**PROCESSES of rDNA

TECHNOLOGY**

I. **Isolation of Desired DNA Fragment**

1. Breaking the cell wall
2. Removal of impurities from the DNA
3. Separation of purified DNA (Spooling)
4. Cutting of DNA at specific location
using Restriction Endonuclease
5. Desired DNA fragment is separated
using Gel electrophoresis
6. Amplification of DNA fragment using
PCR

II. **Ligation of DNA Fragment into a Vector

using Ligase**
- DNA fragment and vector plasmid are cut
with the same restriction enzymes, producing
complementary sticky ends.
- DNA ligase joins them to create rDNA.

III. **Vector through or Vector-less Transfer of

rDNA into the Host (Transformation)**
- Selection of transformants (cells with
recombinant DNA).
- Non-transformants: Cells without rDNA.
- Non-recombinant transformants: Cells
with the vector
- but without inserted DNA.
- Recombinant transformants: Cells with the desired rDNA.

IV. **Culturing the Host Cell in a Medium**

- Bioreactors used to culture cells in large quantities.

V. **Extraction & Purification of Desired Protein or Enzymes (Downstream Processing)**

.

**Biotechnology: Technological Exploitation of Biological Processes**

**Definition:** Biotechnology is the technological exploitation of biological processes for the benefit of mankind.

**Two Aspects of Biotechnology:**

1. **Merits of Genetic Engineering over Traditional
Hybridization:**
- Hybridization Techniques:
- Lead to inclusion and multiplication of undesirable
genes along with desired genes.
- Genetic Engineering:
- Helps to isolate and introduce only desirable
genes into the target organism.
- Example: Stanley Cohen & Herbert Boyer (1972)
constructed the first recombinant DNA.
- Linked a gene of antibiotic resistance from the plasmid of Salmonella typhimurium with a plasmid of E. coli and
transferred it into E. coli.
**Classical Biotechnology:**
- Includes processes based on the natural capabilities of microbes.
- Examples: Making curd, bread, wine.

**Modern Biotechnology / rDNA Technology / Genetic Engineering:**

- Involves modifying organisms genetically for obtaining specific products.
- Examples: In vitro fertilization, preparation of vaccines using rDNA technology.

Note: Biotechnology has diverse applications and is constantly evolving, leading to significant advancements in
various fields, including medicine, agriculture, and industry.
**Biotechnology: Genetic Engineering Process**

**Genetic Engineering Process:**

I. Isolation of a Desired DNA Fragment:

i. Breaking the Cell Wall:
- Different lytic enzymes used based on the cell type to be lysed.
- Bacterial cell → lysozyme
- Plant cells → cellulase
- Fungal cell → chitinase

ii. Removal of Impurities from the DNA:

- RNA removed by treating with ribonuclease.
 - Proteins removed by treating with protease.
- Other molecules can be removed through appropriate treatments.

iii. Separation of Purified DNA:

- Add chilled ethanol to precipitate out the purified DNA (appears as fine threads in the suspension).
- Purified DNA can be separated by spooling.

iv. Cutting of DNA at Specific Locations Using Restriction Endonuclease**

- Purified source DNA and vector DNA are cut with a specific restriction enzyme.
- Restriction Enzymes: Occur in bacteria to restrict replication of attacking bacteriophages by cleaving their
introduced DNA.
- Belong to a class of enzymes called nucleases (exonucleases & endonucleases).
- Endonucleases: Cut at specific recognition sequences, often palindromic, leaving sticky ends.

**Naming of Restriction Enzymes:**

**V. Separation of Desired DNA Fragment Using Gel
Electrophoresis**
- Cleavage by endonuclease produces many different DNA fragments.
- Gel electrophoresis separates DNA fragments based on size using an
electric field.
- Smaller fragments move farther through the gel.
- DNA bands can be visualized by staining with Ethidium Bromide
under UV light.
- E.g.: EcoRI
- 1st letter (capital and italics) - Genus of
the prokaryotic cell (E for Escherichia)
- 2nd & 3rd letters - Species (co for coli)
- 4th letter - Strain name of bacteria (R for
RY 13)
- Following Roman numerals indicate the
order of isolation from the strain (I for
first).

**VI. Amplification of Gene of Interest Using PCR**

- Polymerase Chain Reaction (PCR) amplifies the gene of interest.
- Requirements: DNA template, free nucleotides, primers (complementary to 3' end of template), and DNA
polymerase (e.g., Taq polymerase).

- Steps:
1. Denaturation: Heating DNA to separate strands.
2. Annealing of Primers: Cooling to allow primers to bind to complementary DNA ends.
3. Primer Extension: DNA polymerase adds complementary nucleotides to extend the primers.
4. Amplification: Replication is repeated, resulting in multiple copies of the gene of interest.

**II. Ligation of the DNA Fragment into a Vector (Preparing rDNA)**

- Cloning Vectors: DNA molecules carrying foreign DNA segments and replicating inside host cells.
- Desired DNA fragments are incorporated into the vector, which has been cut with the same restriction endonuclease
as the source DNA.

- Ligase is used to join the DNA fragments and vector, creating recombinant DNA (rDNA). Desired DNA + Vector =
rDNA

**Desirable Features of a Cloning Vector:**

(a) Presence of Ori (Origin of Replication):
- Sequence where replication starts, allowing DNA linked to ori to replicate within host cells.
(b) High Copy Number:
- Vectors that replicate to a number equal to the copy number of the vector when linked with DNA.
(c) Presence of Selectable Markers:
- Genes aiding in differentiating transformants and non-transformants.
- Allows screening for successful transformation.
- Examples: Antibiotic resistance genes (e.g., ampR, tetR), Enzyme-producing genes (e.g., lacZ gene producing β-
galactosidase).
(d) Presence of Unique Recognition Sequence in Cloning Sites:
- Should have at least one recognition site for restriction enzymes to link desired DNA.
- Multiple recognition sites may generate several fragments, complicating gene cloning.

**Types of Vectors:**
1. Plasmids:
- Circular extra-chromosomal DNA of bacteria.
- Examples: pBR322, Ti plasmid.

- pBR322 features:
- 8 restriction enzyme recognition sites.
- ori (origin of replication).
- rop gene (synthesizes proteins for plasmid replication).
- 2 selectable markers: ampicillin and tetracycline resistance
genes.

2. Retroviruses:
- Disarmed retroviruses used to transfer rDNA into animal cells.
- Retroviruses have the ability to transform normal cells into cancerous cells.

3. Bacteriophages:
- Viruses that infect bacteria, used as vectors for gene transfer.

**III. Transferring the rDNA into the Host (Transformation)**

Gene transfer can be done through two ways:
A. Vector through gene transfer
B. Vectorless gene transfer

**Vectorless Gene Transfer:**

- Direct transfer of genes into host cells using specific methods:
1. Heat-shock method: Making host cells competent (able to take up rDNA) by treating with divalent cations,
followed by a brief heat shock.
2. Micro-injection: Injecting DNA directly into the nucleus of an animal cell using a micro-needle.
3. Gene gun method: Coating micro-particles of gold or tungsten with DNA and shooting them at plant cells with
high velocity.
**Selection of Transformants:**
Selection of transformed cells (those carrying the recombinant DNA) from non-transformed ones is achieved using
selectable markers and insertional inactivation. There are two types of selection methods:
**A. Using Antibiotic Resistance Gene as
Marker:**

- Ligation of foreign DNA is carried out at a restriction

site within one of the two antibiotic resistance genes.
- For example, normal E. coli cells (non-transformants)
do not carry resistance against antibiotics.
- When pBR322 plasmid is added to E. coli,
transformants can grow on both ampicillin and
tetracycline medium, allowing differentiation from
non-transformants.
- If foreign DNA is inserted at a specific site (e.g.,
BamHI site) in the tetR gene, it undergoes insertional
inactivation, making the transformant unable to grow
on tetracycline but still able to grow on ampicillin.

**B. Using Enzyme Producing Gene as Marker:**

- This method involves insertional inactivation of the lac-Z gene and is known as the Blue-White selection method.
- Normal bacteria (non-transformants) contain the lac-Z gene on their plasmids, which produces the enzyme β-
galactosidase.
- β-galactosidase: Blue color
- Transformant bacteria with foreign DNA inserted within the lac-Z gene experience inactivation, leading to no β-
galactosidase production and resulting in white colonies. These are identified as recombinant colonies.
**IV. Culturing the Host Cells:**
- Bioreactors are used to produce large quantities of products.
- Bioreactors provide optimal growth conditions (temperature,
pH, substrate, salts, vitamins, oxygen) for achieving the desired
product.
- Types of Bioreactors: Simple Stirred-tank reactor, Sparged
stirred-tank reactor.

**V. Extraction of the Desired Product:**

- The product obtained from the bioreactor is not in pure form.
- Extraction and purification of the desired product from the culture medium is known as Downstream Processing.
- Procedure:
1. Separation and purification of gene products
2. Addition of suitable preservatives.
3. Goes through clinical trials (in the case of drugs).
4. Quality control testing.