Introduction to

Biotechnology
 Identification of Gene of Interest

In genetic engineering the first

step is the gene of interest must
first be Identified and Isolated

The process of identifying the

regions of genomic DNA that
encode genes
 Molecular Markers

Molecular marker is a gene or DNA sequence that

is associated with a certain location within the
genome OR
 Molecular markers associated to a gene are naturally occurring
DNA sequences that are close to the specific genes

Molecular markers are used in molecular biology

and biotechnology to identify a particular sequence
of DNA in a pool of unknown DNA
They are used to 'flag' the position of a particular
gene or the inheritance of a particular characteristic
or desired characteristics
Types of Molecular markers

DNA-based
Markers
Morphological marker
 These are the traditional markers
 Require simple equipment to screen
 Visible to naked eyes
 The are highly influenced by environmental factor
 Often the environmental condition can influence the expression of a
particular characteristics which leads to a selection of false positive
plants

Biochemical marker
 Biochemical markers are proteins produced by gene expression
 The product of a gene can be used as a marker for the presence of a
gene
 Isozymes, different variants of the same enzyme having identical
functions and present in the same individual
 Isozymes are used as biochemical markers in plant breeding
 Common enzyme expressed in the cells of plants
– The enzymes are extracted and run on denaturing
electrophoresis gels
– Denaturing component in the gels unravels the
secondary and tertiary structure of the enzymes
and separated on the basis of mass

DNA-based Marker
– Allowed scanning of the whole genome and
assigning landmarks in high density on every
chromosome in many species
– Not influenced by the environment
– They are more reliable (should be tightly linked to
target loci)
Types of DNA-based Marker

Non-PCR Based (Hybridization based marker)

i. RFLP- Restriction fragment length polymorphism.

PCR Based

ii. RAPD- Random amplification of polymorphic DNA

iii. AFLP-Amplified fragment length polymorphism.
iv. SCAR-Sequence characterize amplified region
v. STS- Sequence tagged sites.
vi. EST-Express sequence tags.
vii. SNP-Single nucleotide polymorphism.
viii. SSR-Simple sequence repeats
ix. CAPS-Cleaved amplified polymorphic sequences.
 Forward and Reverse genetics
 In molecular biology there are number of techniques are available to
understand the function of the gene
 For identification of gene function there are two methods used
commonly
– Forward genetics (Classical genetics)
– Reverse genetics
 Forward genetics
– A traditional approach to the study of gene function
that begins with a phenotype and proceeds to a gene
that encodes the phenotype

– It depends upon the identification and isolation of

random mutation that affect the phenotype of
interest

– Two types of mutations were majorly used in the

forward mutation

A. Spontaneous mutation
B. Creating random mutation
A. Spontaneous Mutation
 It arises spontaneously from natural changes in DNA structure or from
error in the replication
 Mutation results from both internal and external factors
 Where as the changes caused by the radiation or environmental chemicals
are called as induced mutation
B. Creating random mutation
 It depends upon the identification and isolation of random mutation that
affect the phenotype
 Radiation (X rays), chemical mutagen (EMS) and transposable elements
(insert within a coding region and disrupt the amino acid sequence ) are
used to create the mutation
 Reverse genetics
– A molecular approach that begins with a genotype ( a
DNA sequence) and proceeds to the phenotype by
altering the sequence or by inhibiting its expression

– It is possible due to the advancement in the molecular

genetics
 Isolation of gene of interest
In genetic engineering a gene of interest
is first identified and isolated from the
thousands of genes

The isolated gene is then cut from its

source DNA molecule with restriction
enzymes and using a vector, is transferred
into target DNA

There are two main methods for isolating

genes
1. Gene Cloning
2. Using the PCR
Gene Cloning
The insertion of a fragment of DNA carrying a gene into a
cloning vector and subsequent propagation of recombinant
DNA molecules into many copies is known as gene cloning
OR
Gene cloning is a common practice in molecular biology
labs that is used by researchers to create copies of a
particular gene for downstream applications, such as
sequencing, mutagenesis, genotyping etc…

Cloned genes are useful for making copies of a particular

gene and producing a protein product
 Steps in a gene cloning

I. A fragment of DNA, containing the gene to be

cloned, is inserted into a circular DNA
molecule called a vector, to produce a
recombinant DNA molecule.

II. The vector transports the gene into a host

cell, which is usually a bacterium, although
other types of living cell can be used

III. Within the host cell the vector multiplies,

producing numerous identical copies, not
only of itself but also of the gene that it
carries
IV. When the host cell divides, copies of the
recombinant DNA molecule are passed to
the progeny and further vector replication
takes place

V. After a large number of cell divisions, a

colony, or clone, of identical host cells is
produced

Each cell in the clone contains one or more

copies of the recombinant DNA molecule; the
gene carried by the recombinant molecule is
now said to be cloned
 A preview of gene cloning and some uses of
cloned genes
Bacterium
1 Gene inserted into
Cell containing
plasmid
gene of interest

Bacterial Plasmid
chromosome Gene of
Recombinant interest DNA of
DNA (plasmid)
2 Plasmid put into chromosome
bacterial cell (“foreign” DNA)

Recombinant
bacterium
 3 Host cell grown in
culture to form a clone
of cells containing the
“cloned” gene of interest
Gene of Protein expressed from
interest gene of interest
Copies of gene Protein harvested

4 Basic research
Basic and various Basic
research applications research
on gene on protein

Gene for pest Gene used to alter Protein dissolves Human growth
resistance inserted bacteria for cleaning blood clots in heart hormone treats
into plants up toxic waste attack therapy stunted growth
 Bacterium
1 Gene inserted into
Cell containing gene
plasmid
of interest

Bacterial Plasmid
chromosome Gene of
Recombinant interest DNA of
DNA (plasmid)
2 Plasmid put into chromosome
bacterial cell (“foreign” DNA)

Recombinant
bacterium

3 Host cell grown in culture to

form a clone of cells containing
the “cloned” gene of interest

Gene of Protein expressed from

interest gene of interest
Copies of gene Protein harvested

4 Basic research
Basic and various Basic
research applications research
on gene on protein

Gene for pest Gene used to alter Protein dissolves Human growth
resistance inserted bacteria for cleaning blood clots in heart hormone treats
into plants up toxic waste attack therapy stunted growth
Gene cloning requires specialized tools and
techniques

Vehicles: The central component of a gene cloning

experiment is the vehicle, which transport the gene
into the host cell and is responsible for its replication.
To act as a cloning vehicle a DNA molecule must be
capable of entering a host cell and, once inside,
replicating to produce multiple copies of itself

Vector: A DNA molecule, capable of replication in a

host organism, into which a gene is inserted to
construct a recombinant DNA molecule