BCH3245: BIOCHEMICAL GENETICS AND

GENETIC ENGINEERING
COURSE CONTENT

●Review of Genetic materials and genetic expression

● Common genetic terminologies
● Genetic mutation
●Introduction and History of Biotechnology
● Common Molecular techniques
●Application of biotechnology to medicine
 Introduction To Genetic Materials
Genetic material is the hereditary
substance in the cells of living
organisms. It carries all genetic
information specific to an organism. It is
known as ribonucleotide or nucleic
acids. The major genetic materials
include DNA and RNA which are
translated into Protein
 DNA is a double-stranded helix

• James Watson and Francis Crick worked out the

three-dimensional structure of DNA, based on
work by Rosalind Franklin
 DNA
DNA stands for deoxyribose nucleic acid

This chemical substance is present in the nucleus

of all cells in all living organisms
DNA controls all the chemical changes which
take place in cells
The kind of cell which is formed, (muscle, blood,
nerve etc) is controlled by DNA
The kind of organism which is produced (buttercup,
giraffe, herring, human etc) is controlled by DNA
 DNA molecule
DNA is a very large molecule made up of a long
chain of sub-units
The sub-units are called nucleotides

Each nucleotide is made up of

a sugar called deoxyribose
a phosphate group -PO4 and
A Nitrogen-containing organic bases
Each strand of a DNA molecule is composed of a long
chain of monomer nucleotides. The nucleotides of DNA
consist of a deoxyribose sugar molecule to which is
attached a phosphate group and one of four
nitrogenous bases: two purines (adenine and guanine)
and two pyrimidines (cytosine and thymine). The
nucleotides are joined together by covalent bonds
between the phosphate of one nucleotide and the
sugar of the next, forming a phosphate-sugar backbone
from which the nitrogenous bases protrude. One strand
is held to another by hydrogen bonds between the
bases; the sequencing of this bonding is specific—i.e.,
adenine bonds only with thymine, and cytosine only
with guanine.
Joined nucleotides

PO4

A molecule of DNA is
formed by millions of
PO4 nucleotides joined
together in a long
chain
PO4

PO4

sugar-phosphate + bases
backbone
In fact, the DNA usually consists of a double
strand of nucleotides

The sugar-phosphate chains are on the outside

and the strands are held together by chemical
bonds between the bases
2-stranded DNA
PO4
PO4

PO4
PO4

PO4 PO4

PO4
PO4

PO4
PO4

PO4
PO4

PO4
PO4

PO4
PO4
RNA is a single-stranded but not a linear molecule.

The shape is very important for catalytic purposes

• RNA is also a nucleic acid
– different sugar
– U instead of T
– Single strand, usually
Nitrogenous base
(A, G, C, or U)

Phosphate
group

Uracil (U)

Sugar
(ribose)
 Differences Between DNA and RNA
●DNA contains the sugar deoxyribose, while RNA contains the
sugar ribose. The only difference between ribose and deoxyribose
is that ribose has one more -OH group than deoxyribose, which has
-H attached to the second (2') carbon in the ring.
●DNA is a double-stranded molecule, while RNA is a single-
stranded molecule.
● DNA is stable under alkaline conditions, while RNA is not stable.
●DNA and RNA perform different functions in humans. DNA is
responsible for storing and transferring genetic information, while
RNA directly codes for amino acids and acts as a messenger
between DNA and ribosomes to make proteins.
● DNA and RNA base pairing is slightly different since DNA uses the
bases adenine, thymine, cytosine, and guanine; RNA uses adenine,
uracil, cytosine, and guanine. Uracil differs from thymine in that it
lacks a methyl group on its ring.
 RNA types
• mRNA messenger RNA (mRNA) RNA molecule
that specifies the amino acid sequence of a
protein.
• rRNA ribosomal RNA (rRNA) Any one of a
number of specific RNA molecules that form part
of the structure of a ribosome and participate in
the synthesis of proteins
• tRNA transfer RNA (tRNA) Set of small RNA
molecules used in protein synthesis as an
interface (adaptor) between messenger RNA
and amino acids.
 Types of RNA
Type of RNA Functions in Function
Messenger RNA Nucleus, Carries DNA
(mRNA) migrates sequence
to ribosomes information to
in cytoplasm ribosomes

Transfer RNA Cytoplasm Provides linkage

(tRNA) between mRNA
and amino acids;
transfers amino
acids to ribosomes

Ribosomal RNA Cytoplasm Structural

(rRNA) component
of ribosomes
 What Are Proteins?
• Large molecules
• Made up of chains of amino acids
• Are found in every cell in the body
• Are involved in most of the body’s functions
and life processes
• The sequence of amino acids is determined by
DNA
 Structure of Proteins
• Made up of chains of amino acids; classified by
number of amino acids in a chain
– Peptides: fewer than 50 amino acids
• Dipeptides: 2 amino acids
• Tripeptides: 3 amino acids
• Polypeptides: more than 10 amino acids
– Proteins: more than 50 amino acids
• Typically 100 to 10,000 amino acids linked together
• Chains are synthesizes based on specific bodily DNA
• Amino acids are composed of carbon, hydrogen,
oxygen, and nitrogen
Structural Differences Between Carbohydrates, Lipids, and
Proteins
The Anatomy of an Amino Acid
 Peptide Bonds Link Amino Acids
• Form when the acid group (COOH) of one
amino acid joins with the amine group (NH2)
of a second amino acid
• Formed through condensation
• Broken through hydrolysis
Condensation and Hydrolytic Reactions
Essential, Nonessential, and Conditional
• Essential – must be consumed in the diet
• Nonessential – can be synthesized in the body
• Conditionally essential – cannot be
synthesized due to illness or lack of necessary
precursors
– Premature infants lack sufficient enzymes needed
to create arginine
 Structure of the Protein
• Four levels of structure
– Primary structure
– Secondary structure
– Tertiary structure
– Quaternary structure

Any alteration in the structure or sequencing

changes the shape and function of the protein
• REPLICATION, TRASCRIPTION &
TRANSLATION (CENTRAL DOGMA)
– The DNA is transcribed into RNA, which
is translated into the polypeptide

DNA

TRANSCRIPTION

RNA

TRANSLATION

Protein

Figure 10.6A
 Common Genetic Terminologies
Phenotype-: An observable character or characters
in an organism that is specified by genotype; it may
refer to structural or functional characters e.g.
blood group, hair colour, etc.

Genotype-: The genetic make up of an individual

with respect to a given characteristic(s) e.g. an
individual with the phenotype blood group O has
the genotype OO. Genotype can also be used to
refer to the specific alleles present at a particular
genetic location in a given individual. The genes
partly determine the observable characteristics of
an organism, such as hair color, height, etc.
 Haploid-: Describes a cell or individual with a
single copy of each chromosome e.g. an egg or
sperm cell
Diploid-: Describes a cell or individual with two
copies of each chromosome e.g. a normal
human somatic cell
Gene-: A gene is a basic unit of heredity and a
sequence of nucleotides in DNA that encodes the
synthesis of a gene product, either RNA or protein.
During gene expression, the DNA is first copied into
RNA. The RNA can be directly functional or be the
intermediate template for a protein that performs
a function.
Introns
Introns are nucleotide sequences in DNA
and RNA that do not directly code for
proteins, and are removed during the
precursor messenger RNA (pre-mRNA) stage
of maturation of mRNA by RNA splicing.
Introns can range in size from 10’s of base
pairs to 1000’s of base pairs, and can be
found in a wide variety of genes that
generate RNA in most living organisms,
including viruses.
Exons
Exons are nucleotide sequences in DNA and RNA
that are conserved in the creation of mature RNA.
The process by which DNA is used as a template
to create mRNA is called transcription. mRNA
then works in conjunction with ribosomes and
transfer RNA (tRNA), both present in the
cytoplasm, to create proteins in a process known
as translation. Exons usually include both the 5’-
and 3’- untranslated regions of mRNA, which
contain start and stop codons, in addition to any
protein coding sequences.
Genome-: The complete genetic make up
of an individual or species (for all
characteristics)
Locus-: The position of a gene on a
chromosome : for ABO blood group the
locus is chromosome 9, 9q34.1q34.2
Allele-: One or more alternative
variations of a particular gene that can
exist at that gene locus e.g. A, B, and O
alleles for blood groups
Homozygote-: A diploid individual with two
identical alleles at a given locus e.g. (in ABO blood
groups) AA or BB

Heterozygote-: A diploid individual with two

different alleles at a given locus e.g. AO or AB
E.g. for the ABO blood group:
• The A allele causes the production of A antigens
• The B allele causes the production of B antigens
• The O allele causes the production of a non
functioning protein so no antigens are produced.
 Introduction to genetic code
Genetic code is the term we use for the way that
the four bases of DNA--the A, C, G, and Ts--are
strung together in a way that the cellular
machinery, the ribosome, can read them and turn
them into a protein. In the genetic code, each three
nucleotides in a row count as a triplet and code for
a single amino acid The genetic code is the cell’s
‘instruction manual’ for producing a protein from
an mRNA sequence. Three-base-long sections of
mRNA (codons) are ‘read’ in sequence at the
ribosome. Each codon corresponds to a particular
amino acid, which is added to the growing protein
chain.
Notice that many amino acids are
represented in the table by more than one
codon. For instance, there are six different
ways to "write" leucine in the language of
mRNA (see if you can find all six).
An important point about the genetic code
is that it's universal. That is, with minor
exceptions, virtually all species (from
bacteria to you!) use the genetic code
shown above for protein synthesis.
There are 64 possible codons, three of which do
not code for amino acids but indicate the end of
a protein. The remaining 61 codons specify the
20 amino acids that make up proteins. The AUG
codon, in addition to coding for methionine, is
found at the beginning of every mRNA and
indicates the start of a protein. Methionine and
tryptophan are the only two amino acids that are
coded for by just a single codon (AUG and UGG,
respectively). The other 18 amino acids are
coded for by two to six codons. Because most of
the 20 amino acids are coded for by more than
one codon, the code is called degenerate.
Codons
Cells decode mRNAs by reading their nucleotides
in groups of three bases called codons. Here are
some features of codons:
Most codons specify an amino acid. Three "stop"
codons mark the end of a protein. One "start"
codon, AUG, marks the beginning of a protein and
also encodes the amino acid methionine
Codons in an mRNA are read during translation,
beginning with a start codon and continuing until
a stop codon is reached. mRNA codons are read
from 5' to 3' , and they specify the order of amino
acids in a protein from N-terminus (methionine)
to C-terminus.
GENETIC MUTATION
 INTRODUCTION
• The term mutation refers to a heritable change in
the genetic material
• Mutations provide allelic variations
– On the positive side, mutations are the foundation for
evolutionary change needed for a species to adapt to
changes in the environment
– On the negative side, new mutations are much more
likely to be harmful than beneficial to the individual and
often are the cause of diseases

• Since mutations can be quite harmful, organisms

have developed ways to repair damaged DNA

47
 2 Main Types of Mutations

1.) Chromosomal Mutations

2.) Gene Mutations

 Chromosomal Mutations

• Any change in the structure or number

of chromosomes

• Large scale: Affect many genes

 5 Types
1. Deletion
2. Duplication
3. Inversion
4. Translocation
5. NonDisjunction
Chromosomal Deletion

One or more genes are removed

Chromosomal Duplication

A segment of genes is copied twice and added to the

chromosome
 Chromosomal Inversion

a segment of genes flip end-to-end on the chromosome

 Chromosomal Translocation

Material is swapped with another chromosome

 Nondisjunction
• Chromosomes FAIL TO SEPARATE
during meiosis

• Meiosis I Nondisjunction
• Meiosis II Nondisjunction
 Nondisjunction
• Produces gametes (and therefore a
baby) with one missing chromosome
or one extra chromosome
 Chromosomal Mutations
• Most chromosomal mutations are
lethal

• If the fetus survives: Tend to cause

wide-spread abnormalities

• Example: Down Syndrome

 Down Syndrome
• Cause:
Nondisjunction of
chromosome 21

• Three copies of chromosome 21 =

“TRISOMY 21”
Trisomy 21 - Down Syndrome
 Gene Mutations
• Small scale: one gene is
affected

• Any change to the DNA

sequence of a gene:
Nucleotides/Bases may be
added, missing, or changed
 Gene Mutations: 3 Types
Point Mutation Frameshift Mutation

A third type of gene mutation is splice site mutation

 Point Mutation
• One base (A, T, C, or G) is substituted for another
• Major cause of diseases like Sickle-cell anemia
• 3 Possible Consequences:
– nonsense mutations: code for a stop, which can
translate the protein
– missense mutations: code for a different amino acid
– silent mutations: code for the same amino acid
Fig 4.4
Silent. A silent mutation in a coding region is the
substitution of a synonymous codon for the
original codon. Because the amino acid encoded is
the same, such mutations are not likely to have
any phenotypic effect. Substitution of one stop
codon for another is also generally a silent
mutation. We can also think of base substitutions
in non coding regions (98% of the human genome)
as silent mutations, as the overwhelming majority
of such mutations will not have any phenotypic
effect.
Missense. A missense mutation is the
substitution of a non synonymous codon
for the original codon. The amino acid
replacement might be a conservative
substitution (like isoleucine for valine)
where the two amino acids are chemically
similar, or a non conservative substitution
(like cysteine for tryptophan as shown
below) where the two amino acids are very
different chemically.
Nonsense. A nonsense mutation is the substitution of a
stop (nonsense) codon for the original codon. Nonsense
mutations cause the termination of protein synthesis, and
if they occur anywhere but very near the 3' end of the
gene, are likely to cause a complete loss of gene function.
 Frameshift Mutations
In a frameshift mutation, a base is inserted or
deleted, altering the codon in which the insertion
or deletion took place, but also changing the
reading frame so that all codons downstream are
read out of frame. This typically produces a string
of amino acid substitutions before a stop codon is
reached. Stop codons are fairly frequent in coding
sequences read out of frame. Frameshift
mutations can be caused by the insertion or
deletion of any number of bases that is not an
integral multiple of three.
This shows insertion of a single nucleotide
 A frame shift mutation: Deletion

Normal gene Deletion mutation

GGTCTCCTCACGCCA GGTC/CCTCACGCCA
↓ ↓
CCAGAGGAGUGCGGU CCAGGGAGUGCGGU
Codons
↓ ↓
Pro-Glu-Glu-Cys-Gly Pro-Gly-Ser-Ala-Val
Amino acids
Splice Site. Mutations of splice sites account for
about half of all disease-causing mutations in
humans. A splice donor is a GT pair at the 5' end
of an intron that is part of a consensus sequence,
and a splice acceptor is an AG base pair at the 3'
end of an intron that is part of a consensus
sequence. In the example shown here, the splice
donor site mutates via a single base substitution
such that it can no longer be a splice donor. The
consequence in this case is shown in the image
below.
Splice Site. In the original sequence at the top of the
image, exon 9 joins exon 10 as the first intron shown is
spliced out. There is an alternative splice acceptor for the
second intron, producing two slightly different splices of
exon 10 to exon 11. The single splice donor for the second
intron is mutated. Loss of the splice donor for the second
intron causes the splicing machinery to use the prior
splice donor. This causes exon 9 to be spliced to exon 11,
entirely omitting exon 10. The effect on the synthesis of
the protein is the loss of all of exon 10. In addition, it is
possible that the novel splice junction will cause a
frameshift immediately downstream of the new splice.
This is an example of mutation outside the coding region
which still affect gene function.
 Gene Mutations and Their Effects
on Genotype and Phenotype
 In a natural population, the wild-type is the relatively
prevalent genotype. Genes with multiple alleles may
have two or more wild-types.

 A forward mutation changes the wild-type genotype

into some new variation

 A reverse mutation changes a mutant allele back to

the wild-type
 It is also termed a reversion
 Mutations can also be described based on their
effects on the wild-type phenotype
 They are often characterized by their differential
ability to survive
 Deleterious mutations decrease the chances of survival

The most extreme are lethal mutations
 Beneficial mutations enhance the survival or reproductive
success of an organism
 The environment can affect whether a given mutation is
deleterious or beneficial
 Some mutations are conditional
 They affect the phenotype only under a defined set of
conditions
 An example is a temperature-sensitive mutation
 Mutations Can Occur in
Germ-Line or Somatic Cells
 Geneticists classify animal cells into two types
 Germ-line cells

Cells that give rise to gametes such as eggs and sperm
 Somatic cells

All other cells
 Germ-line mutations are those that occur directly in a
sperm or egg cell, or in one of their precursor cells

 Somatic mutations are those that occur directly in a body

cell, or in one of its precursor cells.
 Germ-line
mutation Gametes

Somatic
mutation The size of the patch
Embryo
will depend on the
timing of the mutation
The earlier the mutation,
the larger the patch

Patch of An individual who has

affected somatic regions that are
Mutation is area genotypically different
found Mature from each other is
throughout individual called a genetic mosaic
the entire
Therefore, the body.
mutation can be
passed on to future Therefore, the mutation cannot be
generations passed on to future generations

Half of None of
the gametes the gametes
carry the carry the
mutation. mutation.

(a) Germ-line mutation (b) Somatic cell mutation

 Mutation Rate
 The term mutation rate is the likelihood that a gene
will be altered by a new mutation
 It is commonly expressed as the number of new mutations
in a given gene per cell generation
 It is in the range of 10-5 to 10-9 per generation

 The mutation rate for a given gene is not constant

 It can be increased by the presence of mutagens

 Mutation rates vary substantially between species

and even within different strains of the same species
 Mutation Rates and Frequencies
 Within the same individual, some genes mutate at a
much higher rate than other genes
 Some genes are larger than others

This provides a greater chance for mutation

 Some genes have locations within the chromosome that

make them more susceptible to mutation

These are termed hot spots

 Note: Hot spots can be also found within a single gene


Specific bases or regions that are more likely to be the site of a
mutation within a gene
 Causes of
Spontaneous Mutations
 Spontaneous mutations can arise by three types of
chemical changes

 1. Depurination The most common; We will focus here

 2. Deamination

 3. Tautomeric shift
 Causes of
Spontaneous Mutations
 Depurination involves the removal of a purine
(guanine or adenine) from the DNA
 The covalent bond between deoxyribose and a purine base
is somewhat unstable

It occasionally undergoes a spontaneous reaction with water that
releases the base from the sugar
 This is termed an apurinic site

 Fortunately, apurinic sites can be repaired


However, if the repair system fails, a mutation may result during
subsequent rounds of DNA replication
5′ 3′ 5′ 3′
C G C G
A T A T
T A Depurination T A Apurinic site
C G C
G C G C
3′ 5′ 3′ 5′

(a) Depurination

5′ 3′
C G Three out of four (A, T
A T and G) are the
T A
5′ 3′ C G incorrect nucleotide
C G G C There’s a 75% chance
A T DNA replication 3′ 5′ of a mutation
T A
C 5 3′
G C C G
3′ 5′ A T
T A X could be
X A, T, G, or C
G C
3′ 5′
(b) Replication over an apurinic site

Spontaneous depurination
 Mutations Due to Trinucleotide
Repeats
 Several human genetic diseases are caused by an
unusual form of mutation called trinucleotide repeat
expansion (TNRE)
 The term refers to the phenomenon that a sequence of 3
nucleotides can increase from one generation to the next
 These diseases include
 Huntington disease (HD)
 Fragile X syndrome (FRAXA)
 Certain regions of the chromosome contain
trinucleotide sequences repeated in tandem
 In normal individuals, these sequences are transmitted from
parent to offspring without mutation
 However, in persons with TNRE disorders, the length of a
trinucleotide repeat has increased above a certain critical size

Disease symptoms occur

In some diseases, it also becomes prone to expansion

This phenomenon is shown here with the trinucleotide repeat CAG

CAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAG

n = 11
CAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAG
n = 18
 In some cases, the expansion is within the coding
sequence of the gene
 Typically the trinucleotide expansion is CAG (glutamine)
 Therefore, the encoded protein will contain long tracks of
glutamine

This causes the proteins to aggregate with each other

This aggregation is correlated with the progression of the disease

 In other cases, the expansions are located in

noncoding regions of genes
 Some of these expansions are hypothesized to cause
abnormal changes in RNA structure
 There are two particularly unusual features that
some TNRE disorders have in common

 1. The severity of the disease tends to worsen in future

generations

This phenomenon is called anticipation

 2. Anticipation usually depends on whether the disease is

inherited from the father or mother

In Huntington disease, the TNRE is more likely to occur if inherited
from the father

In myotonic muscular dystrophy, the TNRE is more likely to occur if
inherited from the mother

This suggests that TNRE can occur more frequently during
oogenesis or spermatogenesis, depending on the gene involved.
 Types of Mutagens
 Apart from spontaneous mutations described
above, induced mutations are caused by mutagens
and are is the most common type of mutation. An
enormous array of agents can act as mutagens that
permanently alter the structure of DNA
 The public is concerned about mutagens for two
main reasons:
 1. Mutagens are often involved in the development of
human cancers
 2. Mutagens can cause gene mutations that may have
harmful effects in future generations
 Mutagenic agents are usually classified as
chemical or physical mutagens
 Mutagens Alter DNA Structure in
Different Ways
 Chemical mutagens come into three main types

 1. Base modifiers

 2. Intercalating agents

 3. Base analogues
 Base modifiers covalently modify the structure of a
nucleotide
 For example, nitrous acid, replaces amino groups with
keto groups (–NH2 to =O)
 This can change cytosine to uracil and adenine to
hypoxanthine

These modified bases do not pair with the appropriate nucleotides
in the daughter strand during DNA replication

 Some chemical mutagens disrupt the appropriate pairing

between nucleotides by alkylating bases within the DNA

Examples: Nitrogen mustards and ethyl methanesulfonate (EMS)
 Template strand After replication

H NH2 H
O H N H
N
H N HNO2
N
N H N
N
Sugar
O N N
Sugar
Sugar O H
These mispairings
Cytosine Uracil Adenine create mutations
in the newly
H replicated strand
H N NH2 H N O H N H
HNO2
N N N
N N H H
Sugar Sugar
N N N
H H O Sugar
Adenine Hypoxanthine Cytosine

Mispairing of modified bases

 Intercalating agents contain flat planar structures
that intercalate themselves into the double helix

 This distorts the helical structure

 When DNA containing these mutagens is replicated, the

daughter strands may contain single-nucleotide additions
and/or deletions resulting in frameshifts

 Examples:

Acridine dyes

Proflavin
 Base analogues become incorporated into daughter
strands during DNA replication
 For example, 5-bromouracil is a thymine analogue

It can be incorporated into DNA instead of thymine

H N H
Br O H O
N H
Br O H N
N
N H N
N
N H N Sugar
Sugar N N
N N Sugar O H N
Sugar O H
H
5-bromouracil Adenine 5-bromouracil Guanine
(keto form) (enol form)

Mispairin
This tautomeric
Normal g
shift occurs at a
pairing relatively high
rate
(a) Base pairing of 5BU with adenine or guanine
 In this way, 5-bromouracil can promote a change
of an AT base pair into a GC base pair

5′ 3′

A T 5′ 3′
5′ 3′
DNA
replication
3′ 5′ G C
A 5BU
5′ 3′
DNA
replication
3′ 5′
3′ 5′
G 5BU 5′ 3′

3′ 5′ or A 5BU
G or
G

3′ 5′

(b) How 5BU causes a mutation in a base pair during DNA replication
 Physical mutagens come into two main types
 1. Ionizing radiation
 2. Nonionizing radiation

 Ionizing radiation
 Includes X-rays and gamma rays
 Has short wavelength and high energy
 Can penetrate deeply into biological molecules
 Creates chemically reactive molecules termed free radicals
 Can cause

Base deletions

Oxidized bases

Single nicks in DNA strands

Cross-linking

Chromosomal breaks
 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

H
O
O N O
 Nonionizing radiation O P
O–
O CH2
O
N
H H CH3
 Includes UV light H H
H
H Thymine
 Has less energy CH3
O
 Cannot penetrate deeply O P O CH2
O
H O

into biological molecules O–

H
H H
H
N N
H
O
 Causes the formation of H Thymine

cross-linked thymine Ultraviolet

light
H
dimers O
O
P O CH2
O N O
O
 Thymine dimers may O–
H H
H
N
CH3
H
cause mutations when that H

H
DNA strand is replicated
O CH3
H O
O P O CH2
O
O– N N
H H H
H H
O
H Thymine dimer
 Types of human variation
Now that we have surveyed how mutations
can arise and the different kinds of
mutations, it is useful to survey variation in
humans to see what we have learned from
our study of the human genome. Human
variants can be classified into types, some of
which are point mutations.
 Single Nucleotide Polymorphisms (SNPs).
Single nucleotide polymorphisms (SNPs) are
sites of variation in the genome that exhibit
single-base differences in the population.
Where the reference sequence of the
human genome has a T, there may be
alleles with an A, C, or G at that position in
the population. There is a SNP about every
1000 base pairs in the human genome. Any
two individuals differ at about 6,000,000
SNPs from each other. Most SNPs are
selectively neutral and represent ancient
variation in our species.
SNPs can be in non coding sequence (as are
the vast majority) or in coding sequence,
where they may either be synonymous
substitutions or other alleles that are
tolerated. Because of the way that SNPs are
identified (survey sequencing of individuals),
some mutations that are under negative
selection may be identified as SNPs that have
very low allele frequencies in the population.
 Copy Number Variants.
Copy number variants are insertions or deletions of
DNA anywhere from tens or hundreds of bases up
to large duplications or deletions that are visible
cytologically. While these are a significant
component of human genetic variation, copy
number variants above a certain size are not easily
detected by current sequencing technology.
The most important human copy number repeat
variant is short tandem repeat (STR) loci, also
known as variable number of tandem repeat
(VNTR) loci, that are used in the forensic analysis of
human DNA.
The human genome has a large number
of locations of short sequences that are
repeated a number of times, flanked by
unique DNA. The number of repeats at
any particular locus is highly variable.
These are typically not in coding regions.
Different alleles of this particular STR are very different.
Most individuals are heterozygous, with each of their
alleles carrying a different number of copies of the repeat.
GENETIC ENGINEERING/MOLECULAR BIOLOGY

INTRODUCTION AND
HISTORICAL BACKGROUND
 INTRODUCTION/DEFINITIONS
-Biotechnology is the name given to the generic
technology of the 21st century.
-It means any technological application that uses
biological systems, living organisms, or derivatives
for specific use.
-Molecular biology is the study of biology at molecular
level. The field overlaps with other fields of biology and
chemistry particularly genetics and biochemistry.
-Molecular biology chiefly concerns itself with
understanding the interactions between the various
systems of a cell including the interrelationship of DNA,
RNA and protein synthesis and learning how these
interactions are regulated.
History of biotechnology
There are many important discoveries that have
played big roles in the evolution of the biotechnology
industry. Modern biochemistry and microbiology
techniques utilize a number of molecular techniques
that have developed in the past couple of decades as
a result of the discovery of PCR, DNA fingerprinting,
restriction enzymes, sequencing and
cloning techniques. However, before we ever knew
what a gene was, humans were manipulating cells in
some very industrious ways, to produce foods,
chemicals or improved crops. The list below outlines
some of the more historical biotechnological
techniques that laid the groundwork for this area of
study, before the term "biotechnology" was ever
used.
Fermentation is perhaps the most
ancient biotechnological discovery. Over
10,000 years ago mankind was
producing wine, beer, vinegar and bread
using microorganisms, primarily yeast.
Yogurt was produced by lactic acid
bacteria in milk and molds were used to
produce cheese. These processes are
still in use today for the production of
modern foods. However, the cultures
that are used have been purified and
often genetically refined to maintain the
most desirable traits and highest quality
of products.
In 1897 the discovery that enzymes
from yeast can convert sugar to
alcohol lead to industrial processes
for chemicals such as butanol,
acetone and glycerol. Fermentation
processes are still in use today in
many modern biotech
organizations, often for the
production of enzymes to be used
in pharmaceutical processes,
environmental remediation and
other industrial processes.
Drying, salting and freezing
foods to prevent spoilage by
microorganisms were
practiced long before anyone
really understood why they
worked or even fully knew
what caused the food to spoil
in the first place.
The practice of quarantining to
prevent the spread of disease
was in place long before the
origins of disease were known.
However, it demonstrates early
acceptance that illness could
be passed from an infected
individual to another healthy
individual, who would then
begin to have symptoms of the
disease.
Crop improvement, by selecting seeds from the
most successful or healthiest plants, to obtain a
new crop having the most desirable traits, is a
form of early crop technology. Farmers learned
that using only the seeds from the best plants
would eventually enhance and strengthen the
desired traits in subsequent crops. In the mid-
1860's, Gregor Mendel's studies on inheritable
traits of peas improved our understanding of
genetic inheritance and lead to practices of
cross-breeding (now known as hybridization).
• Other important discoveries include:

• In 1590 AD the microscope was invented

by Zacharias Janssen.
• In 1675, Microorganisms were
discovered using the first microscope.
• In 1856, Greggor Mendel discovered the
laws of inheritance.
• In 1975, methods for producing
monoclonal antibody.
• In 1980, modern biotechnology was
characterized by recombinant DNA
technology. The Bacteria E. Coli was used to
produced insulin.
• In 1984, FDA approves the first genetically
modified food i.e. FLAVR TOMATO produced
from calgene.
• In 1997, British scientist from the Roseline
Institute reported cloning a sheep called Doli.
• Future perspective: Designer babies, drug
development, gene therapy, etc
 Techniques in Molecular Biology

Molecular biology is an area of biology

concerned with the process of gene
transcription to yield RNA, the translation
of RNA into proteins and the role those
proteins play in cellular function. Since
around 1960, molecular biologists have
developed methods to identify, isolate, and
manipulate molecular components in cells
including DNA, RNA, and proteins. Several
techniques used in the field of molecular
biology are described below.
●Polymerase chain reaction (PCR) – This is one of the
most important techniques used in molecular biology
and is basically used to copy DNA. PCR allows a single
DNA sequence to be amplified into millions of DNA
molecules. PCR can also be used to introduce mutations
within the DNA or introduce special restriction enzyme
sites. In addition, PCR is used to determine whether a
certain DNA fragment exists in a cDNA library. Different
types of PCR include reverse transcription PCR (RT-PCR)
for amplification of RNA and quantitative PCR (QPCR) to
measure the amount of RNA or DNA present. PCR can
be used for many purposes, including genetic
fingerprinting in forensics, paternity testing, mutation
detection for disease and cloning genes for research.
•Expression cloning – This technique helps
scientists understand the protein function. The DNA
that codes for a particular protein is cloned or
copied using PCR into an expression vector called
a plasmid. The plasmid is introduced to either an
animal cell or a bacterial cell. This plasmid has
promoter elements that can stimulate high
expression of the desired protein so that its
enzymatic activity can then be examined.
•Gel electrophoresis – This is another important
technique used in molecular biology to separate
DNA, RNA, and proteins based on their size by
applying an electric field as the DNA is run through
agarose gel.
•Macromolecule blotting and probing – Processes
such as southern blotting, northern blotting, western
blotting and eastern blotting are used to transfer DNA
or RNA and proteins onto a blotting membrane (often
after gel electrophoresis) so they can be stained or
radioactively labelled and then visualized.
•Arrays – A DNA microarrays or DNA chip is a
collection of DNA spots mounted on a solid surface
such as a microscope slide that can be used to
simultaneously quantify protein expression levels
across a large number of genes. The technique can
also be used to genotype various different genomic
regions.
The blotting techniques
The Southern, Northern and Western blots
are used to detect DNA, messenger RNA
(mRNA) and protein, respectively. Blotting
refers to the actual technique, where
molecules that have been separated on a gel
are transferred or blotted onto a type of paper
called nitrocellulose. The naming of the
different blots originated with the DNA blot,
developed by Edward Southern, and the
Northern and Western blots followed.
Southern blot
Before the blot itself can be done, DNA that has been cut up
with restriction enzymes is separated by gel electrophoresis.
For the blotting step, the gel is placed on a sponge which is
sitting in a buffer solution. The nitrocellulose paper, where the
DNA will be transferred to, is placed on top of the gel and then
covered with paper towels and a weight. The transfer of the
DNA from the gel to the paper happens by capillary action as
the buffer moves toward the dry paper towels. After several
hours, the transfer is complete and the paper will have the
DNA fragments on it in the same pattern as they were in the
gel.
The paper can then be incubated with a probe that
is specific to a DNA fragment of interest. The probe
is radioactively labelled and once the incubation is
complete, it can be detected by autoradiography.
Controls must be used to ensure that the
electrophoresis and the blot were successful. A
comparison of the band patterns by autoradiography
shows the presence or absence of the DNA of
interest.

Northern blot
A Northern blot is done in the same way as a Southern blot, but it uses
mRNA instead of DNA.
Western blot
A Western blot also follows the same method
as a Southern blot, but is used to detect
proteins instead of DNA. After the proteins are
blotted onto the paper, antibodies are used to
detect their presence. The primary antibody
binds to the protein on the paper and the
secondary antibody binds to the primary
antibody. The secondary antibody is either
tagged with a colour or is attached to an
enzyme that can produce a colour in order to
detect where it is on the paper.
Enzyme-Linked Immunosorbent Assay (ELISA)
ELISA is a technique used to detect the presence of
specific antibodies or antigens in a sample. It is a
very simple test that can analyse a large number of
samples at once, which makes it a very important
diagnostic technique.
The ELISA method takes advantage of the natural
property of antigens and antibodies to bond together.
A plastic dish with many wells in it is coated with an
antibody for a particular antigen. Then a different
sample is added to each well - for example, blood
samples from different people. Several wells will
contain positive and negative control samples. If the
antigens in the blood match the antibody in the well,
they will bind.
Those that do not bind will be washed off. A second
antibody is then added to the wells, which will only
attach to the antigens. This second antibody is
attached to an enzyme ("enzyme-linked") that will
produce a colour when a solution is added to it. The
entire dish can then be read by a scanner that looks
for the presence of the enzyme's colour. If a colour is
present, it means that sample contained the antigen
of interest. If there is no colour, there was no antigen
to bind to the first antibody. The control samples are
used to make sure the procedure was successful -
the positive control should be coloured and the
negative control should not.