INTROCTION TO DIAGNOSTIC

MOLECULAR BIOLOGY.
Molecular biology.
Molecular biology is a branch of biology that deals with
molecular basis of biological activity. Molecular biology deals
with the study of gene structure and functions at molecular
level to understand the molecular basis of hereditary, genetic
variation and expression patterns of genes.
So, Molecular Biology diagnostic involves a lot of gene
manipulations, such as, gene knock outs, gene insertions, gene
duplication, etc, to study behaviors of cells.
 Gene
• A gene is a basic functional and structural unit of genetic
information. In meaning of molecular genetics gene is segment
of DNA, that is transcribed and contain regulatory and coding
parts. According to the genetic information that they carry, we
can divide genes into three groups: structural genes, regulatory
genes, and genes for the RNA molecules, except mRNA. A
Structural Gene is a part of the DNA chain (sometimes an RNA
chain as in RNA viruses), which codes for the primary structure
of the proteins. The size of the genes is expressed in the
number of base pairs (bp) in the DNA, it contains. The larger
units are called kilobases (kb) or megabases (Mb).
Cont…
The longest human gene (coding for dystrophine) is 2.4 Mb
long and makes up 0.1 % of the entire human genome. The
structural gene consists of two parts: regulatory and coding
part. The regulatory part is called the promoter. It contains
important sequences (parts of base sequences, the so called
boxes), for example TATA box, CAAT box etc. Their task is
to bind regulatory proteins, the so called transcription
factors. These proteins must bind on the mentioned sequences
of the nitrogenous bases in the right order for transcription
(syntheses of mRNA) to begin, which is carried out by RNA
polymerase.
Cont…
• The prokaryotic promoter contains two important functional parts. It
is the sequence in the area of the nucleotide in the -35 location (before
the start codon), which is called the GACA box and has the following
primary structure:
5´– T T G A C A T – 3´
• The second important sequence is in the area of the nucleotide -10,
which is called the TATA box (Pribnow box) and has a sequence:
5´– T A T A A T – 3´
 Cont…
• In the area between -75 and -80 is another important box called a CAAT
box, to which the NF1 transcription factor binds, strengthening the
promoter.
5´– G G C C A A T C T – 3´
• The third box is called the GC, it is in the area - 90 and is bonded by the
transcription factor SP1, which also strengthens the promoter.
5´– G G G C G G – 3´
In the coding areas the eukaryotic genes often contain non-coding parts
(introns) in between the coding parts (exons). The coding part of the gene
starts with the untranslated region (UTR) on the 5´ end, which serves as a
connection of the mRNA to a small ribosomal subunit. Immediately after it
follows the first exon, which starts with the so called start triplet (ATG).
Cont…
• The first exon is followed by the first intron. Then proceeds another exon
etc. This area therefore contains alternating exons and introns. The last
exon ends with the so called stop triplet (TAA, TAG or TGA).
Transcription finishes on the 3´ UTR, that that continues. After it
proceeds a sequence activating the poly(A) polymerase (forming a so
called poly-A tail). In most cases the introns are longer then the exons and
the length of the gene is measured from the start triplet to the stop triplet.
 Cont…
The regulatory gene is a part of the DNA which codes for the primary
structure of the regulatory protein, which function is usually the
induction or repression of the other genes expression.
The RNA coding genes are responsible for the primary structure of the
ribosomal and transfer RNA and other types of smaller molecules of
RNA.
 GENOME
• Genome is the totality of the genetic information and encoded in the DNA
or RNA. Cells: the fundamental working units of every living organism. •
Metazoa: multicellular organisms. E.g. humans: trillions of cells. •
Protozoa: unicellular organisms. E.g. yeast, bacteria. Each cell contains a
complete copy of an organism’s genome, or blueprint for all cellular
structures and activities. Cells are of many different types (e.g. blood, skin,
nerve cells), but all can be traced back to a single cell, the fertilized egg.
 Cont…
• Cell composition: 90% water. Of the remaining molecules, dry weight are: 50%

protein, 15% carbohydrate, 15% nucleic acid, 10% lipid, 10% miscellaneous. By

element: 60% H, 25% O, 12%C, and 5%N.

• The genome is distributed along chromosomes, which are made of compressed

and entwined DNA. A (protein-coding) gene is a segment of chromosomal DNA

that directs the synthesis of a protein. The human genome is distributed along

23 pairs of chromosomes 22 autosomal pairs and 1 pair of the sex chromosome

pair, XX for females and XY for males. In each pair, one chromosome is

paternally inherited and the other is maternally inherited.

Chromosome.
• Chromosome refer to the thread like structure located in the nucleus for
storage of genetic information. All the living cells contains DNA as genetic
material (viruses can have DNA or RNA as genetic material). Genetic
material of all living organisms including viruses is made up of nucleoprotein
because DNA (nucleic acid) is found to be associated with specific proteins
hence called as nucleoprotein. It is actually this binding of DNA with
proteins, due to which DNA exists in condensed state. Packaging of DNA
helps to conserve space in the cells. Approximately, two meters of the
human DNA can fit into a cell that is only a few micrometers wide.
 Cont…
The chromosomes are found in the nucleus of the cell. In prokaryotic
organisms, the DNA is not present in the nucleus; the DNA floats in the
cytoplasm in an area called the nucleoid. Nuclear DNA of eukaryotes (plants
and animals) is present in the form of chromosomes.

Chromosomes are made up of DNA segments and each chromosome comprises

of single long duplex of DNA. Chromosomes carry all the information that
helps a cell to grow, survive and reproduce. DNA segments with specific
patterns are called genes. Chromosomes carry genes which transmit genetic
information from one generation to another. Each chromosome can contain
large number of genes. The position on which genes are present on
 Cont…
• Every species contains a fixed number of chromosomes pairs. However,
chromosomes of eukaryotes are much more complex as compared to that
of prokaryotes and viruses.

• The chromosomes vary widely between different organisms. Eukaryotic

cells have large number of linear chromosomes and cells of prokaryotes
have smaller and circular DNA. Cells may contain more than one type of
chromosome, like in most eukaryotic cells. The mitochondria and the
chloroplasts in plant cells possess their own set of chromosomes. In
nucleus of eukaryotic organism, the chromosomes are packed by proteins
to form a compact structure called chromatin.
 Cont…
• This condensation allows long molecules of DNA to fit into the cell nucleus.
Chromosomes are more condensed than the chromatin and they are essential
for cell division. They are replicated, divided and passed on to the daughter
cells, to ensure genetic diversity and survival of the progeny.

• Duplicated chromosomes contain two identical copies, known as chromatids or

sister chromatids; they are joined by a Centromere. Compaction of the
chromosomes during the cell division process, results in the four-arm structure.
Recombination of chromosome plays a vital role in genetic diversity. Incorrect
multiplication of the chromosomes may lead to mitotic failure or death of the
cell; it may lead to apoptosis and sometimes may be cancerous.
Cont….
• The term chromosome is mainly used to describe the chromosome of
eukaryotic cell. The naked DNA of prokaryotes and DNA or RNA of
viruses is sometimes broadly called prokaryotic chromosome and viral
chromosome, respectively, due to their similarity in fundamental
properties with eukaryotic chromosomes.

• But the morphology and the organization of eukaryotic chromosome is

much more complex. The morphology of chromosomes in all eukaryotes
is essentially similar-except some variations in number and size. Most of
the chromosomes in a eukaryotic cell are called autosomes which control
Cont…
• But, in addition, there are some other chromosomes which control
some specialized characteristics of an organism and are called
allosomes. Sex chromosome (X and Y) for determination of sex, B-
chromosomes, L-chromosomes, Mchromosomes, S-chromosomes
and E-chromosomes are examples of allosomes. Autosomes are
universally present in all eukaryotic-organisms, but allosomes may or
may not be present in all organisms.
Structure of Chromosome Basic Structure
of Chromosomes
1.Chromosomes are thread like bodies present in nuclei of animals and
plants.

2. They are covered with a sheath made of proteins.

3. Inside this sheath is present granular matter referred as matrix.

4. Inside the matrix, there are two threads called Chromonemata which
are the subunits of chromatids and are present during Prophase.
Cont…
5. At Metaphase, the chromosome consists of two symmetrical strands
called Chromatids.
6. Each chromosome possesses a distinct constriction called
Centromere (Primary constriction), divides into two parts and it gets
attached to the spindle network.
7. The ends of chromosome are termed as Telomeres and it protects
from deterioration or from being destroyed. 8. Some chromosomes have
another constriction called Secondary constriction.
Cont…
 Cont…
• Chromosome Number: The number of chromosomes in a given species
is usually constant containing diploid number of chromosomes in their
somatic cells and haploid (gamete or reduced) number of chromosomes in
their sex cells (sperms and ova). The number of chromosomes is variable
from one to several hundred among different species. For example- In
Ascaris megalocephalia (horse roundworm) it is 2, while in certain
protozoan (group - Aggregata), there are more than 300 chromosomes.
Cont…
Species names of some Plant & Animals Diploid (2N)
Chromosome Number
• Zea mays (Corn or maize) 20
• Triticum vulgare (common wheat) 42
• Ascaris lumbricoides (Giant roundworm) 4
• Musca domestica ( Housefly) 12
• Drosophila melanogaster (fruit fly) 8
• Homo sapiens ( Man) 46
• Macaca mulatta ( Rhesus monkey) 42
 Cont…
• Chromosome Shape: The shape of the chromosomes is changeable from
phase to phase in the continuous process of the cell growth and cell division.
During cell division chromosomes may appear in different shapes, they can
be rod shaped, twisted or spiral curved or filamentous. In the resting phase
or interphase stage of the cell, the chromosomes occur in the form of thin,
coiled, elastic and contractile, thread-like stainable structures, the chromatin
threads.
Cont…
• In the metaphase and the anaphase, the chromosomes become thick
and filamentous. Based upon the position of centromere in anaphase,
chromosomes may appear as rod-shaped, J-shaped and V-shape. Each
chromosome contains a clear zone, known as centromere or
kinetochore, along their length. The centromere divides the
chromosomes into two parts, each part is called chromosome arm.
Cont…
• Chromosome size: The size of chromosomes varies greatly in size in
organisms, it means that different organism has chromosomes of different
size. Also, in same species, different chromosome pairs have different
size. Chromosomes range, on an average from 0.5 to about 30µ in length
and from 0.2 to Зµ in diameter. The relative number of chromosomes
generally differs in the nucleus but at a time all chromosomes of a cell
may be of the same size.–
Cont…
• Plant cells normally possess larger chromosomes than animal cells.
Trillium spp. has chromosomes which may reach up to the length of
32µ at metaphase. Monocotyledon plants usually have larger
chromosomes than the dicotyledon which contain greater number of
chromosomes. Among the animals, grasshoppers, crickets, mantids,
newts and salamanders have large chromosomes
 Cont…
• Functions of chromosome.
• As carrier of genes they transmit characters from generation to generation,
i.e. parents to offspring. Chromosomes form a link between the offspring
and the parents.
• The chromosomes control the physiological and biochemical processes in
the body of the organisms.

• It is universally accepted that DNA is the genetic material, and that in

eukaryotes almost all the DNA is present in chromosomes.
Cont…
• Another very important function of chromosomes is to protect the
genetic material (DNA) from being damaged during cell division.

• The properties of chromosomes ensure a precise distribution of DNA

(genetic material) to the daughter nuclei during cell division.

• The chromosomes are capable of self-duplication. During duplication

process the DNA strands unwind.
Nucleosomes
• Nucleosomes is the basic structural unit of the DNA in the eukaryotic
chromosomes. Nucleosomes are considered as fundamental unit of
chromatin. Each nucleosome consists of core particle made up of octamer
proteins and a spacer or linker DNA made up of histone H1. Generally,
these proteins are similar in different species. Each of these proteins is
made up of 102-135 amino acids and is rich in basic amino acids like
lysine and arginine. Positively charged R group of amino acids of
proteins binds to the negatively charged phosphate group of DNA in the
ratio of 1: 1 to form deoxyribonucleic proteins (DNP).
Cont…
• There are 5000 types of non-histone proteins. Figure below shows packaging
of eukaryotic DNA along with structure of single nucleosome. If the DNA
from all 46 chromosomes in a human cell nucleus was laid out end to end, it
would measure approximately two meters; however, its diameter would be
only 2 nm. Considering that the size of a typical human cell is about 10 µm
(100,000 cells lined up to equal one meter), DNA must be tightly packaged
to fit in the cell‘s nucleus. At the same time, it must also be readily
accessible for the genes to be expressed. During some stages of the cell
cycle, the long strands of DNA are condensed into compact chromosomes.
Cont…
• There are a number of ways that chromosomes are compacted. In the first
level of compaction, short stretches of the DNA double helix wrap around a
core of eight histone proteins at regular intervals along the entire length of the
chromosome

• The DNA-histone complex is called chromatin. The bead-like, histone DNA

complex is called a nucleosome, and DNA connecting the nucleosomes is
called linker DNA. A DNA molecule in this form is about seven times shorter
than the double helix without the histones, and the beads are about 10 nm in
diameter, in contrast with the 2-nm diameter of a DNA double helix.
Cont…
• The next level of compaction occurs as the nucleosomes and the linker
DNA between them are coiled into a 30-nm chromatin fiber. This
coiling further shortens the chromosome so that it is now about 50
times shorter than the extended form. In the third level of packing, a
variety of fibrous proteins is used to pack the chromatin. These fibrous
proteins also ensure that each chromosome in a non-dividing cell
occupies a particular area of the nucleus that does not overlap with
that of any other chromosome.
Cont…
Nucleic Acid
• Nucleic Acid is the chain of nucleotides which store genetic information
in biological systems. It creates DNA and RNA, which stores
information needed for protein synthesis. Nucleic were discovered by
Friedrich Miescher in 1869.

• In the early 1880s Albrecht Kossel further purified the substance and
discovered its highly acidic properties. He later also identified the
nucleobases. In the 1920's nucleic acids were found to be major
components of chromosomes, small gene-carrying bodies in the nuclei of
complex cells.
Cont…
• Elemental analysis of nucleic acids showed the presence of phosphorus, in
addition to the usual C, H, N & O. We now know that nucleic acids are found
throughout a cell, not just in the nucleus, the name nucleic acid is still used
for such materials. A nucleic acid is a polymer in which the monomer units
are nucleotides. In 1889 Richard Altman created the term nucleic acid

• In 1938 Ashbury and Bell published the first X-ray diffraction pattern of
DNA. In 1953 Watson and Cric determined the structure of DNA (double
helix structure). The heredity material found in cells. Large molecules that are
acidic in nature. Associated with the nuclear material of cells.
Cont…
Two types of nucleic acid:

• Deoxyribonucleic Acid (DNA). Found within cell nucleus for storing

and transferring of genetic information that are passed from one cell to
other during cell division

• Ribonucleic Acid(RNA). Occurs in all parts of cell serving the

primary function is to synthesize the proteins needed for cell
functions.
Cont…
Deoxyribonucleic acid (DNA).
• DNA is the molecule composed of two chains which coil each other to
form double helix carrying genetic information used in growth,
development, functioning and reproduction of all living organisms and
many viruses. DNA is made up of deoxyribose sugar, phosphoric acid
and nitrogenous bases. DNA is responsible for all cellular activity.
Directs the production of proteins. DNA is double stranded and helical.
DNA is maintained by hydrogen bonds (weak bonds). DNA is very stable
and can survive at temperatures as high 70 C, High salt concentrations
and Acid environments.
Cont…
• Within eukaryotic cells, DNA is organized into long structures called
chromosomes. Before typical cell division these chromosomes are
duplicated in the process of DNA replication, providing a complete set
of chromosomes for each daughter cell. Eukaryotic organisms (animals,
plants, fungi and protists) store most of their DNA inside the cell
nucleus and some of their DNA in organelles, such as mitochondria or
chloroplasts.
Cont…
• In contrast, prokaryotes (bacteria and archaea) store their DNA only in
the cytoplasm. Within eukaryotic chromosomes, chromatin proteins
such as histones compact and organize DNA. These compact
structures guide the interactions between DNA and other proteins,
helping control which parts of the DNA are transcribed.
Cont…
• Components of DNA
There are three components; a pentose sugar, phosphoric acid and
nitrogenous bases which combine to form monomer unit called as nucleotide.
Large number of nucleotides joins to form polynucleotide chain. Each
nucleotide is composed of one of four nitrogen-containing nucleobases i.e.,
cytosine (C), guanine (G), adenine (A) or thymine (T), a sugar called
deoxyribose, and a phosphate group. The nucleotides are joined to one
another in a chain by covalent bonds between the sugar of one nucleotide and
the phosphate of the next, resulting in an alternating sugar-phosphate
backbone.
Cont…
• The nitrogenous bases of the two separate polynucleotide strands are
bound together, according to base pairing rules (A with T and C with G),
with hydrogen bonds to make double-stranded DNA. The three
components of DNA are:

• (1) Sugar: The sugar present in DNA is a pentose sugar (Fig. 5.2) called
as deoxyribose sugar. Its name indicates that it is derived from ribose
sugar by loss of an oxygen atom. Deoxyribose sugar joins with a
nitrogenous base to form nucleoside.
Cont…
• (2) Phosphoric acid: Phosphoric acid along with sugar molecule
forms the backbone of polynucleotide chain. The bond formed by a
phosphate between the sugar molecules of two different nucleotides is
called phosphodiester bond. In one strand of DNA helix the
phosphodiester bond is formed in the direction 3ˈ-5ˈ direction and in
the other strand of the helix phosphodiester bonds are formed in 5ˈ-3ˈ
direction.
Cont…
• (3) Nitrogenous bases: There are four nitrogenous bases
present in structure of DNA which are grouped into two
classes called as purines and pyrimidines. The complementary
nitrogenous bases are divided into two groups, pyrimidines
and purines. In DNA, the pyrimidines are thymine and
cytosine; the purines are adenine and guanine.
Cont…
Cont…
• Pyrimidines: Pyrimidines are simple aromatic compounds composed
of carbon and nitrogen atoms in a six membered, heterocyclic ring
system. The name also refers to a specific compound (composition
C4H4N2), not found in nature that can be regarded as the parental
structure of a wide range of naturally occurring chemical species. The
most abundant naturally occurring pyrimidines are uracil (2, 4-
dihydroxypyrimidine), cytosine (2-hydroxy-4-aminopyrimidine), and
thymine (2, 4- dihydroxy-5-methyl pyrimidine).
Cont…
 Cont…
• Purines. A purine is a heterocyclic aromatic organic compound that
consists of a pyrimidine ring fused to an imidazole ring. Purine gives its
name to the wider class of molecules, purines, which include substituted
purines and their tautomer’s, are the most widely occurring nitrogen-
containing heterocycle in nature. Purine is water soluble, are found in high
concentration in meat and meat products, especially internal organs such as
liver and kidney.
Cont…
• All purines contain a double-ringed structure that consists of a six-
membered ring fused to a five-membered ring; think of a honeycomb
cell attached to a pentagon. Purines are double ringed nitrogenous
bases found to be present in nucleic acids. Both DNA and RNA
contain two types of purines which are adenine and guanine.
Cont…
Ribonucleic acid (RNA)
• Ribonucleic acid (RNA). RNA is the molecule of single stranded which
has roles in coding, decoding, regulation and expression of genes. RNA
is made up of ribose sugar, nitrogenous bases and phosphate group. RNA
is a single-stranded nucleic acid polymer of the four nucleotides A, C, G,
and U joined through a backbone of alternating phosphate and ribose
sugar residues. It is the first intermediate in converting the information
from DNA into proteins essential for the working of a cell. Some RNAs
also serve direct roles in cellular metabolism.
 Cont…
• RNA is made by copying the base sequence of a section of double-
stranded DNA, called a gene into a piece of single-stranded nucleic acid.
This process, called transcription, and is catalysed by an enzyme called
RNA polymerase. RNA is very similar to DNA, but differs in a few
important structural details: in the cell, RNA is usually single-stranded.
Three types of RNA:
• mRNA messenger RNA
• tRNA transfer RNA
• rRNA ribosomal RNA
STRUCTURE OF RNA
Complementary DNA (cDNA) and Synthesis

• Complementary DNA (cDNA). Is the DNA copy of the messenger RNA

(mRNA) molecule produced by reverse transcriptase enzyme. In genetics,
complementary DNA (cDNA) is DNA synthesized from a mature
mRNA template. Eukaryotic DNA has introns, these have to be removed
and exons spliced. This spliced DNA is called complementary DNA.
• Synthesis. cDNA is most often synthesized from mature (fully spliced)
mRNA using the enzyme reverse transcriptase. This enzyme operates
on a single strand of mRNA, generating its complementary DNA based
on the pairing of RNA base pairs (A, U, G, C) to their DNA complements
(T, A, C, G).
Cont…
• To obtain eukaryotic cDNA whose introns have been spliced:

• A eukaryotic cell transcribes the DNA (from genes) into RNA (pre-
mRNA). The same cell processes the pre-mRNA strands by removing
out introns, splicing the exons and adding a poly-A 3’end and 5’ Methyl-
Guanine cap. This mixture of mature mRNA strand is extracted from the
cell. A Poly-T oligonucleotide primer is hybridized onto the poly-A
3’end of the mature mRNA template (Reverse transcriptase requires this
double stranded segment as a primer to start its operation).
Cont…
• Reverse transcriptase is added, along with deoxynucleoside
triphosphates (A, T, G, C). The reverse transcriptase scans the mature
mRNA and synthesizes a sequence of DNA that complements the
mRNA template. This strand of DNA is complementary DNA (cDNA)
Cont…

Applications

• Complementary DNA is often used in gene cloning or as gene probes

or in the creation of a cDNA library.

• Partial sequences of cDNAs are often obtained as expressed sequence

tags.
 cDNA library

 cDNA library refers to a complete, or nearly complete, set of cDNA

of all the mRNAs contained within a cell or organism.

 Because working with mRNA is difficult (as mRNA is unstable and

is easily degraded by RNases which can be found even on the skin),

 researchers use an enzyme called reverse transcriptase which will

produce a DNA copy of each mRNA strand.

 Referred to as cDNA these reverse transcribed mRNAs are

collectively known as the library.
DNA CLONING
 Cloning is the creation of an exact genetic replica of a small segment
of DNA, a cell or a whole organism.

 Identical twins are an example of human clones that are created

naturally.
 Three types of cloning technologies will be discussed:

(1) Recombinant DNA technology or DNA cloning,

(2) Reproductive cloning, and
(3) Therapeutic cloning.
Cont…
• To "clone a gene," a DNA fragment containing the gene of interest is
isolated from chromosomal DNA using restriction enzymes and then
united with a plasmid that has been cut with the same restriction
enzymes.

• When the fragment of chromosomal DNA is joined with its cloning

vector in the laboratory, it is called a "recombinant DNA molecule."

• Following introduction into suitable host cells, the recombinant DNA

can then be reproduced along with the host cell DNA.
Cont…
• Cloning is essential to enable enough copies of the gene to be
analyzed.

• This may lead to human genetic testing to diagnose genetic conditions

and predictive or pre-symptomatic genetic testing for genetic
conditions in asymptomatic individuals or prenatally.

• Gene cloning is also important for the development of drugs and

treatments such as in pharmacogenetics and gene therapy.
 Branched DNA(bDNA)
• Branched DNA is the signal amplification technology used in clinical
research laboratories to quantitatively detect nucleic acids. An overnight
incubation is a significant drawback of high sensitive (bDNA) assay.
Branched DNA (bDNA) Technology.
• Introduction
• The branched DNA (bDNA) assay provides a unique and powerful tool for
reliable quantification of nucleic acid molecules. Fundamentally different
from target amplification methods such as PCR, the bDNA assay directly
measures nucleic acid molecules at physiological levels by boosting the
reporter signal, rather than replicating target sequences as the means of
detection, and hence avoids the errors inherent in the extraction and
amplification of target sequences.
 Cont…
• The bDNA assay employs linear signal amplification using synthetic
oligonucleotide probes and bDNA molecules, and can accurately and
precisely measure between approximately 500 and 10,000,000 molecules.
• History of bDNA technology
• Well suited to routine use in a clinical or research setting, the bDNA assay
has been applied successfully in a number of areas, including the
prognosis and monitoring of patients with viral diseases. Providing a
reliable means for direct quantification of viral load in clinical specimens,
bDNA assays have been developed to measure hepatitis B virus DNA,
hepatitis C virus RNA), human immunodeficiency virus type 1 RNA, and
cytomegalovirus DNA.
Cont…
• With the custom design of oligonucleotide probes, the potential
applications of the bDNA assay reach far beyond viral nucleic acid
quantification. By creating oligonucleotide probes for specific sequences
of target nucleic acid molecules, the bDNA assay has been adapted to a
wide variety of applications. For example, researchers have designed
probes for the bDNA assay to measure cellular mRNA levels.
• Outline
• The bDNA assay uses a 96-well microplate format, and is based on a series
of specific hybridization reactions and chemiluminescent detection of
hybridized probes. Attached to the surface of each microwell are “capture”
probes that contain a specific nucleotide sequence.
Cont…
• These capture probes bind to a subset of “target probes” which are bound to
specific nucleotide sequences in the target nucleic acid molecule. This series
of hybridizations anchors the target nucleic acid molecule to the microwell
surface. Detection of the target nucleic acid and amplification of the signal is
accomplished through another series of hybridizations.

• A second subset of target probes links the target nucleic acid molecule to
bDNA amplifier molecules. With the addition of preamplifier molecules to
provide an additional layer of enhancement between the target probes and the
bDNA amplifier molecules, even greater signal amplification can be achieved.
Cont…
• Each bDNA amplifier molecule has been designed to contain 15 arms,
each of which contains three binding sites for alkaline phosphatase-
conjugated “label probes”. A chemiluminescent signal is generated
upon introduction of a dioxetane substrate which is activated by the
alkaline phosphatase. This signal is easily quantified by counting the
number of photons emitted in a luminometer. The bDNA assay is
inherently quantitative since the number of photons emitted is directly
related to the amount of target nucleic acid in the specimen.
 Cont…
• Materials
• Equipment – Chiron plate heater equipped with 8×12 microwell holder –
Chiron plate-reading luminometer
• Reagents for day 1
• Oligonucleotide-modified microwellsLysis diluentLysis reagent (proteinase
K)Target probes for captureTarget probes for labelSpecimensStandards and
controls (optional)
• Reagents for day 2
• bDNA amplifier concentrateLabel diluentLabel probe concentrateDioxetane
substrate (LumiphosPlus)10% sodium dodecyl sulphate (SDS)Wash A
(0.015 M NaCl, 0.0015 M sodium citrate, 0.1% SDS, 0.05% sodium azide,
0.05% Proclin 300Wash B (0.015 M NaCl, 0.0015 M sodium citrate, 0.05%
sodium azide, 0.05% Proclin 300)
 Cont…
• Application

• Viral quantification or viral load testing has become part of the routine
management of patients infected with HIV-1 or hepatitis C virus (HCV).
There are currently several molecular technologies that are available for
use in the clinical laboratory setting. Of these, only the branched DNA
(bDNA) assays are FDA-approved for HIV-1 and HCV viral load testing.
This signal amplification technology is built on a series of hybridization
reactions that are highly amenable to full automation and thus lessen the
amount of labor required to perform this type of analysis.
Cont…
• Molecular diagnostic assays using bDNA technology for detection of
nucleic acid target molecules are sensitive, specific, and reliable tools in
the diagnosis of viral and bacterial infections and for monitoring disease
progression during the course of therapy. bDNA tests have evolved from
developmental stages in the research laboratory to US Food and Drug
Administration–approved quantitative assays with valuable clinical
applications. The bDNA assays are less labor-intensive than many
molecular-based procedures because they are highly amenable to total
automation.
Nucleotide
• Nucleotide. This is the monomer which linked to one another to form a
polynucleotide chain in DNA molecule. Nucleotide compose of three
molecules nitrogenous base, five-carbon sugar (deoxyribose or ribose),
and one phosphate group. Nucleotides are the building blocks of nucleic
acids; they are composed of three subunit molecules: a nitrogenous base,
a five-carbon sugar (ribose or deoxyribose), and at least one phosphate
group. Nucleotide is formed by addition of phosphoric acid to
nucleoside. Hence it can be said that a nucleotide is nucleoside
monophosphate.
Cont…
Cont…

• The phosphate group gets attached to C-5 of deoxyribose sugar. Because

there are four types of nitrogenous bases, hence, there exist four types of
nucleotides which are: Deoxyadenosine monophosphate (dAMP),
Deoxyguanosine monophosphate (dGMP), Deoxycytidine
monophosphate (dCMP) and Deoxythymidine monophosphate (dTMP)
Cont…
• Deoxyadenylic acid or (dAMP) = Adenine + Deoxyribose +
Phosphoric acid
• Deoxyguanylic acid or (dGMP) = Guanine + Deoxyribose +
Phosphoric acid
• Deoxycytidylic acid or (dCMP) = Cytosine + Deoxyribose +
Phosphoric acid
• Deoxythymidylic acid or (dTMP) = Thymine + Deoxyribose +
Phosphoric acid
Nucleoside
• Nucleoside. This is the molecule formed by association of nitrogenous
base and pentose sugar. Nitrogenous bases get attached to C-1 of
pentose sugar. When a glycosidic bond is formed between sugar and
nitrogenous base, C-1 of sugar is always involved. If the nitrogenous
base is a purine, the nitrogenous base is linked to the sugar via its N-9
atom, while if it‘s a pyrimidine, it is linked via its N-1 atom. Depending
upon the type of sugar present, nucleosides are of two types;
Ribonucleosides and Deoxyribonucleosides. Because the pentose sugar
present in DNA is deoxyribose, hence, nucleoside present in DNA called
Cont…
• Deoxyribonucleosides. There are four types of deoxyribose
nucleosides present in DNA molecule:
• Deoxyadenosine = Adenine + Deoxyribose
• Deoxyguanosine = Guanine + Deoxyribose
• Deoxycytidine = Cytosine + Deoxyribose
• Deoxythymidine = Thymine + Deoxyribose
Oligonucleotide
• Oligonucleotide. These are short single strand of DNA or RNA that
serves as the starting point for molecular biology and synthesize biology
applications. Oligonucleotide primers are artificially synthesized one-
stranded DNA sections with an approximate length of 20 nucleotides,
which are complementary to the 3’ ends of both replicated strands of the
DNA. The primers determine a DNA sequence which we want to
amplify, and serve as starting points for the Taq polymerase
Cont…
• . They are chosen in a way, to ensure a specific hybridization of primer
with a unique sequence in the genome – not to bind on to multiple
places. It is also necessary to choose such sequence of primers bases,
which prevents their mutual pairing and the formation of primer
dimers. To fulfill all mentioned conditions for successful
amplification, the accurate primers are chosen in computer databases
of human genome.
Codon

• Codon. This is the DNA or RNA sequence of tree nucleotides that

contain genetic information to encode for a particular amino acid. The
genetic code is the set of rules by which information encoded in genetic
material (DNA or RNA sequences) is translated into proteins (amino
acid sequences) by living cells. The genetic code, once thought to be
identical in all forms of life, has been found to diverge slightly in certain
organisms and in the mitochondria of some eukaryotes.
Cont…
• Nevertheless, these differences are rare, and the genetic code is
identical in almost all species, with the same codons specifying the
same amino acids. The genetic code consists of 64 triplets of
nucleotides. These triplets are called codons. With three exceptions,
each codon encodes for one of the 20 amino acids used in the
synthesis of proteins. This produces some redundancy in the code;
most of the amino acids being encoded by more than one codon
Organization and Concept of Genetic Code
• Arrangement of nitrogenous bases in DNA is said to determine by the
sequence of amino acid in a protein molecule. Since, there are only 4
nitrogenous bases Adenine (A), Cytosine (C), Guanine (G) and
Thymine (T). It is obvious that sequence of these four nitrogenous
bases on DNA stand directs the synthesis of proteins. Different theories
have been proposed to predict the mechanism through which sequence
of nitrogenous bases present in DNA transcribed into mRNA, which
eventually determines the position of specific amino acids in protein.
Cont…
• Theory proposed by F.H.C Crick remains widely accepted. The theory
explains existence of genetic code and its smallest unit codon which
codes for one amino acid. A codon is defined as nucleotide sequence in
mRNA which codes for particular amino acid. When it became clear
that codon is a sequence of nucleotide which codes for amino acid, the
next question arise was how many nucleotides are present in one codon
i.e., it has to be determined the exact number of nucleotides in a codon
which codes for an amino acid. Since, there are 20 amino acids and four
nitrogenous bases in mRNA.
Cont…
• Hence, there was requirement of sufficient number of codons which could
code for 20 amino acids. If you consider that a codon consists of 1
nucleotide i.e., singlet code.

• Now in this case, since we have 4 nitrogenous bases the total number of
codon becomes 4, but how can four codons code for 20 amino acids.

• Hence, it was clear that each codon consists of more than one nucleotide.
Singlet Code = 4x1 = 4 Codons (how can 4 codons code for 20 amino
acids).
Cont…
• If we consider that a codon consists of 2 nucleotides each i.e., doublet
code for each amino acid. Now, in this case the total number of codons
becomes 16. Still the problem was not solved because by considering
a codon to be a set of two nucleotides only 16 possible codons could
be figured out and the amino acids were still 20. Doublet Code = 4x4
= 16 codons. Hence, it become clear that codon with one or two
nitrogenous bases could not provide sufficient number of codon
combinations to code for 20 known amino acids.
Cont…
• Gamow (1954) proposed possibility of a three letter code i.e., each codon
consisting of three nitrogenous bases (Triplet code). Now, in this case the
total possible combination of codon becomes 4x4x4= 64 (Table 2) Since
there are only 20 amino acids, hence, 64 codons are more than enough to
code for amino acids. Here you should also note that now total no. of
codons becomes 64 and amino acids for which they will code is 20.
Cont…
• Hence, several different codons will code for same amino acids.
Hence, genetic code is a nucleotide base sequence consisting of
codons, each codon made up of three nitrogenous bases. Genetic code
is translated into a sequence of amino acids which combine to form
proteins.
Table of composition of different codons to comprising for genetic
code
Types of Codon
• (a) Sense Codon: Those codons that code for amino acids are called
sense codons. There are 61 sense codons in the genetic code which code
for 20 amino acids.
• (b) Signal Codons: Those codons that code for signals during protein
synthesis are known as signal codons. There are four codons which code
for signal. These are AUG, UAA, UAG and UGA.
• Signal codons are of two types:
• 1. Start codons,
• 2. Stop codons.
Cont…
• 1. Start Codon (Chain Initiation Codons): Codon with nucleotide
sequence ―AUG is called as start codon. It codes for amino acid
methionine in most organisms. The process of translation (protein
synthesis) always begins with expression of start codon AUG. However,
codon AUG can occur later in mRNA also, then it will simply code for
amino acid methionine. The triplets AUG and GUG, plays double roles in
E. coli. When they occur in between the two ends of a cistron
(intermediate position), they code for the amino acids methionine and
valine, respectively in an intermediate position in the protein molecule.
Cont…
• 2. Stop Codon (Chain Termination Codons): Out of total 64 genetic
codes, the 3 triplets UAA, UAG, UGA do not code for any amino acid.
They were originally described as non-sense codons, as against the
remaining 61 codons, which are termed as sense codons. During the
process of protein Synthesis these codons function to terminate the
process.
Cont…

• When any of the three codon is read, the ribosomes pause and gets
separated from mRNA and hence the process of protein synthesis is
terminated. Due to this reason these codons are also called as
termination codons. UAA, UAG and UGA are also known as ochre,
Amber and Umber respectively, are believed to be used as signals
which end the synthesis of a protein chain.
Anticodon
• Anticodon. This refer to the sequence of nucleotides that are
complementary to the codon. They are found in tRNA. An anticodon
pairs complementary nitrogenous bases with mRNA. For example, if
mRNA has a codon AUC, it will pair with anticodon sequence UAG of
tRNA. The tRNA molecules with same anticodon sequence will always
carry the same amino acids, ensuring the consistency of the proteins
coded for in DNA.
Messenger RNA (mRNA).
• Messenger RNA (mRNA). This is the molecule formed inside the
nucleus by the transcription process from DNA. mRNA carries genetic
information from the nucleus to the cytoplasm for protein synthesis.)
Messenger RNA (m-RNA): The term messenger RNA was coined by
Francois Jacob (French biologist) and Jacques Monad (French
biochemist). Messenger RNA is a linear molecule formed inside the
nucleus by the process of transcription from DNA. The sequence of
mRNA is complementary to the template DNA strand.
Cont…
• The nitrogenous bases on mRNA strand are arranged in form of codons,
made up of three nitrogenous bases. Messenger RNA carries genetic
information from nucleus (chromosomal DNA) to cytoplasm for protein
synthesis. After being transported to cytoplasm m-RNA combines with
ribosomes to form polyribosomes or polysomes. Each polysome contains
several ribosomes (normally five) which form a complex with m-RNA.
Generally, each gene transcribes its own m-RNA therefore there are
approximately as many types of m-RNA molecules as there are genes.
The characteristic feature of m-RNA is its heterogeneous nature.
Cont…
• Messenger RNA significantly differs in their size and molecular
weight. This difference arises due to difference in size and number of
cistrons.
• Messenger RNA formed in eukaryotes is much more stable and has a
longer life span as compared to the m-RNA formed in prokaryotes
which survive for a very short time period probably for about one
minute or even less than a minute. Messenger RNA might have short
life span but to compensate it they possess high turnover number.
Messenger RNA comprises about 10% of total cellular RNA.
Immediately after formation it is transported from nucleus into
cytoplasm where it gets deposited on ribosomes.
Cont…
• Messenger RNA has been classified as monocistronic and
polycistronic. Monocistronic m-RNA is a type of m-RNA which codes
for only one protein whereas polycistronic m-RNA can synthesis more
than one type of protein from same m-RNA molecules. This is because
monocistronic m-RNA contains codons of single cistron whereas
polycistronic m-RNA contains codons for more than one cistron.
Monocistronic m-RNA is characteristic feature of eukaryotes while
prokaryotes contain polycistronic m-RNA.
Cont…
• Components of structure of m-RNA

• Messenger RNA molecule comprises of several segments namely 5ˈ cap,

non-coding region, initiation codon, coding region, termination codon,
another non coding region and 3ˈpoly(A). Messenger RNA strand begins
with a cap region present at 5ˈend of m-RNA strand. Presence of cap has
been found to be associated with binding of m-RNA molecules to
ribosomes.
• The next segment present in m-RNA sequence is a non-coding region.
The length of this region is about 10-100 nucleotides. As its name
suggests it is non coding region i.e., it does not codes for any protein.
Cont…
• Non-coding region is followed by initiation codon (AUG). Initiation
codon marks the beginning of protein synthesis. Next segment in m-
RNA strand is coding region which is translated to form a protein.

• A termination codon is present at end of each coding segment. When

this termination codon is reached the process of protein synthesis is
terminated as these codons do not code for any amino acids.
Termination codon is also called stop codon. There are three
termination codons, UAA, UAG and UGA.
Cont…
• Coding region is followed by another non coding segment. This
segment is about 50-150 nucleotides long and contains a specific
sequence AAUAAA.

• The last segment in m-RNA strand is poly (A) sequence i.e., large
number of AAA..... (About 200-250) nucleotides are present. The
addition of poly (A) sequence in m-RNA occurs inside nucleus before;
m-RNA is transported from nucleus to cytoplasm.
Cont…
Transfer RNA (tRNA)
• Transfer RNA (tRNA). This is the molecule of RNA that carries amino
acid from the cytoplasm to the ribosome for protein synthesis. Also
transfer mRNA codon to the amino acid. Transfer RNA (soluble RNA)
molecule contains 71-80 nucleotides (mostly 75) with a molecular weight
of about 25,000. There are at least 20 species of tRNA corresponding to
20 amino acids present in protein structure. The structure of tRNA (for
alanine) was first elucidated by Holley. The structure of tRNA depicted
in resembles that of a clover leaf. Transfer RNA molecule consists of a
single polynucleotide chain folded to form 5 arms.
Cont…
• These five arms are named as acceptor arm, D-arm, anticodon arm, TΨC
arm and Variable arm. Transfer RNA contains mainly four arms, each
arm with a base paired stem. Except acceptor arm, other arms have a
stem and loop it means that this arm has two segments one called as stem
in which complementary base pairing occurs and another part as a loop
in which there is base pairing. As it has already been stated that acceptor
arm has only stem and no loop. Beside this variable arm may or may not
have a stem.
• 1. The acceptor arm: This arm is capped with a sequence CCA (5′ to
3′). The amino acid is attached to the acceptor arm.
Cont…

• 2. The anticodon arm: This arm, with the three specific nucleotide
bases (anticodon), is responsible for the recognition of triplet codon
of mRNA. The codon and anticodon are complementary to each other.
3. The D arm: It is so named due to the presence of dihydrouridine.
4. The TΨC arm: This arm contains a sequence of T, pseudouridine
(represented by psi, Ψ) and C.
Cont…
• 5. The variable arm: This arm is the most variable in tRNA. Based
on this variability, tRNAs are classified into 2 categories: (a) Class I
tRNAs: The most predominant (about 75%) form with 3-5 base pairs
length. (b) Class II tRNAs: They contain 13-20 base pair long arm.
Cont…
Cont…
• Base pairs in tRNA: The structure of tRNA is maintained due to the
complementary base pairing in the arms. The four arms with their
respective base pairs are given below:
1. The acceptor arm – 7 bp
2. The TΨC arm – 5 bp
3. The anticodon arm – 5 bp
4. The D arm – 4bp.
Cont….
• Transfer RNA differs from m-RNA and r-RNA as it contains some
unusual bases beside the normal bases A, G, C and U. Many of the
unusual bases present in m-RNA are formed by methylation of usual
bases. For example Methylation of guanine forms methylguanine,
Methylation of cytosine forms methylcytosine.
• During t-RNA synthesis, first a precursor t-RNA is formed which is
processed to form mature t-RNA. Precursor t-RNA contains normal
bases and it is when precursor mRNA is modified to mature RNA,
some usual bases is also modified to unusual bases. Presence of
unusual bases is considered important because they protect t-RNA
molecule from degradative action of enzymes.
Deoxyribonucleic acid (DNA) in relation to molecular
biology.

• Molecular biology chiefly concerns itself with understanding the

interactions between the various system of the cell, including the
interactions between DNA, RNA and protein biosynthesis as well as
learning how these interactions are regulated.

• DNA is the molecule that carries information for the development and
functioning of an organism. DNA is made up of two linked strands that
wind around each other to resemble twisted ladder a shape known as a
double helix.
Cont…

• Each strand has a backbone made of alternating sugar (deoxyribose)

and phosphate group. Attached to each sugar is one of four bases:
adenine (A), cytosine(C), guanine (G), or thymine (T). Adenine bonds
with thymine and cytosine bonds with guanine. The sequence of the
bases along DNA's backbone encodes biological information, such
instructions for making a protein or RNA molecule.
Deoxyribonucleic Acid
(DNA)
Ribonucleic acid (RNA) in relation to molecular
biology.

• RNA is a nucleic acid present in all living cells that has structural
similarities to DNA. Unlike DNA, however, RNA is most often single
stranded. A RNA molecule has a backbone made of alternating phosphate
groups and the sugar, ribose rather than the deoxyribose found in DNA.
Attached to each sugar is one of the four bases:
Cont…
• : Adenine (A), uracil (U), cytosine(C), or guanine (G).

• Different types of RNA exist in cells: Ribosomal RNA (rRNA),

messenger RNA (mRNA), and transfer RNA (tRNA). In addition,
some RNAs are involved in regulating gene expression. Certain virus
uses RNA as their genomic materials.
RIBONUCLEIC ACID(RNA)
.
.Differentiate DNA and RNA structures

DNA STRUCTURE RNA STRUCTURE

Has deoxyribonucleotides monomer Has ribonucleotide monomer

Contain deoxyribose sugar Contain ribose sugar

Nitrogenous bases; Adenine, cytosine, Nitrogenous bases; Adenine, cytosine,

guanine and thymine. guanine and uracil.
Double stranded Single-stranded.

Located in the nucleus. Located in the cytoplasm.

Cont…
• The basic principle under laying this technique is that when a double
stranded DNA molecule is heated to a high temperature, the two DNA
strands separate giving rise to single stranded DNA molecules which
can be made to hybridize with small oligonucleotides primer (single
stranded) by bringing down the temperature.

• Nowadays it is one of the most significant and perspective method in

molecular genetics. The using of the PCR in practice is very broad.
PCR reaction is simulation of the DNA replication process in vitro.
Polymerase Chain Reaction (PCR).
• PCR is the laboratory technique used for amplification billions of copies
of a specific segment of DNA. PCR (polymerase chain reaction) is a
method which allows the in vitro enzymatic synthesis of specified DNA
segments, of defined length and border. It results to multiplication
(amplification) of replicated DNA sequence. The size of the DNA region
amplified during the PCR reaction typically falls within the range of 100
bp to 10 kbp. In 1983, Kary Mullis (American biochemist) invented the
polymerase chain reaction (PCR).
Cont…
• Here does not take place the synthesis of the entire DNA molecule, but
only of its small defined part - with length to 23kb. For demarcation of
replicated part, two short (oligonucleotide) chains of DNA – primers –
are required. On the base of complementarity with a target sequence,
primers hybridize with complementary sequences (sc. primer binding
sites – PBSs) denatured single strands of target DNA, which enables
the start of replication.
• The attached primers are detected by Taq DNA polymerase, which –
under rules of complementarity – synthesize the complementary
strand, until the end of the single strand. The essential condition for
PCR is that the sequence of bases on both ends of the amplified DNA
(PBSs) must be known – so it is possible pointed use of proper
primers.
Cont…
• It is important, that the complete sequence of the inner part of replicated
DNA (i.e. between PBSs) doesn’t need to be known. For instance, in case
of degraded DNA it is enough if only one of the two DNA strands is
complete.

• The process of DNA amplification by PCR works on a base of exact

changes of temperature in the environment of reaction mixture. PCR
method consists of repetition of cycles, of which each of them has three
steps: denaturation, hybridization of primers into single-stranded DNA
(annealing), and the synthesis of complementary DNA strands.
Cont…
• In each cycle the amount of the amplified segment of DNA doubles.
The growing number of repetitions leads to an exponential
multiplication of the amplified sequence. In practice, by 25-35 cycles
of repetitions it is possible to achieve a 10 4 – 106 fold amplification
(multiplication) of a specified sequence. The PCR takes place in three
steps:

• • DNA denaturation takes place under the temperature of 94° - 96° C

and lasts between 30 seconds and 2 minutes.
Cont…
• Hybridization with primers (annealing) – primers connect to the single-stranded
DNA sections on the principle of complementarity. Hybridization takes place under
the temperature 55oC - 65oC, lasting between 30 seconds and 1 minute. The
temperature and length of the step depends on the sequence, length and concentration
of primers;

• Polymerization (extension) – The synthesis of the complementary DNA strand,

bounded by the primer. The optimal temperature for this step – activity of Taq-
polymerase is 72oC. The amplification of the DNA segment is ascertained by
electrophoresis in agarose gel or polyacrylamide gel. Visualization is done by
ethidium bromide.
Cont…
• Primers. These are short, artificial DNA strands which determine the
fragment to be amplified. The fragment to be amplified is determined by
selecting primers. Primers are short, artificial DNA strands, usually 18
to 25 base pairs long. They are complementary to the beginning or the
end of the target DNA fragment to be amplified. Primers anneal by
adhering to the DNA template at the starting and ending points. Then
DNA polymerase binds and begins the synthesis of the new DNA strand.
The primers are selected based on the DNA region you want to amplify.
There are computer programs to help design primers.
Cont…
Mutation
• Mutation refer to the change to a gene sequence to produce something
different. It creates permanent change DNA sequence. A mutation is a
permanent change in the DNA sequence of a gene or the genetic code.
Mutations in a gene's DNA sequence can alter the amino acid sequence of
the protein encoded by the gene.

• How does this happen? Like words in a sentence, the DNA sequence of
each gene determines the amino acid sequence for the protein it encodes.
The DNA sequence is interpreted in groups of three nucleotide bases,
called codons. Each codon specifies a single amino acid in a protein
Cont…
• Mutations can lead to changes in the structure of an encoded protein or
to a decrease or complete loss in its expression. Because a change in
the DNA sequence affects all copies of the encoded protein, mutations
can be particularly damaging to a cell or organism. In contrast, any
alterations in the sequences of RNA or protein molecules that occur
during their synthesis are less serious because many copies of each
RNA and protein are synthesized.
Cont…
• Geneticists often distinguish between the genotype and phenotype of
an organism. Strictly speaking, the entire set of genes carried by an
individual is its genotype, whereas the function and physical
appearance of an individual is referred to as its phenotype.
Recombinant gene.

• Recombinant of gene refer to the process by which the pieces of DNA

are broken and recombined to produce new combinations of alleles. This
recombination process creates genetic diversity at the level of gene that
reflects differences in the DNA sequence of different organisms. In
eukaryotic cells, recombination occurs during meiosis. Meiosis is a form
of cell division that produces gametes or egg and sperm cells. During the
first phase of meiosis the homologous pair of maternal and paternal
chromosomes align.
Cont…
• During the alignment, the arms of the chromosomes can overlap and
temporarily fuse causing a crossover. Crossover result in
recombination and the exchange of the genetic material between the
maternal and paternal chromosomes. As a result, offspring can have
different combination of genes that their parents. Genes that are
located further apart on the same chromosomes have a great likelihood
of undergoing recombination, which means they have a great
recombination frequency.
Strand.
• Strand. This refer to a chain or single unit of the multimeric molecule
such as DNA. A DNA strand means a single chain of polynucleotide.
Template. Template refer to the RNA or single stranded DNA upon
which the complementary nucleotide strand is synthesized.
• Leading strand. This is the strand of the nascent DNA which is
synthesized in the same direction as the growing replication fork and is
synthesized almost continuously. Because DNA polymerase moves along
the parent strand in the 5' to 3' direction, replication can occur very easily
on the leading strand. Addition of nucleotides occurs in the 5' to 3'
direction.
Cont…

• Lagging strand. This is the single strand that during DNA replication
is replicated in opposite direction of the replication fork. The strand
that is being copied in the direction away from the replication fork and
is synthesized discontinuously, with small fragments (Okazaki
fragments) of DNA being copied near the replication fork.
Okazaki fragments.

• These refer to the short sequence of DNA which formed at the time of
discontinuously synthesis of the lagging strand during DNA replication.
These short stretches of discontinuous DNA, termed Okazaki fragments,
are eventually joined to become a single, continuous strand. These
fragments are stretches of 100 to 200 nucleotides in humans (1000 to
2000 in bacteria) that are synthesized in the 5' to 3' direction away from
the replication fork.
The Concept of the Central dogma
• The central dogma of molecular biology that attempt to explains the key
processes inside the nucleus of the cell, whereby the theory stating that
genetic information flows only in one direction from the replication of
DNA, later transcribed to RNA, translated directly to protein. The
information flows in one direction. The central dogma take place in two
stages:
Cont…
• 1.Transcription. Transcription is the process of creating an equivalent
RNA (mRNA) copy from a sequence of DNA in double helix by RNA
polymerase enzyme.

• The enzyme RNA polymerase transfer information from one strand of

DNA to another strand of RNA during transcription. Three part of the
DNA strand are involved in this process: the promoter, RNA structural
molecules and DNA molecules. Transcription process involve three
stages:
 Cont…

• Initiation. RNA polymerase binds to the promoter region of DNA near

the beginning of a gene, separating the double helix near the promoter.

• Elongation. RNA polymerase travels along the DNA template strand,

catalyzing the addition of ribose nucleotides into the RNA molecules.
The nucleotides in the RNA complementary to the template strand of
DNA.
Cont…
• Termination. At the end of a gene, RNA polymerase encounters a
sequence of DNA called termination signal. The RNA polymerase
detaches from the DNA and release the RNA molecule.

• After termination the DNA completely rewinds into double helix.

RNA molecules formed is free to move from the nucleus to the
cytoplasm for translation. RNA polymerase may move to another gene
and begin transcription process once again.
2. Translation.
• Translation. Translation is the process by which the genetic code
contained within the mRNA is decoded to produce a specific sequence of
amino acid in a polypeptide chain.
• Initiation begin with the tRNA bearing methionine attaching to one of the
ribosomal units. The codon of methionine is a universal "start" codon for
reading the mRNA strand. The ribosomal unit binds to mRNA where the
code for methionine (AUG). The anticodon (UAC) of the tRNA matches
the "start" codon on the mRNA. The large ribosomal subunit now binds
to the smaller unit forming a ribosomal complex. The tRNA binds first to
the active site on the ribosome. Translation may now begin.
Cont…
• The second codon in mRNA (GUU) matches the anticodon of a tRNA
carrying the amino acid valine (CAA). The second tRNA binds to the
second active site on the large subunit. A catalytic site on the large
subunit binds the two amino acids using dehydration synthesis
forming peptide bond between them.

• The first tRNA now detaches and goes to find another methionine in
the cytoplasm. The mRNA chain shifts over one codon, placing the
second codon (CAU) over the second active site.
Cont…
• A tRNA with another anticodon (GUA) matches the exposed codon
(CAU) moves onto the ribosome. This tRNA carries histidine. A new
peptide bond is formed between valine and histidine on the catalytic
site. The tRNA that carries valine detaches and find another valine.
The process continues until the ribosome finds the "stop" codon. The
subunits detach from one another, the mRNA is released, and the
polypeptide chain moves to the endoplasmic reticulum for further
processing. The methionine is removed and the chain is folded into its
final shape.
Cont…
List of enzymes involved in the DNA replication

• DNA gyrase/topoisomerase. unwinds and rewinds DNA strands to

prevent the DNA from becoming tangled or supercoiled.

• DNA Helicase. DNA helicase: Unwinds and separates double stranded

DNA as it moves along the DNA. It forms the replication fork by
breaking hydrogen bonds between nucleotide pairs in DNA.

• DNA polymerase. DNA polymerases: Synthesize new DNA molecules

by adding nucleotides to leading and lagging DNA strands.
Cont…
• DNA Primase. DNA primase: A type of RNA polymerase that generates RNA
primers. Primers are short RNA molecules that act as templates for the starting point
of DNA replication

• DNA Ligase. DNA ligase: joins DNA fragments together by forming phosphodiester
bonds between nucleotides.

• Taq Polymerase. Is the enzyme which is thermo-stable used in the amplification of

DNA copies in the Polymerase Chain Reaction (PCR). Thermo-stable DNA
polymerase, eg, Taq polymerase - Thermus aquaticus (or any other thermo-stable
polymerase). This bacteria normally lives in hot springs so can tolerate temperatures
above 90⁰C.
DNA Replication process.

• Replication. Replication refer to the process by which the double strand

DNA molecule is copied to produce two identical DNA molecules.

• DNA replication is a process in which original DNA molecule (present

in nucleus of eukaryotes and cytoplasm of prokaryotes) called as parent
DNA is replicated to form two new DNA molecules called as daughter
DNA molecules. Parental strands act as template (guide) strand for the
synthesis of daughter strands. Three different hypotheses had been
proposed for the mode of DNA replication:
Cont…
• 1. Conservative Mode: In this model, the two strands of DNA unwind
from each other, and each acts as a template for synthesis of a new,
complementary strand. This results in two DNA molecules with one
original strand and one new strand. An entirely new molecule is
synthesized from a DNA template and no change occurs in template
(parent) DNA i.e. it remains conserved.
Cont…

• 2. Semi-Conservative Model: In this model, DNA replication results

in one molecule that consists of both original DNA strands (identical
to the original DNA molecule) and another molecule that consists of
two new strands (with exactly the same sequences as the original
molecule). It means after replication each DNA molecule has one
parental strand and one newly synthesized strand.
Cont…
• 3. Dispersive Model: In the dispersive model, DNA replication results in
two DNA molecules that are mixtures, or hybrids, of parental and
daughter DNA. In this model, each individual strand is a patchwork of
original and new DNA. New molecules are made up of segments of new
and old DNA.
The complete process of DNA replication can be divided into three
steps:
1.Initiation
• The first step occurs when DNA helicase unwinds the double helix by
breaking the hydrogen bonds between the parent strands of DNA at
locations called replication origins. This opens up the DNA molecule to
form a Y-shape structure, which is called as replication fork.
Cont…
• Single stranded binding proteins (SSB) work with helicase to keep the
parental DNA helix unwound. Before the synthesis of daughter DNA
strands, an RNA primer is made by the enzyme called RNA Primase.
RNA Primase is the enzyme that builds an RNA primer on the parent
strand to initiate DNA replication. Once the RNA primer is built, then the
next enzyme, DNA polymerase, begins synthesis of DNA strands. During
the process of replication DNA polymerase is positioned behind the RNA
primer. The Topoisomerase proteins surround the unzipping strands and
relax the twisting that might damage the unwinding DNA.
Cont…
2. Elongation

• After separation of helix separated and primer synthesis DNA polymerase

starts adding complementary nucleotides and synthesis of daughter strand
begin. Addition of complementary nucleotides means that if A is present on
parental strand then nucleotide with base T will be added on daughter strand,
if G is present on daughter strand then nucleotide with base C will be added
on daughter strand and vice versa. Now at this stage where two parent strands
of DNA are unwound or separated, one strand is oriented in the 5' to 3'
direction and the other strand has opposite orientation in the 3' to 5' direction.
Cont…
• Single DNA polymerase will catalyze replication of both the strands.
DNA polymerase functions only in 5' to 3' direction. This feature
makes synthesis of daughter strands through different methods, one
adding nucleotides one by one in the direction of the replication fork,
the other able to add nucleotides only in chunks. The first strand,
which replicates nucleotides one by one, is called the leading strand;
the other strand, which replicates in chunks, is called the lagging
strand.
Cont…
• (a) The Leading Strand: Because DNA polymerase moves along the
parent strand in the 5' to 3' direction, replication can occur very easily on
the leading strand. Addition of nucleotides occurs in the 5' to 3' direction.
Triggered by RNA Primase, which adds the initial nucleotides (in form
of primer) to the new chain, the DNA polymerase moves along the fork
and keeps on adding complementary nucleotides one after the other
according to the sequence of nucleotides present on parental strand.
Synthesis of leading strand is a continuous process.
Cont…
• (b) The Lagging Strand: Whereas the DNA polymerase III on the
leading strand can simply follow the replication fork, because DNA
polymerase III must move in the 5' to 3' direction, on the lagging
strand the enzyme must move away from the fork. The lagging strand
replicates in small segments, called Okazaki fragments. These
fragments are stretches of 100 to 200 nucleotides in humans (1000 to
2000 in bacteria) that are synthesized in the 5' to 3' direction away
from the replication fork.
Cont…
• For synthesis of each Okazaki fragment a new primer is made before
the segment is replicated. After replication of over these primers are
degraded. Now in the vacant spaces DNA is synthesized by DNA
polymerase I. These fragments are then stitched together by DNA
ligase, creating a continuous strand. The synthesis of lagging strand is
called discontinuous

• 3. Termination. After elongation is complete, two new double helices

have been made from the original parental DNA molecule.
Cont…
• DNA REPLICATION
Significance of the molecular diagnostic techniques.

• The rate of diseases gene discovery is increased exponentially, which

facilitates the understanding diseases at molecular level.

• To introduce essential concepts in the molecular diagnostics that has

impact on the identification of human diseases.

• To develop and apply useful molecular assays to monitor diseases,

determine appropriate treatment strategies and predict diseases outcome.
Cont…

• Molecular understanding of diseases is translated into diagnostic

testing, therapeutics and eventually preventive therapies.

• To expand scientific knowledge of the natural history of diseases,

identifying people who are at risk for acquiring specific diseases and
diagnosis of human diseases at nucleic acid level.
Recombinant gene and its clinical utility
• Recombinant DNA is the exchange of genetic information between
DNA segments of the same species. With the advancement of
technology one can transfer genes of one species to another artificially.
The technology of recombinant DNA was developed in 1973 by Boyer
and Cohen.

• Recombinant DNA is the DNA from two distinct species injected into
the host organism to create new genetic combinations useful in science,
medicine, agriculture and industry. Laboratory genetics their primary
purpose is to identify, define and modify genes.
Cont…
• Consider each human cell has about 2metres of DNA. As a result, even
a small piece of tissue can contain thousands of kilometers of DNA.
Recombinant DNA technology has made possible to isolate a single
gene or other section of DNA, allowing researchers to establish its
nucleotide sequence, investigate its transcripts, and reintroduce the
transformed sequence into a living creature. The recombinant DNA
technology is also called genetic engineering.
 Cont…
• Tools of rDNA Technology

Recombinant DNA technology has the implementation tools which are:

restriction enzymes aids in cutting, polymerase aid in synthesizing, and
ligase aid in binding. In recombinant DNA technology, restriction enzymes
play an important role in deciding where the desired gene is inserted into the
vector genome. Endonucleases and Exonucleases are types of Restriction
enzymes. Exonucleases remove the nucleotides off the end of the strand
while endonucleases cut within the DNA strand. Restriction endonucleases
are sequence specific enzymes that cleave DNA at specified locations and are
Cont…
• They examine the length of DNA and cut it at a specified location known
as the restriction site. As a result, the sequence has a stickly end the
complimentary stickly notes are obtained by cutting a desired gene and
vectors with the same restriction enzymes, enables the work of the ligases
to bind the desired gene to the vectors much easier.

• The vectors aid in the transport and integration of the desired gene. These
are the ultimate vehicles that transfer the desired gene into the host
organism hence there are a crucial part of the recombinant DNA
technology toolkit.
Cont…
• Because of their large copy number, plasmids and bacteriophages are the
mostly common employed vectors in the recombinant DNA Technology.
These vectors are made up of origin of the replication, which the
nucleotide sequence from which replication begins, a selectable marker,
which are genes that shows antibiotic resistance and cloning sites which
are sites recognized by restriction enzymes where desired DNAs are
inserted. The recombinant DNA is injected to the host organism.
Cont…
• The host is the most powerful instrument in the recombinant DNA
technology as it uses enzymes to take in the vectors that has been
modified with the desired DNA.

• These recombinant DNAs are injected into the host in a variety of ways
including microinjection, biolistic or gene gun, alternate cooling and
heating, calcium ion usage and soon
 Cont…
• Steps of Recombinant DNA Technology

1. DNA isolation. DNA is isolated in its pure form, which means they are
devoid of other macromolecules. In rDNA technology, the initial step is to
extract the desired DNA in its purest form , that is free of extractaneous
macromolecules. Because DNA coexists with other macromolecules such as
RNA, polysaccharides, protein, and lipids within the cell membrane, it must be
separated and purified using enzymes such as lysozomes, cellulase, chitinase,
ribonuclease, and proteases. Other enzymes or treatments can remove other
macromolecules. The DNA eventually precipitates out as fine thread as a result
of the presence of ethanol. After that the pure DNA is spooled out.
Cont…
2. cutting of DNA/ Restriction enzymes. The restriction enzymes are quite
vital. It helps to identify the location wherein a designated gene is introduced into
the vector genome. The reaction known as restriction enzymes digestions. The
entail incubating the pure DNA with the restriction enzyme of choice at a
condition that are appropriate for that enzyme. The Agarose Gel Electrophoresis
technology displays the restriction enzyme digestion’s progress. This method
entails passing the DNA across an agarose gel. When current is applied
negatively charged DNA flows to the positive electrode and is divided into
different sizes. This permits the digested DNA fragments to be separated and
snipped out. The same method is used to process the vector DNA.
 Cont…
• Joining of DNA. The vector and a section of DNA are joined in this step. It is
achieved with the help of the enzyme DNA ligase. With the same restriction
enzymes, the pure DNA and the vector DNA of interest are cut. This yield the cut
DNA fragment and the cut vector, both of which are now open. Ligation is the
process of putting r=these two parts together with the enzyme DNA ligase. The
resulting DNA molecule is a hybrid of the interest molecule and the vector DNA
molecules. Recombination is the term used in genetics to describe the merging of
different DNA strands. As a result, this new hybrid DNA molecule is known as a
recombinant DNA molecule, and the process in known as recombinant DNA
technology.
 Cont…
• Insertion of rDNA into the Host. Here rDNA is added to the recipient
host cell, and the entire process is called transformation. Post insertion,
the recombinant DNA multiplies and manifests as manufactured protein
under favorable conditions. The recombinant DNA is then transferred
into the recipient host cell, commonly a bacterial cell in this stage. The
term of the procedure is transformation. Bacterial cells have a hard time
to accept foreign DNA. As a result, the treatment are given to make them
capable of accepting new DNA. Thermal shock, Ca++ ion therapy,
electroporation, and other procedures may be applied
Cont…
• Recombinant DNA Technology
 Cont…
Application of Recombinant DNA Technology.

• To produce recombinant HB vaccines

• For producing human insulin

• To facilitate better crop production

• For producing growth hormones in human to treat dwarfism

• For better gene therapy

• To acquire DNA fingerprinting

• To diagnose several types of diseases.

 Nucleic Acid amplification-based techniques
for pathogen detection and identification

• Nucleic acid amplification technique has become a common place in clinical

settings; the resulting rapid identification allows the patient to be placed on
a specific antimicrobial therapy and avoid prolonged management on
empiric, potentially inappropriate drugs.

• Nucleic acid amplification-based techniques are generally specific and

highly sensitive and can be used for all categories of microbes.
Cont…

• Results can be provided rapidly, because each test is typically specific to a

single organism. The clinician must know the diagnostic possibilities and
request test accordingly.

• For example, if a patient has symptoms suggesting influenza but the

influenza season is over, doing a more general viral diagnostic test (eg,
viral culture) rather than a specific flu test is better because another virus
(eg, parainfluenza, adenovirus) may be the cause.
Cont…

• Nucleic acid amplification techniques take tiny amount of DNA or

RNA, replicate them many times, and thus can detect minute traces of
an organism in a specimen. These techniques are particularly useful
for organisms that are difficult to culture or identify using other
methods (eg, viruses, obligate intercellular pathogens, fungi,
mycobacteria, some other bacteria) or that are in low numbers.
Cont…
Nucleic acid amplification techniques for pathogen detection and
identification includes:

• Polymerase Chain Reaction (PCR)

• Ligase Chain Reaction (LCR)

• Nucleic acid sequence-based amplification (NASBA)

• Transcription-mediated amplification (TMA)

• Strand displacement amplification (SDA).