generation to the next.
• RNA: RNA is synthesized through a process called transcription, which involves the synthesis of
an RNA molecule using DNA as a template. This RNA molecule can be further processed and
modified to perform its specific functions within the cell.
5. Occurrence:
• DNA: DNA is found in the nucleus of eukaryotic cells and the mitochondria and chloroplasts. It
is the primary genetic material in most organisms.
• RNA: RNA is found in the nucleus, cytoplasm, and other cellular compartments. It is present in
all living organisms, and certain viruses exclusively contain RNA as their genetic material.

The Watson-Crick model of DNA

The Watson-Crick model of DNA, proposed by James Watson and Francis Crick in 1953, describes the structure
of the DNA molecule and its mechanism of replication. Their model is based on X-ray crystallography data
obtained by Rosalind Franklin and Maurice Wilkins, which revealed the helical nature of DNA. The key features
of the Watson-Crick model are as follows:
Double Helix Structure: The Watson-Crick model describes DNA as a double-stranded helix, where two DNA
strands are twisted around each other in a right-handed manner. The helix resembles a twisted ladder or a spiral
staircase.
Complementary Base Pairing: The DNA strands are held together by hydrogen bonds between specific
nitrogenous bases. Adenine (A) forms two hydrogen bonds with thymine (T), while cytosine (C) forms three
hydrogen bonds with guanine (G). This complementary base pairing ensures that A always pairs with T, and C
always pairs with G.
Antiparallel Arrangement: The two DNA strands in the double helix run in opposite directions, referred to as
antiparallel. One strand runs in the 5' to 3' direction, while the other runs in the 3' to 5' direction. This arrangement
ensures that the base pairing occurs in a precise manner.
Sugar-Phosphate Backbone: The DNA strands are composed of a sugar-phosphate backbone, with the sugar
(deoxyribose) and phosphate groups alternating along the length of the strands. The nitrogenous bases project
inward, forming the rungs of the ladder.
Major and Minor Grooves: The helical structure of DNA gives rise to two grooves along the length of the double
helix. The larger groove is called the major groove, while the smaller groove is called the minor groove. These
grooves provide access points for proteins and other molecules that interact with DNA.
DNA Replication
DNA replication is the process by which a cell makes an exact copy of its DNA. It is a crucial step in cell division,
ensuring that each daughter cell receives a complete set of genetic information. The process of DNA replication
can be summarized in the following steps:
1. Initiation: DNA replication begins at specific sites on the DNA molecule called the origins of replication.
Enzymes called helicases unwind and separate the two DNA strands, forming a replication fork.
2. Primer Binding: Primase, an enzyme, synthesizes a short RNA primer that provides a starting point for
DNA synthesis. The primer binds to the DNA template strand at the replication fork.
3. Elongation: DNA polymerase enzymes catalyze the addition of new nucleotides to the growing DNA
strands. The enzyme responsible for the bulk of DNA synthesis is DNA polymerase III in prokaryotes and
DNA polymerase α, δ, and ε in eukaryotes. These enzymes read the DNA template strand and add
complementary nucleotides to the new DNA strand.
• Leading Strand: The leading strand is synthesized continuously in the 5' to 3' direction, matching
the template strand's 3' to 5' direction. DNA polymerase synthesizes this strand continuously.
• Lagging Strand: The lagging strand is synthesized discontinuously in small fragments called
Okazaki fragments. DNA polymerase synthesizes each Okazaki fragment separately, moving away
from the replication fork. Primers are required for the synthesis of each Okazaki fragment.
4. Primer Removal and DNA Repair: Once the new DNA strands are synthesized, the RNA primers need
to be removed and replaced with DNA nucleotides. An enzyme called DNA polymerase I in prokaryotes
(DNA polymerase δ in eukaryotes) removes the RNA primers and fills the resulting gaps with DNA
nucleotides.
5. Proofreading and Editing: DNA polymerases have proofreading capabilities, allowing them to detect
and correct errors in DNA replication. Mismatched nucleotides are removed, and the correct nucleotides
are inserted.
6. Strand Joining: The gaps between the Okazaki fragments on the lagging strand are sealed by an enzyme
called DNA ligase. DNA ligase forms phosphodiester bonds between adjacent nucleotides, joining the
fragments into a continuous DNA strand.
7. Termination: The replication process continues bidirectionally until the entire DNA molecule is
replicated. At the termination stage, specific DNA sequences called termination sites signal the completion
of replication. The newly synthesized DNA molecules separate from the template DNA.

By the end of DNA replication, two identical DNA molecules are produced, each containing one original strand
and one newly synthesized strand. This process ensures the accurate transmission of genetic information from
parent cells to daughter cells during cell division.
Transcription
Transcription is the process by which genetic information encoded in DNA is converted into an RNA molecule.
It is a key step in gene expression, where the information stored in DNA is used to produce functional RNA
molecules, such as messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). The process
of transcription involves several stages:
1. Initiation: Transcription begins with the binding of an enzyme called RNA polymerase to a specific region
of DNA called the promoter. The promoter sequence signals the starting point for transcription. RNA
polymerase separates the DNA strands, creating a transcription bubble.
2. Elongation: Once the DNA strands are separated, RNA polymerase synthesizes an RNA molecule using
one of the DNA strands as a template. It adds complementary RNA nucleotides to the growing RNA chain
in the 5' to 3' direction. The RNA molecule is synthesized in an antiparallel manner to the template DNA
strand, resulting in a complementary RNA strand.
3. Termination: Transcription continues until a termination signal is reached on the DNA template.
Termination signals cause the RNA polymerase to detach from the DNA template, and the newly
synthesized RNA molecule is released.
4. Post-transcriptional Processing: In eukaryotic cells, the RNA molecule undergoes additional processing
steps before it becomes functional. These processes include:
• Capping: A modified guanine nucleotide (5' cap) is added to the 5' end of the RNA molecule. The
cap protects the RNA from degradation and is involved in various cellular processes.
 • Polyadenylation: A stretch of adenine nucleotides (poly-A tail) is added to the 3' end of the RNA
molecule. This poly-A tail also helps in stabilizing the RNA and regulating its transport and
translation.
• Splicing: In eukaryotes, many genes contain non-coding regions called introns within the coding
regions called exons. RNA splicing removes the introns and joins the exons together to produce a
mature, processed RNA molecule called messenger RNA (mRNA) that carries genetic information
to the ribosomes for protein synthesis.
5. mRNA Export and Translation: Once processed, mRNA is transported out of the nucleus and into the
cytoplasm. There, it serves as a template for protein synthesis during the process of translation, where the
mRNA is read by ribosomes to produce a specific protein.
Transcription is a vital process that allows the information stored in DNA to be transcribed into functional RNA
molecules, which in turn play critical roles in various cellular processes, including protein synthesis and gene
regulation.

Translation
Translation is the process by which the genetic information carried by an mRNA molecule is used to synthesize
a protein. It occurs in the ribosomes, cellular structures where proteins are assembled. The process of translation
involves several steps:
1. Initiation: Translation begins with the binding of the mRNA molecule to the small ribosomal subunit.
This interaction is facilitated by a specific sequence on the mRNA called the start codon, which is usually
AUG (adenine-uracil-guanine). The start codon specifies the amino acid methionine and signals the
beginning of protein synthesis.
2. Elongation: Once the mRNA is bound to the small ribosomal subunit, the large ribosomal subunit joins
to form a functional ribosome. The ribosome moves along the mRNA in a 5' to 3' direction, reading the
codons (three-nucleotide sequences) on the mRNA. Transfer RNA (tRNA) molecules carry specific amino
acids to the ribosome. Each tRNA molecule has an anticodon that pairs with the codon on the mRNA,
 ensuring the correct amino acid is added to the growing protein chain. The ribosome catalyzes the
formation of peptide bonds between adjacent amino acids, resulting in the elongation of the protein chain.
3. Termination: The process of elongation continues until the ribosome encounters a stop codon on the
mRNA. Stop codons (UAA, UAG, or UGA) do not code for any amino acids but instead, signal the
termination of protein synthesis. When a stop codon is reached, release factors bind to the ribosome,
causing the release of the completed protein chain.
4. Protein Folding and Modifications: After translation, the newly synthesized protein may undergo
additional modifications. These modifications can include folding into a specific three-dimensional
structure, post-translational modifications such as phosphorylation or glycosylation, or the addition of
prosthetic groups. These modifications are crucial for the protein to attain its functional conformation and
perform its specific role in the cell.
The process of translation enables the genetic code carried by mRNA to be decoded into a specific sequence of
amino acids, resulting in the synthesis of a functional protein. The sequence of amino acids determines the
structure and function of the protein, playing a vital role in various cellular processes and functions.

Types of RNA
There are several types of RNA (ribonucleic acid) molecules that play different roles in the cell. The major types
of RNA include:
Messenger RNA (mRNA): mRNA carries the genetic information from DNA to the ribosomes, where it serves
as a template for protein synthesis. It is transcribed from a DNA template during the process of transcription and
undergoes further processing, such as the addition of a 5' cap and a poly-A tail before being translated into protein.
Transfer RNA (tRNA): tRNA molecules play a key role in protein synthesis by bringing amino acids to the
ribosomes. Each tRNA molecule has a specific sequence of nucleotides that binds to a complementary codon on
the mRNA, carrying the corresponding amino acid. This process ensures the correct assembly of amino acids
during protein synthesis.
Ribosomal RNA (rRNA): rRNA is a major component of ribosomes, which are the cellular machinery
responsible for protein synthesis. Multiple rRNA molecules combine with proteins to form ribosomes, where they
provide the structural scaffold for protein synthesis and catalyze the formation of peptide bonds between amino
acids.
Small Nuclear RNA (snRNA): snRNA molecules are involved in the splicing of pre-mRNA, which is the
removal of non-coding introns and the joining of coding exons. They form complexes with proteins called small
nuclear ribonucleoproteins (snRNPs) to carry out these splicing reactions.

MicroRNA (miRNA): miRNAs are small RNA molecules that regulate gene expression by binding to specific
mRNA molecules. They can inhibit translation or promote degradation of the target mRNA, thereby controlling
the levels of specific proteins in the cell. miRNAs have important roles in development, cell differentiation, and
various cellular processes.
Small Interfering RNA (siRNA): siRNAs are similar to miRNAs in structure and function. They are involved
in the regulation of gene expression by targeting and degrading specific mRNA molecules. siRNAs are also
utilized as research tools for gene silencing and gene function studies.
Long Non-coding RNA (lncRNA): lncRNAs are RNA molecules that are longer than 200 nucleotides and do
not encode proteins. They have diverse functions, including the regulation of gene expression, chromatin
organization, and the formation of cellular structures. lncRNAs play important roles in development, disease
processes, and epigenetic regulation.

Recombinant DNA technology

Recombinant DNA technology, also known as genetic engineering or gene cloning, is a powerful set of techniques
used to manipulate and modify DNA molecules. It involves the artificial combination of DNA fragments from
different sources to create novel genetic combinations that do not occur naturally. This technology has had a
profound impact on various fields, including medicine, agriculture, and biotechnology. Here are the key steps and
applications of recombinant DNA technology:
1. Isolation of DNA: The first step in recombinant DNA technology is to isolate the DNA of interest from
its source. This can involve extracting DNA from cells, tissues, or organisms using various extraction
methods.
 2. Cutting DNA: Once the DNA is isolated, specific DNA fragments of interest are generated by using
restriction enzymes. These enzymes recognize specific DNA sequences (restriction sites) and cleave the
DNA at those sites, producing fragments with sticky ends or blunt ends.
3. Plasmid or Vector Preparation: A plasmid or vector is a small, circular DNA molecule that can replicate
independently within a host cell. It is used to carry and replicate the recombinant DNA construct. The
plasmid is isolated and prepared by cutting it with the same restriction enzyme used to generate the DNA
fragments. The DNA fragments and the cut plasmid have complementary sticky ends, allowing them to
be joined together.
4. DNA Ligation: The cut DNA fragments and the cut plasmid are mixed with DNA ligase, an enzyme that
catalyzes the joining (ligation) of DNA fragments. The sticky ends of the DNA fragments hybridize with
the complementary sticky ends of the plasmid, forming a recombinant DNA molecule.
5. Transformation: The recombinant DNA molecule is introduced into host cells, usually bacteria or yeast,
through a process called transformation. The host cells take up the recombinant DNA, and some of the
cells become transformed and contain the recombinant DNA molecule.
6. Selection and Screening: Host cells containing the recombinant DNA molecule are selected and screened
to identify those that have taken up and successfully replicated the recombinant DNA. This is often done
by incorporating selectable markers, such as antibiotic-resistance genes, into the plasmid. Only the
transformed cells that carry the recombinant DNA will survive in the presence of the selective agent.
Applications of Recombinant DNA Technology:
1. Production of Recombinant Proteins: Recombinant DNA technology allows for the production of
valuable proteins, such as hormones, enzymes, and antibodies, in large quantities. These proteins can be
used for medical, diagnostic, and industrial applications.
2. Genetic Engineering of Plants: Recombinant DNA technology has been used to genetically modify
plants for various purposes, such as improving crop yield, pest resistance, and nutritional content.
3. Gene Therapy: Recombinant DNA technology plays a crucial role in gene therapy, which aims to treat
genetic disorders by introducing functional genes into the patient's cells.
4. Production of Transgenic Animals: Recombinant DNA technology enables the introduction of foreign
genes into animals, creating transgenic animals that can serve as models for studying human diseases or
producing valuable substances in their milk or eggs.
5. DNA Forensics: Recombinant DNA techniques, such as DNA profiling or fingerprinting, are widely used
in forensic science for identifying individuals and solving criminal cases.
6. Vaccine Development: Recombinant DNA technology has revolutionized vaccine development by
enabling the production of safer and more effective vaccines. It allows for the expression of specific viral
or bacterial antigens, which can stimulate an immune response without causing disease.
Recombinant pharmaceutical products and their uses
Recombinant pharmaceutical products are medications or therapeutic substances that are produced through
recombinant DNA technology. This technology involves inserting specific genes into host organisms, such as
bacteria, yeast, or mammalian cells, to produce large quantities of proteins with therapeutic value. Here are some
examples of recombinant pharmaceutical products and their uses:

Insulin: Recombinant human insulin is widely used in the treatment of diabetes. It is produced by inserting the
human insulin gene into bacteria or yeast cells, which then produce the insulin protein. Recombinant insulin helps
regulate blood sugar levels in individuals with diabetes who cannot produce sufficient insulin naturally.

Growth Hormones: Recombinant human growth hormone (rhGH) is used to treat growth hormone deficiency
in children and adults. It is produced by introducing the human growth hormone gene into host cells. rhGH
promotes skeletal and muscular growth, and its therapeutic use extends to conditions such as Turner syndrome
and short bowel syndrome.

Erythropoietin (EPO): Recombinant erythropoietin is used to stimulate red blood cell production in individuals
with anemia, particularly those with chronic kidney disease or undergoing chemotherapy. It is produced by
introducing the EPO gene into mammalian cells. Recombinant EPO helps increase red blood cell count,
improving oxygen-carrying capacity and reducing the need for blood transfusions.

Factor VIII and IX: Recombinant factors VIII and IX are used in the treatment of hemophilia A and B,
respectively. Hemophilia is a genetic disorder characterized by a deficiency in clotting factors. Recombinant
clotting factors are produced by introducing the corresponding genes into host cells, enabling the production of
the missing clotting factors.

Monoclonal Antibodies: Monoclonal antibodies are used in various therapeutic applications, including the
treatment of cancer, autoimmune diseases, and inflammatory disorders. They are produced by introducing genes
that code for specific antibodies into host cells, typically mammalian cells. Examples of recombinant monoclonal
antibodies include trastuzumab (Herceptin®) for HER2-positive breast cancer and adalimumab (Humira®) for
rheumatoid arthritis.

Vaccines: Recombinant DNA technology is employed in the production of certain vaccines. For instance,
recombinant hepatitis B vaccines are developed by introducing the hepatitis B virus surface antigen gene into
yeast cells. Recombinant vaccines offer a safer and more scalable alternative to traditional vaccine production
methods.
These are just a few examples of pharmaceutical products produced through recombinant DNA technology. The
field of biotechnology continues to advance, leading to the development of novel recombinant products with
diverse therapeutic applications.

The application of gene sequencing and polymerase chain reaction

Gene sequencing and polymerase chain reaction (PCR) are two powerful techniques that have revolutionized
molecular biology and have numerous applications in various fields. Here's a discussion of their applications:
Gene Sequencing:
Genomic Research: Gene sequencing is used extensively in genomic research to decipher the complete DNA
sequence of an organism's genome. It enables the identification and study of genes, regulatory elements, and
genetic variations associated with diseases and traits.
Medical Diagnostics: Gene sequencing plays a crucial role in diagnosing genetic disorders. It helps identify
disease-causing mutations, enabling early detection, personalized medicine, and genetic counseling.
Cancer Genomics: Sequencing cancer genomes helps in understanding the genetic alterations driving cancer
development, progression, and drug resistance. It aids in the development of targeted therapies and precision
medicine approaches.
Evolutionary Studies: Comparative genomics and sequencing of ancient DNA provide insights into evolutionary
relationships, migration patterns, and genetic adaptations of species over time.
Microbial Genomics: Sequencing the genomes of microbes helps in understanding their pathogenicity, and
antibiotic resistance, and developing diagnostic tools.

Polymerase Chain Reaction (PCR):

DNA Amplification: PCR allows the amplification of specific DNA regions from a small amount of starting
material. It has applications in research, diagnostics, and forensic analysis, where a limited DNA sample needs to
be amplified for further analysis.
Genetic Testing: PCR-based techniques, such as allele-specific PCR and reverse transcriptase PCR (RT-PCR),
are used in genetic testing for identifying specific genetic variations associated with diseases or genetic traits.
Infectious Disease Diagnosis: PCR-based assays are widely used for the rapid and sensitive detection of
pathogens, including viruses, bacteria, and parasites. PCR enables early detection, accurate diagnosis, and
monitoring of infectious diseases.
Forensic Analysis: PCR is a critical tool in forensic DNA analysis. It enables the identification and comparison
of DNA profiles from crime scenes, missing persons, and suspects.
Environmental Studies: PCR-based techniques, such as quantitative PCR (qPCR), are used to detect and
quantify microbial communities in environmental samples. It helps in studying microbial diversity, tracking
pollutants, and monitoring ecosystem health.
Both gene sequencing and PCR have significantly advanced our understanding of genetics, disease mechanisms,
and biological processes. They have become indispensable tools in research, clinical diagnostics, forensics, and
various other fields, enabling precise and targeted analysis of DNA and RNA molecules.