Overview

Molecular Biology and Genetics ●

Central Dogma of Molecular Biology
●
DNA, RNA and Protein structure and
function
Module 2 ●
Mendelian Genetics
●
rDNA Technology and Genome Editing

… in other
Central Dogma of Molecular Biology
words
• “The central dogma deals with
the detailed residue-by-residue • Protein information
transfer of sequential cannot flow back to
information. It states that such nucleic acids
information cannot be
transferred back from protein to
either protein or nucleic acid.” • Fundamental
framework to
Francis Crick,
1958 understanding the
transfer of sequence
information between
biopolymers
The flow of
information Molecular Biology
• Each species has a uniquely fundamental set of
genetic information, its genome.
• The genome is composed of one or more DNA
(deoxyribonucleic acid) molecules (46 in human
beings), each organized as a chromosome.
• Prokaryotic genomes are mostly single circular
chromosomes.
• Eukaryotic genomes consist of usually two sets
of linear chromosomes confined to the nucleus.
• A gene is a segment of DNA that is transcribed
into a RNA molecule used to make proteins.
• Introns interrupt many eukaryotic genes.
• Viral genomes consist of either DNA or RNA.

Central Dogma of Molecular Biology (in flowchart form)

The Human Genome Chromosomes & Genes

DNA
double
helix
(2-nm • A gene is certain region of
diameter)
DNA which is converted
Histones
during a process called
“Beads on transcription into an
a string”
intermediate sequence of
chemically distinct
nucleotides called an RNA
Nucleosome (different types such as
(10-nm diameter)
mRNA, tRNA, etc.) In a
process called translation,
RNA is then used to
produce proteins that can
be used by the cell to
Tight helical fiber
(30-nm diameter)Supercoil
maintain its activity. The
• 22 pairs of chromosomes called autosomes (200-nm diameter) entire process is sometimes
• Two sex chromosomes (X,Y): XY in males and XX in females called the “central dogma”
700
of molecular biology.
nm

Metaphase chromosome
 The “Central Dogma” in
Prokaryotes and Eukaryotes
• Prokaryotic and Eukaryotic gene structure is different
Structure of
Genes
• Prokaryotic and
Eukaryotic gene
structure is
different

Introns and Exons in The “Central Dogma” of Molecular Biology

Genes • Replication: DNA copies itself
into two identical strings
although copying errors may
occur called mutations.
• Transcription: A gene is
converted into an intermediate
sequence of chemically distinct
nucleotides called an RNA
(different types such as mRNA,
tRNA, rRNA, etc.).
• Translation: RNA is further
decoded to produce the
functional activity of a gene
which usually takes the form of a
protein.
•Exons: coding regions of genes
•Introns: noncoding regions (“junk” DNA)
 DNA has four kinds of bases: A, T, C,
DNA Basics and G
 DNA molecules consist of double helix strands, which are antiparallel
 complementary base pairing rules:
 adenine (A) only pairs with thymine (T), and guanine (G) only pairs with cytosine (C)
 thepairs of bases form base pairs (bp)
 reversecomplementation of s = AGCTAAC in the 5’ → 3’ direction is =
GTTAGCT

Thymine (T) Cytosine (C) Adenine (A) Guanine (G)

Pyrimidines Purines

DNA and RNA are polymers of nucleotides

DNA Helix molecule
• General - double stranded
structure: - complementary
Nitrogenous
base
(A, G, C, or T)
- helical
Phosphat
e - antiparallel
group

Thymine
(T)
• Strands: - backbone of alternating
phosphate and deoxyribos units

• Double helix: - due to base pairing: A=T

and G – C (triple bond)
Sugar • Major and Minor groove
(deoxyribos
e)

DNA
Polynucleotid nucleotide
e Sugar-phosphate
backbone
 RNA is also a nucleic acid DNA
double

The Basics: Additional Points

helix
(2-nm
●
RNA has a slightly different sugar: ribose rather than deoxyribose diameter)
●
RNA has U instead of T to bind with A
●
RNA does not form a double helix; three-dimensional structure of • Almost always read in 5' and 3' Histones
“Beads on
RNA is far more varied than that of DNA direction a string”

• DNA and RNA are dynamic - 2°

Nitrogenous base
(A, G, C, or U) structure
Nucleosome
Phosphate • Not all DNA is found in (10-nm diameter)
group
chromosomes
• Mitochondria
• Chloroplasts
Uracil (U) • Plasmids Tight helical fiber
(30-nm diameter)Supercoil
• BACs and YACs (200-nm diameter)

• Some extrachromosomal DNA can

be useful in Synthetic Biology 700
nm
Sugar
(ribose)
Metaphase chromosome

DNA replication

The process of copying double-

Initiation of replication
stranded DNA molecules

• Start point: -only one (1) on the chromosome (300 bp)

- origin (ori)
• Semi -new DNA molecules contain:
conservative: 1 old strand and
ori
1 new strand

• use a ’template’:
- one of the strand is used

• ’primers’: -usually a piece of RNA

- DNA-polymerase unable
to start replication • bidirectional: - both directions
 DNA replication

Synthesis of DNA
• ’leading’ and ’lagging’ strands:
- leading: continous synthesis
- lagging: dis-continous synthesis

• proof-reading:
- checking if any mitakes has been made
- pol. III removes the wrong nucleotides
(3’ → 5’)

DNA Replication: Enzymes involved Transcription Basics

- DNA helicase Unwinding the molecule • RNA molecule is synthesized from a segment of DNA that includes a gene
- DNA gyrase Open up (cut) the • RNA nucleotides are similar to DNA nucleotides but have a (slightly)
(topoisemerase II) strands different backbone. In particular, DNA and RNA are composed of repeating
units of nucleotides. Each nucleotide consists of a sugar, a phosphate and a
- DNA-binding Protect ss-DNA from nucleic (nitrogenous) acid base. The sugar in DNA is deoxyribose. The sugar
enzymes nucleases in RNA is ribose, the same as deoxyribose but with one more OH (oxygen-
- Synthesises the RNA hydrogen atom combination called a hydroxyl).
Primase primer
- DNA-plymerase I Removes the primer GATTACA
Repair any missing bp in DNA

- DNA-plymerase II cleans up Okazaki fragments

- DNA-plymerase III Synthesis in direction 5’→ 3’

CUAATGU
There are 3 enz. in E. coli; pol I, II and III
•T is replaced with U (U =
- DNA ligase Makes a phospho-di-ester bond Uracil) in DNA to RNA
(glueing)
 RNA polymerase

Transcription DNA of gene

Promoter
DNA Terminator
DNA

• Process of copying DNA to RNA Initiation

• Differs from DNA synthesis in that only

one strand of DNA, the template
strand, is used to make mRNA Elongation Area shown
in Figure 10.9A
• Does not need a primer to start
• Can involve multiple RNA polymerases
• Divided into 3 stages
• Initiation Termination
Growing
• Elongation RNA

• Termination
Completed RNA

RNA
polymerase

Eukaryotic RNA is processed before leaving

the nucleus
 Noncoding segments ExonIntron Exon Intron Exon
DNA

called introns are Cap

Transcription
Addition of cap and tail

spliced out - Splicing RNA

transcript
 A cap and a tail are with cap Introns removed Tail
and tail

added to the ends –

PTM (Post Exons spliced together

translational mRNA

Modifications) Coding sequence

NUCLEUS

CYTOPLASM
 Translation Role of different RNAs
RNA  protein • Messenger RNA (mRNA): Encodes protein sequences. Carries
coded instructions for protein synthesis (translation). Each three-
nucleotides group, called a codon, translates to an amino acid (the
protein building block).
• Transfer RNA (tRNA): Decodes the mRNA molecules to amino
acids. It connects to the mRNA with one side and holds the
appropriate amino acid on its other side.
• Ribosomal RNA (rRNA): Part of the ribosome, a machine for
translating mRNA to proteins. It catalyzes (like enzymes do) the
reaction that attaches the hanging amino acid from the tRNA to the
amino acid chain being created.

The genetic code

• Codons
The Codon
• It is instructions for making a protein, a series • Notice that several codons
of three nucleotides on the mRNA, called a
codon. code for the same amino
• Each codon signifies: start, stop, or an amino acid.
acid. • For example, Proline has 4
• The first codon is the start codon, and codons.
usually coincides with the Amino Acid • AUG is the START codon.
Methionine. (M which has codon code ‘ATG’)
• The last codon is the stop codon and does • This signals the start of a
NOT code for an amino acid. It is sometimes protein.
represented by ‘*’ to indicate the ‘STOP’ • UAA, UAG, & UGA are the
codon.
3 STOP codons.
• A coding region (abbreviation CDS) starts at
the START codon and ends at the STOP
• These signal the end of a
codon. protein.
• There are 64 possible codons
 The Genetic Code
Of the 64 codons, 61 specify one of the 20 amino Transfer RNA (tRNA)
acids. The other 3 codons are chain-terminating
codons and do not specify any amino acid. AUG,
one of the 61 codons that specify an amino acid, is
• Brings amino acids to the ribosome so
used in the initiation of protein synthesis. it can build proteins
Note the degeneracy of the genetic code. Each • It has Anticodons at the bottom
amino acid might have up to six codons that specify
region
it.
• Different organisms have different frequencies of • 3 nucleotide sequence complementary
codon usage. to the mRNA codon
How do tRNAs recognize to which codon to bring an • The anticodon binds to a codon to
amino acid? The tRNA has an anticodon on its
mRNA-binding end that is complementary to the
hold an amino acid in place for
codon on the mRNA. Each tRNA only binds the binding to other amino acids.
appropriate amino acid for its anticodon. • The anticodon has an mRNA sequence
complementary to the codon.
• For example, the codon for Leucine is
UUA, so its anticodon is AAU.

The Translation Process

Role of the Ribosome
1. A ribosome attaches to the AUG
(start) codon on an mRNA chain.
• Ribosomes are made of rRNA and either 2. A tRNA with the anticodon UAC binds
float in the cytoplasm or stick to the ER. to the start codon.
• The tRNA is carrying the amino acid
• Free floating ribosomes produce protein for Methionine.

use inside the cell. 3. Enzymes help form peptide bonds

between amino acids
• ER Ribosomes make proteins that will be 4. The ribosome moves down the mRNA
exported to other cells. and tRNA keeps bringing amino acids.
• Ribosomes have 3 binding sites; 1 to bind to 5. This continues until a stop codon is
reached
mRNA and 2 to bind to tRNAs so their 6. When the ribosome reaches a stop
anticodons can pair with mRNA codons. codon translation stops.
7. mRNA is released from the ribosome
and the new protein is sent out for
use.
 Overview
●
Central Dogma of Molecular Biology
●
DNA, RNA and Protein structure and Molecules
function of
Life
●
Mendelian Genetics
●
rDNA Technology and Genome Editing

37 38

Composition of Living Matter Percentage

compositions
 All living organisms are made up of of chemical
chemical substances of both organic
and inorganic types. elements in
 Except, water, all major molecules living matter
found in living system contain carbon
atom and these complex carbon-
containing molecules are called as
organic molecules such as protein,
lipids etc.
 Of the 118 known elements, only the
first 98 are known to occur naturally
on Earth.
 Out of known 98 naturally occurring
elements only about 30 elements are
essential to living organisms.

39 40

39 40
 Composition of Living Matter
The six most abundant elements in living organisms
which together make up over 99% of the mass of most
cells include carbon, hydrogen, oxygen, nitrogen,
phosphorous and sulphur.
When these six major elements combined in various
ways, form virtually all known organic biomolecules.
Covalently linked carbon atoms in biomolecules can
Nucleic Acid
form linear chains, branched chains, and cyclic
structures. To these carbon skeletons are added groups
of other atoms, called functional groups, which confer
specific chemical properties on the molecule.
There are four general classes of macromolecules within
living cells: nucleic acids, proteins, polysaccharides, and
lipids.
41 42

42

Nucleic Acid Nucleic Acid

Types
Nucleic acids were discovered by Friedrich
Deoxyribose nucleic acid (DNA):
Miescher in 1869.  Double stranded contain deoxy ribose sugar.

Nucleic acids are the most important biological Ribose nucleic acid (RNA) :
 Single stranded contain ribose sugar.
macromolecules which is essential for all living Size
organisms. Nucleic acids can vary in size, but
They function in encoding, transmitting and are generally very large molecules.
expressing genetic information. Nucleic acid molecules range in size
from 21 nucleotides (small
Nucleic acids constitute the genetic material interfering RNA) to large
present in all living systems. chromosomes (human chromosome
This genetic material carries the information about 1 is a single molecule that contains
247 million base pairs).
the reconstruction of living organism which is in the Biopolymer:
form of a code, called Genetic Code.
Nucleic acid is the largest
This code is essential to regulate and propagate life.
biopolymer consists of monomeric
43 units called nucleotides. 44

43 44
 Nucleotide Polymer
Nucleotides
 Nucleic acids are long chain molecules formed of several
 Pyrimidines are monocyclic nitrogenous bases
while Purines are bicyclic bases.
repeating units called Nucleotides.  The base pairing of the nitrogen bases are
 The Nucleotides are composed of 3 important chemical specific.
components:  Adenine (A) pairs with Thymine (T) in DNA while in
 Sugar: It is a pentose sugar (5 carbon atoms) having a RNA it pairs with Uracil (U) and
pentagonal ring structure. It is Deoxyribose sugar in DNA  Cytosine (C) pairs with Guanine (G).
while in RNA it is Ribose sugar.  Nucleotides are joined together by a
 Phosphate: It is present as Phosphoric acid (H3PO4). Condensation Reaction between the
 Nitrogen Bases: Based on their chemical structure they Phosphate Group of one and the Sugar Group of
are grouped into 2 classes: another.
 Purines which include Adenine (A) and Guanine (G) and
 The bond between the phosphate from the 5 ́ ́
 Pyrimidines which include Cytosine (C), Thymine (T) and Uracil
(U).
position of one nucleotide and the 3 ́ ́ hydroxyl
of the preceding nucleotide called a
Phosphodiester Bond.
 The ribose-phosphate backbone has the same
shape for both A:T pairs and G:C pairs.
 Consequently, all nucleic acids have a free end
composed of the 5’ phosphate group and a 3’
hydroxyl group. The nucleic acid strands have a
5’ to  3’ orientation.
45  Nucleosides: referred to a molecule composed 46

45 of a sugar and a nitrogenous base having a 46

glycosidic linkage. It includes Adenosine,
Guanosine, Thymidine and Cytidine.

DNA & RNA Structure

Deoxyribonucleic acid (DNA)
 DNA containing the genetic instructions used in
the development and functioning of all known
living organisms
 The DNA segments carrying this genetic
information are called genes.
 DNA consists of two long polymers of simple
units called nucleotides, with backbones made of
sugars and phosphate groups joined by ester
bonds.
 These two strands run in opposite directions to
each other and are therefore anti-parallel.
 This information is read using the genetic code,
which specifies the sequence of the amino acids
within proteins.
 Within cells DNA is organized into long
structures called chromosomes.
 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.
 In contrast, prokaryotes (bacteria and archaea)
store their DNA only in the cytoplasm.
47 48

47 48
 Ribonucleic acid (RNA) RNA Types
 Messenger RNA (mRNA) are of
RNA contains the bases A, G, C, and U variable size, depending on the
instead of A, G, C, and T. protein to be manufactured, and
RNA exists as a single strand instead of contain the information that specifies
which protein should be made.This
a double strand. message is a sequence of RNA
 There are three primary types of RNA. nucleotides that is complementary too
They are the template strand of DNA.
 Ribosomal RNAs (rRNA) are
1. messenger RNA (m-RNA)
relatively long RNA strands (hundreds
2. ribosomal RNA (r-RNA) or thousands of nucleotide residues)
3. transfer RNA (t-RNA). that combine with proteins to form
The different kinds of RNA perform ribosomes, the multisubunit
complexes in which protein synthesis
different functions
takes place.
RNA makes protein synthesis  Transfer RNA (tRNA) are the
possible: The three RNA make it smallest of the three types (73-93
possible for the encoded information nucleotide residues), and they carry
carried by the DNA to be put to use in the correct amino acid to the site of
49 protein synthesis. 50
the synthesis of proteins.
49 50

Transfer RNA (t-RNA) RNA makes protein synthesis possible

 Transfer RNA are the smallest of other
RNA molecules
 They have 74-95 nucleotide residues
 Forms 15 % of total RNA
 They transfer the amino acids from
cytoplasm to the protein synthesizing
machinery, hence the name tRNA
 It acts as Acceptor for proper activated AA
and adaptors for carrying AA.
 They are also called Adapter molecules,
since they acts as adapters for the
translation of the sequence of nucleotides
of the mRNA in to specific amino acids.
 There are at least 20 species of tRNA
where one corresponding to each of the 20
AA required for protein
51 52

51 52
 RNA Functions DNA V/S RNA
 The central dogma of molecular biology suggests that DNA maintains
the information to encode all of our proteins, and that three different
types of RNA rather passively convert this code into polypeptides, Characteristic RNA DNA
through the process of transcription and translation.
 However, in the half-century (1953) since the structure of DNA was Bases RNA does not contain the DNA does not contain the
first elaborated, scientists have learned that RNA does much more base thymine; instead, it base uracil; instead it
than simply play a role in protein synthesis. substitutes the base uracil substitutes the base
 For example, many types of RNA have been found to be catalytic--that is, thymine
they carry out biochemical reactions just like enzymes do.
 Furthermore, many other varieties of RNA have been found to have
complex regulatory roles in cells. Strands of RNA is typically single DNA is typically double
 Thus, RNA molecules play numerous roles in both normal cellular Nucleotides stranded stranded
processes and disease states. Generally, those RNA molecules that do
not take the form of mRNA are referred to as noncoding, because Oxygens RNA has one more oxygen in DNA has one less oxygen
they do not encode proteins. the ribose than DNA in the ribose than RNA
 The involvement of noncoding mRNAs in many regulatory processes,
their abundance, and their diversity of functions has led to the
hypothesis that an "RNA world" may have preceded the evolution of
DNA and proteins (Gilbert, 1986).
53 54

53 54

PROTEINS
 Proteins are organic molecules consisting of
multiple amino acids bonded together by
peptide bond. Thus, Proteins are biopolymer of

Proteins
amino acids.
 They are formed by the condensation
polymerization of monomeric units; α-amino
acids which bonded by peptide linkage.
Biomolecules  Proteins differ from one another primarily in
their sequence of amino acids, which is
dictated by the nucleotide sequence of their
genes, and which usually results in protein
folding into a specific three-dimensional
structure that determines its activity.
 Peptide: When amino acids join together they
create a biopolymer, called as peptide.
 The polypeptide will always have NH2 at one
end, the N terminus and COOH at the other,
the C terminus
 The nature of the 'R' group determines how
hydrophilic or hydrophobic the amino acid is.
55  There are total twenty α-amino acids which 56

55
involve in the formation of proteins in living 56
systems
 20 Amino Acids

Why Proteins Important? Amino Acids (AA)

1. Alanine
2. Arginine
3. Asparagine
 After carbohydrates, proteins are essential biomolecules for living beings. They often constitute the
4. Aspartic acid
 There are 20 different amino acids found in nature. 5. Cysteine
majority share (60%) of the dry weight of cells.
 Amino acids are building block of all the proteins 6. Glutamic acid
 The main sources of protein are milk, cheese, pulses, fish etc.
molecules. 7. Glutamine
 They are vital chemical substances which are essential for the growth and maintenance of the life. 8. Glycine
 An amino acid structure composed of a central chiral
 They perform variety of functions in living organisms, some are given below: 9. Histidine
 Enzyme catalysis - carbon atom (c-alpha) to which attached the following 10. Isoleucine
 faciliates/speeds up certain chemical reactions; eg. enzymes three groups: 11. Leucine
 Amylase converts starch to simple sugar.
a) Amino group (NH2) and 12. Lysine
 Defense - 13. Methionine
 recognizes foreign microbes; forms the center of the immune system; b) Carboxyl group (COOH) 14. Phenylalanine
 eg. immunoglobulins, toxins, antibodies (Globular proteins that "recognize" foreign microbes. ) c) R-group: variable - 20 known R-groups, so only 20 15. Proline
 Transport -
AA 16. Serine
 moves certain small molecules/ions; eg. hemoglobin, proton pump
 The sequence or the chain of joined amino acids is called 17. Threonine
 Hemoglobin is red blood cell protein.
18. Tryptophan
 Structure/ support - as Peptide. 19. Tyrosine
 structural role; eg. fibers, collagen (most abundant protein in vertebrates), keratin, fibrin
 Naming Conventions: 20. Valine
 Collagen, which forms the matrix of skin, ligaments, tendons and bones.
 Motion -  Dipeptide: When 2 amino acids joined by peptide bond or
 a muscle protein responsible for muscle contraction linkage.
 contracting muscles; eg. actin, myosin  Tri-peptide: When a peptide composed of 3 amino acids .
 Regulation -  Oligopeptide: When a peptide composed of 3 to 10 amino
 receives/sends information to regulate body functions; ex. hormones
acids .
 Hormones which serve as intercellular messengers; eg. Insulin (blood sugar regulation).
 Polypeptide: When a peptide composed of more than 10
 Storage -
 holds molecules such as calcium and iron; ex. ferritin 57 amino acids. 58

57 58

Amino acid Properties

 These 20 different amino acids arrange in
specific orders to form proteins. The AA
sequence is very crucial for functional Structure
assignment.
 The side chain decides the property of a given of
protein
 Nonpolar amino acids have CH2 or CH3 as side Amino acid
group
 Polar amino acids have oxygen or hydrogen as side
group
 Charged amino acids have acids/bases as side group
 Aromatic amino acids have organic rings w/
alternating single/double bonds as side group

59 60

59 60
 Classification of Amino Acids Essential and Non-Essential Amino Acids
 Amino acids are classified into different ways based on  Out of the twenty amino acids, nine amino acids cannot
Non-
polarity, structure, nutritional requirement, metabolic fate, be synthesized by human body and must be supplied by Essential
essential
food to body and called as essential amino acids and amino acids
etc. amino acids
those amino acids which can be synthesized in our body
 Generally used classification is based on polarity. Based on in adequately amount are called as Non-essential amino
polarity, amino acids are classified into four groups as follows: acids.
 These essential amino acids are required for the growth Histidine Alanine
1. Non-polar amino acids (Hydrophobic)
 Glycine, Alanine, Valine, Leucine, Isoleucine, Proline ( Hydrophobic- of the body and their deficiency causes diseases like Arginine
Isoleucine
aliphatic)
kwashiorkor in which the water balance of living body
 Phenylalanine, Tryptophan, Methionine ( Hydrophobic-aromatic) get disturbed and some of the organs of the body
Leucine Asparagine
become watery and bloated.
2. Polar amino acids with no charge (Hydrophilic)  Some essential amino acids such as Arginine is Lysine Aspartic acid
 Serine, Threonine, Glutamine, Cysteine, Aspargine, Tyrosine synthesized in body but with a very low rate which is not Methionine Cysteine
3. Polar amino acids with positive charge (Basic and sufficient for the necessary amount of needed amino
acids. Phenylalanin
Hydrophilic) e
Glutamic acid
 Histidine, Lysine and Arginine  Some amino acids are complementary to each other,
hence one required for the growth of another one. Like Threonine Glutamine
4. Polar amino acids with negative charge (Acidic and
Methionine is required to produce Cysteine and Tryptophan Glycine
Hydrophilic) Phenyalanine is needed to form tyrosine.
 Aspartic acid and Glutamic acid. Valine Proline
61 Serine 62

61 Tyrosine 62

Protein structure - shape determines function

 Protein Structure (Shape) is determined
Protein Structure
through x-ray diffraction or NMR. The complexity of protein structure is
 Internal amino acids are generally nonpolar
best analyzed by considering the
 Most polar/charged amino acids are found on
molecule in terms of four
the surface
 Different levels of structure - primary, secondary,
organizational levels, namely primary,
motifs, tertiary, domains, quaternary secondary, tertiary and quaternary.
Primary structure
Factors of protein shape
 Hydrogen bonds between amino acids
Secondary
 Disulfide bridges between side chains structure
 Ionic bonds ( Electrostatics)
Tertiary structure
 Van der Waals attractions (weak attractions due
to electron clouds) Quaternary
 hydrophobic exclusion
 polar portions gather on the outside, nonpolar
structure
portions go towards the interior

63 64

63 64
Ramachandran Plot or [φ,ψ] plot
 Ramachandran plot is a way to visualize energetically
allowed regions for backbone dihedral angles ψ against
Properties of Proteins
φ of amino acid residues in protein structure.  Solubility in Water
 G N Ramachandran used computer models of small  The relationship of proteins with water is complex. The secondary structure of proteins
polypeptides to systematically vary phi and psi with the
depends largely on the interaction of peptide bonds with water through hydrogen
objective of finding stable conformations.
bonds.
 For each conformation, the structure was examined for
 Hydrogen bonds are also formed between protein (alpha and beta structures) and
close contacts between atoms. Atoms were treated as
hard spheres with dimensions corresponding to their van water. The protein-rich static ball are more soluble than the helical structures.
der Waals radii. Therefore, phi and psi angles which  At the tertiary structure, water causes the orientation of the chains and hydrophilic
cause spheres to collide correspond to sterically radicals to the outside of the molecule, while the hydrophobic chains and radicals tend
disallowed conformations of the polypeptide backbone. to react with each other within the molecule (i.e. hydrophobic effect).
 In the diagram the red regions correspond to
 The solubility of proteins in an aqueous solution containing salts depends on two
conformations where there are no steric clashes, ie
these are the allowed regions namely the alpha- opposing effects on the one hand related to electrostatic interactions ("salting in") and
helical and beta-sheet conformations. other hydrophobic interactions (salting out).
 The yellow areas show the allowed regions if slightly
shorter van der Waals radi are used in the calculation, ie  Denaturation
the atoms are allowed to come a little closer together.  A protein is denatured when its specific three-dimensional conformation is changed by
This brings out an additional region which corresponds
to the left-handed alpha-helix. breaking some bonds without breaking its primary structure. It may be, for example,
 The white areas correspond to conformations where the disruption of helix areas.
atoms in the polypeptide come closer than the sum of  The denaturation may be reversible or irreversible. It causes a total or partial loss of
their van der Waals radi. These regions are sterically biological activity.
disallowed for all amino acids except glycine which is  There are a number of Denaturing agents as follows:
unique in that it lacks a side chain.  Physical agents: Heat, radiation, pH
 Glycine has no side chain and therefore can adopt phi
 Chemical agents: Urea solution which forms new hydrogen bonds in the protein, organic
and psi angles in all four quadrants of the
solvents, detergents
Ramachandran plot. 65 66

65 66

Overview Mendelian Genetics

●
Central Dogma of Molecular Biology ●
Mendelian genetics is the branch of genetics
that outlines how traits and inherited
●
DNA, RNA and Protein structure characteristics are passed from one
and function generation to the next, based largely on the
foundational work of Gregor Mendel in the
●
Mendelian Genetics 19th century.
●
rDNA Technology and Genome ●
Mendel's experiments with pea plants
established many fundamental principles of
Editing heredity.
67 68
Key Concepts of Mendelian Genetics Key Concepts of Mendelian Genetics

●
Mendel's Laws of Inheritance: ●
Alleles:
– Law of Segregation: Each organism carries two – Alleles are different versions of a gene. For
alleles for each trait (one from each parent), and example, a gene for flower color may have a purple
these alleles segregate during gamete formation. allele (dominant) and a white allele (recessive).
This means that offspring receive one allele from – Dominant Alleles: Mask the expression of recessive
each parent. alleles in heterozygous conditions (e.g., if an
– Law of Independent Assortment: Genes for individual has one purple allele and one white allele,
different traits are inherited independently of one the purple color will be expressed).
another, assuming the genes are located on different – Recessive Alleles: Only express their effect when
chromosomes. This leads to genetic variation. two copies are present (homozygous recessive).

69 70

Key Concepts of Mendelian Genetics Key Concepts of Mendelian Genetics

●
Genotype and Phenotype: ●
Monohybrid vs. Dihybrid Crosses:
– Genotype: The genetic makeup of an organism (e.g., PP, Pp, pp, – Monohybrid Cross: Examines the inheritance of a single trait
where “P” may indicate a dominant allele and “p” a recessive allele). (e.g., flower color).
– Phenotype: The observable traits or characteristics of an organism
(e.g., purple or white flowers).
– Dihybrid Cross: Examines the inheritance of two traits
simultaneously (e.g., flower color and seed shape).
●
Punnett Squares:
– A tool used to predict the genotypic and phenotypic ratios of offspring
●
Test Cross:
from a particular genetic cross. For instance, a cross between two – A breeding experiment used to determine the genotype of an
heterozygous purple flower plants (Pp x Pp) would yield a Punnett individual showing a dominant phenotype. By crossing it with
square showing the possible combinations of alleles (PP, Pp, Pp, pp) a homozygous recessive individual, the offspring’s
and their respective phenotypic ratios (3 purple : 1 white). phenotypes reveal whether the dominant individual is
homozygous or heterozygous.

71 72
 Importance of Mendelian Genetics Overview
●
Foundation of Genetic Science: Mendel's principles laid ●
Central Dogma of Molecular Biology
the groundwork for modern genetics, influencing
subsequent research on DNA, the structure of genes, and
genetic variation.
●
DNA, RNA and Protein structure and
●
Understanding Heredity: These principles are crucial for function
predicting inheritance patterns in both plants and animals,
aiding in breeding practices and genetic counseling. ●
Mendelian Genetics
●
Basis for Genetic Disorders: Mendelian genetics helps in
understanding how certain genetic disorders are passed
●
rDNA Technology
from parents to offspring, allowing for better diagnosis and
treatment.
●
Genome Editing
73 74

Genetic Engineering.....
 A molecular genetic technique used for the direct manipulation,
alteration or modification of genes or genome of organisms in order to
manipulate the phenotypes is called genetic engineering
Genetic Engineering /  In this technique, a recombinant DNA is constructed and inserted into
Recombinant DNA (rDNA) the host genome using a vector. Or we can delete some mutant

Technology
sequences from a genome. The first recombinant DNA was constructed
by Paul Berg in 1972.

In conjunction with his studies of the tumor virus SV40, in

1972, Paul Berg succeeded in inserting DNA from a
bacterium into the virus' DNA. He thereby created the first
DNA molecule made of parts from different organisms.
This type of molecule became known as "hybrid DNA" or
"recombinant DNA".

Paul Berg
 Genetic Engineering techniques.... Process and Method: rDNA Technology
 Recombinant DNA- A recombinant DNA technology is a type of
genetic engineering technology in which an artificial DNA molecule is
constructed by ligating two different DNAs using physical methods.
For that, the gene of interest is inserted into the plasmid vector and
used for gene transfer experiments.

 Gene delivering- Gene delivering technique is

employed for the insertion of a gene of interest
into the host genome.
 Electrophoration, sonication and viral vector-
mediated gene transfer, liposome-mediated
gene transfer, transposon-mediated gene
transfer are some of the methods used for that.

 Gene editing- A gene-editing technique is used to edit the genome in which

an undesired DNA sequence is removed or a new gene can be inserted into
the host genome. CRISPR-CAS9, TALEN and ZFN are some known gene-
editing tools used in gene therapy experiments.

Selecting and isolating the candidate gene Selection and construction of plasmid:
 Special character: The gene must contain a sequence of DNA that we want to  The plasmid DNA is a circular, double-stranded cytoplasmic DNA of the bacteria that
study and for that, a gene has some special characteristics. A candidate gene replicate independently.
should have high GC content and a lower repetitive DNA sequence.
 Size: The gene of interest must not be too long- only a few kb genes can be  Selecting plasmid for the genetic engineering experiment is one of the crucial steps in
successfully inserted. Longer the gene higher the chance of failure. The candidate the entire experiment. Before selecting the plasmid, we must understand why the
gene must have a start and stop codon in it. plasmid is used in the gene transfer experiments.

 Scientists are using it as a vehicle for transferring the gene of interest to the target
 Isolation: Now, the gene of interest
location in the genome. It can efficiently transfer the gene at the target location. The
can be isolated from the rest of the
structure of plasmid is explained in the figure below.
DNA using either Restriction
Digestion or PCR.

 Artificial Synthesis: If the gene of

interest is well studied, previously, then
the information of a gene is accessible
in the genetic library and we can use it
for the artificial synthesis of a gene of
our interest. (using the genetic library
information, the gene can also be
artificially synthesized)
 Preparation of plasmid
Preparation of
Select the plasmid which suits your experiment. plasmid
 The plasmid must have the origin of replication, promoter region, antibiotic
resistance gene and other important sequences. Using the restriction
digestion method, an insertion site is introduced in the plasmid at which our
gene of interest is ligated.
 Utilizing the T4 DNA ligase like power sealer, the DNA of our interest in
inserted and ligated in the plasmid. Along with the plasmid, a selectable
marker is also introduced in the plasmid DNA to identify the recombinant
DNA.
 In addition to this, a promoter region and terminator sequences are also
included in the plasmid for the effective expression of a gene of our interest.
A plasmid with our gene of interest and some other important sequences is
now referred to as a recombinant DNA molecule. Now our recombinant DNA
is ready for expression.
 If we are performing gene cloning than the plasmid is inserted in the
bacterial host, for that generally E.coli are commonly used. Once the
bacteria starts dividing, our recombinant plasmid DNA is also replicated
along with it.
 Now we have the multiple copies of our plasmid DNA which are extracted
using the plasmid DNA extraction kit and used for the transformation
experiments.

Transformation into the host genome Confirmation of insert

 Various methods for recombinant DNA insertion is used for various cell  In the traditional culturing method, the presence or absence of a selectable
types because a single method can’t used for all cell types. marker is used to differentiate transformed cells from the untransformed cells.
 Using stress- bacteria easily uptake the plasmid DNA using some stress  PCR based detection method - widely accepted & trusted method. DNA is
factors such as heat or electrical sock. extracted from the transformed cell and amplified using the primers
 Microinjection- a sharp needle is used for insertion of DNA directly into the complementary to our gene of interest or our recombinant DNA. If the
nucleus of a cell, however, the method is less effective and required a recombinant DNA is present it surely amplified otherwise no amplification
higher level of expertise for that. obtained. For the two factor conformation, one primer set complementary to
 Electroporation- one of the best methods having a great success rate. recombinant DNA specific and one set of primer complementary to the
Recombinant DNA is inserted into the host genome by permeabilizing the selectable marker sequence are taken and multiplex PCR is performed. For
cell with electrical current. validating results, amplification must be obtained in both the reaction.
 Sonication- sonication is yet another good method sometimes used in the  What happened if any mutation occurred during the experiment in our gene
gene transfer experiment in which the recombinant DNA is inserted into the of interest? Because the PCR can only amplify the DNA. We must need
target cell using ultrasonic waves. The ultrasonic waves also increase the sequence information to detect the mutation. For that, the DNA sequencing
permeability of cells. method is used.
 Liposome mediated gene transfer- Using an artificial cell-like outer coat  DNA is extracted from the transformed cells and the gene of interest is
known as a liposome- recombinant DNA can be inserted in the host amplified using the PCR. Now the PCR amplicons are used for DNA
genome. sequencing in which using the fluorescent chemistry the sequence of our gene
 Gene transfer using bacterial infection- This method is one of the popular of interest is orderly determined.
methods and routinely used in plant genetic engineering experiments. Here,  Once all the parameters for determining the gene of interest fulfilled, our cells
the plant species is infected with the transformed bacteria for inserting a are now ready to inject in the host organism or for tissue culture experiments.
gene of interest.
 Applications of Genetic engineering - Medical
Applications of Genetic engineering
 Low-cost drugs, hormones, enzymes, and vaccines are created using genetic engineering tools

 Genetic engineering has great industrial and agricultural value. It is practiced in medicine, e.g. -Anti-blood-clotting factor is the best example of it in which the plasminogen activating
enzyme which is capable of dissolving the blood clot is artificially designed and used in the
genetic research, agriculture, crop improvement, and for production of therapeutic drugs. It is
patients with coronary artery disease or heart attack.
also used in the development of genetically modified organisms.
 The recombinant DNA technology is used in the crop improvement and development of new
economically important traits -
 Herbicide resistance
 Therapeutic proteins somatostatin and lymphokines which
 Virus resistance
are worked against several disease conditions and can be
 Delayed fruit ripening
synthesized artificially.
 Altered oil content
 Insulin is yet a classic example of a therapeutic protein
 Pollen control designed using genetic engineering technology.
 Development of cold and drought-tolerant plant species.  Recombinant vaccines against smallpox, herpes simplex virus
 A classical example of it is the BT cotton- one of the types of genetically modified species and hepatitis.
 Created a unique vaccines that only contains the DNA for viral
provides resistance to the plant against Bacillus thuringiensis and many types of moths and
coat protein thus the pathogen can never be activated again.
butterflies can be controlled.

Genetic Engineering techniques.... Limitations of genetic engineering

 There are ethical issues associated with the use of gene therapy and genetically
 The purpose of developing the genetic engineering or genetic manipulating
engineered products.
technique is to produce organisms or phenotypes which are useful to us.
Genetic engineering techniques are used for -  Also, to provide an economic value to the food product or any GM product, the
 nutritional values are compromised.
Construction of genetically modified plant species
 Abiotic and biotic stress resistant plant species  Because of the adverse effect of it, new resistant pathogenic strains are evolved
 Economically important plant species
 faster. Also, the side effects of gene therapy and the use of viruses in it may
Commercially valuable organism
 sometimes harmful to the target organism.
For the production of therapeutic drugs
 Prevention of genetic abnormalities  The technology is not so cost effective.

 Playing with the embryo or fetus is against the natural law, people strongly believe
in it, thus genetically modified food and plant products are always becoming a
center of controversy.

 Overall, positive use of genetic engineering techniques can change the fate
of mankind.
 Gene and Genome Editing
Overview ●
Gene-editing techniques are powerful tools used to modify the
genome of an organism by adding, removing, or altering specific
●
Central Dogma of Molecular Biology DNA sequences. One of the most notable methods for editing
undesired DNA sequences is CRISPR-Cas9.
Besides, TALEN and ZFN are also some known gene-editing tools
DNA, RNA and Protein structure and
●
●
used in gene therapy experiments.
function Importance of Gene Editing
●

– Precise Targeting: CRISPR-Cas9 offers high precision in targeting

specific DNA sequences, allowing for accurate modifications
●
Mendelian Genetics without affecting other parts of the genome.
– Accessibility: Compared to earlier gene-editing methods, CRISPR
●
rDNA Technology is relatively easy and cost-effective, making it accessible for
research and potential clinical applications.
●
Genome Editing – Ethical Considerations: While the potential of gene editing is
immense, it also raises ethical concerns, particularly regarding
germline editing (changes that can be inherited). Ongoing
discussions focus on the implications for human genetics and
89 biodiversity.

CRISPR-Cas9 Mechanism CRISPR-Cas9 Applications

● Guide RNA: CRISPR utilizes a short RNA molecule that is ● Therapeutic Uses: CRISPR-Cas9 is being explored for
designed to be complementary to the target DNA treating genetic disorders by correcting mutations. For
sequence. This guide RNA directs the Cas9 enzyme to the example, it has potential applications in conditions like
exact location in the genome that needs to be edited. sickle cell anemia and cystic fibrosis.
● Cas9 Enzyme: The Cas9 protein acts as a molecular ● Agricultural Improvements: This technique can enhance
"scissors" that creates a double-strand break in the DNA crop resistance to pests and diseases, improve yield, and
at the specified location. create plants that can withstand environmental stresses.
● Repair Process: After the break, the cell's natural repair ● Research: Scientists use CRISPR to study gene function and
mechanisms kick in. Researchers can harness these disease mechanisms, providing insights that can lead to
processes to either insert new DNA (using a donor new therapies or understandings of biological processes.
template) or disable a gene by causing errors in the repair.