UNIT 1 NOTES — Basic Principles of rDNA Technology

Subject: rDNA Technology (ABT0405)

Based on Unit-1 syllabus and PYQs.

1. Recombinant DNA (rDNA) Technology

Definition

Recombinant DNA technology is the technique of joining DNA from different organisms to form
a new DNA molecule.

It is used to insert a desired gene into a host organism so that the host can produce useful
proteins.

Example:

 Human insulin gene inserted into bacteria for insulin production.

Uses of rDNA Technology

1. Medicine

 Insulin production

 Vaccine production

 Gene therapy

2. Agriculture

 Bt cotton

 Golden rice

 Disease resistant plants

3. Industry

 Enzyme production

 Biofuel production

4. Research

 Gene cloning
  DNA sequencing

 Genetic engineering

2. Basic Steps of rDNA Technology

Step 1: Identification of Desired Gene

The useful gene is identified from donor DNA.

Example:
Insulin gene from human DNA.

Step 2: Cutting DNA

Restriction enzymes cut:

 Desired gene

 Vector DNA

These enzymes act as molecular scissors.

Step 3: Preparation of Vector

A vector carries foreign DNA into host cell.

Common vector:

 Plasmid

Step 4: Insertion of Gene into Vector

DNA ligase joins:

 Vector DNA

 Foreign DNA

Result:
Recombinant DNA molecule.
Step 5: Transformation

Recombinant DNA enters host cell.

Example:
E. coli bacteria.

Step 6: Expression

Host cell produces desired protein.

Step 7: Purification

Protein is collected and purified.

Example:
Insulin purification.

3. Gene Cloning

Definition

Gene cloning is the process of making many copies of a desired gene.

It involves:

 Isolation of gene

 Insertion into vector

 Transfer into host cell

 Multiplication

Applications

 Insulin production

 Vaccine production

 Research studies
4. Molecular Tools of Genetic Engineering

Main tools:

1. Restriction enzymes

2. DNA ligase

3. T4 DNA polymerase

4. Reverse transcriptase

5. Alkaline phosphatase

5. Restriction Enzymes

Definition

Restriction enzymes are enzymes that cut DNA at specific nucleotide sequences called
recognition sites.

They break phosphodiester bonds.

Recognition Site

Usually palindromic sequence.

Example:

5'\text{-}GAATTC\text{-}3' \ 3'\text{-}CTTAAG\text{-}5'

Recognized by EcoRI.

6. Types of Restriction Enzymes

Type I Restriction Enzymes

Features

 Cut DNA far from recognition site

 Require ATP, Mg²⁺ and SAM

 Restriction and methylation together

 EcoKI

Uses

Not useful in genetic engineering because cutting is random.

Type II Restriction Enzymes

Features

 Cut at recognition site

 Highly specific

 Require only Mg²⁺

Examples

 EcoRI

 HindIII

 BamHI

Importance

Most commonly used in:

 Gene cloning

 rDNA technology

 DNA fingerprinting

Type III Restriction Enzymes

Features

 Cut 25–30 bp away from recognition site

 Require ATP, Mg²⁺ and SAM

Example

 EcoPI
Uses

Rarely used in cloning.

7. Comparison of Restriction Enzymes

Feature Type I Type II Type III

Cutting site Far away At site 25–30 bp away

ATP required Yes No Yes

Precision Low High Moderate

Used in cloning No Yes Rare

8. Restriction–Modification System

Bacteria protect their own DNA using methylation.

Two components:

1. Restriction enzyme → cuts foreign DNA

2. Methyltransferase → protects bacterial DNA

9. Blunt Ends and Sticky Ends

Blunt Ends

Straight cut without overhangs.

Features

 No single stranded tail

 Less efficient ligation

Examples

 SmaI

 EcoRV
Sticky Ends

Staggered cut with overhangs.

Features

 Better ligation

 More stable

 Directional cloning possible

Examples

 EcoRI

 HindIII

10. Difference Between Blunt and Sticky Ends

Feature Blunt Ends Sticky Ends

Overhang Absent Present

Ligation efficiency Low High

Specificity Low High

Stability Less More

11. Nomenclature of Restriction Enzymes

Restriction enzymes are named according to source bacteria.

Example:
EcoRI

 E → Escherichia

 co → coli

 R → strain

 I → first enzyme isolated

Definition

Different enzymes that recognize and cut same DNA sequence.

Example

 HpaII

 MspI

Both recognize:
CCGG

13. Neoschizomers

Definition

Enzymes recognizing same sequence but cutting at different positions.

Example

 SmaI

 XmaI

14. Heterohypokomers

Definition

Enzymes recognizing different sequences but producing same sticky ends.

Example

 BamHI

 BglII

Both produce:
5′–GATC–3′ overhang.

15. Unit of Restriction Enzyme

Definition
One unit of restriction enzyme is the amount needed to completely digest 1 µg DNA in 1 hour
under optimal conditions.

16. Restriction Digestion

Definition

Restriction digestion is the cutting of DNA using restriction enzymes.

Applications:

 Cloning

 DNA mapping

 Genetic engineering

17. Complete Digestion

Definition

All recognition sites are cut.

Conditions

 Excess enzyme

 Proper temperature

 Proper buffer

 Enough incubation time

18. Partial Digestion

Definition

Only some recognition sites are cut.

Causes

 Less enzyme

 Short incubation
  Poor conditions

Importance

Used for genomic library preparation.

19. Star Activity

Definition

Star activity is non-specific cutting of DNA by restriction enzymes under abnormal conditions.

Example:
EcoRI normally cuts:
GAATTC

During star activity it may cut similar sequences.

Causes

 High enzyme concentration

 Long incubation

 Wrong buffer

 High glycerol

 Low ionic strength

Effects

 Wrong DNA fragments

 Cloning problems

 Experimental errors

20. Homopolymer Tailing

Definition

Addition of same nucleotide repeatedly at 3′ end of DNA.

Terminal deoxynucleotidyl transferase (TdT)

Principle

TdT adds nucleotides without template.

Example:

 Poly A tail

 Poly G tail

Uses

 Formation of sticky ends

 Cloning blunt-ended DNA

21. Linkers

Definition

Short synthetic double stranded DNA containing restriction enzyme site.

Functions

 Convert blunt ends into sticky ends

 Improve ligation efficiency

Steps

1. Ligate linker to blunt DNA

2. Cut with restriction enzyme

3. Sticky ends formed

Definition

Short oligonucleotides having:

 One sticky end

 One blunt end

Uses

 Join blunt DNA to sticky vector

 Increase cloning efficiency

23. Difference Between Linkers and Adaptors

Feature Linkers Adaptors

Ends Both blunt One blunt, one sticky

Restriction digestion needed Yes No

Efficiency Lower Higher

Self ligation Higher Lower

24. DNA Ligase

Definition

DNA ligase joins DNA fragments by phosphodiester bond formation.

Functions

 Joins insert and vector

 Forms recombinant DNA

 Repairs DNA
25. T4 DNA Polymerase

Functions

1. 5′ → 3′ Polymerase Activity

Adds nucleotides.

2. 3′ → 5′ Exonuclease Activity

Removes nucleotides.

Uses in rDNA Technology

 Converts sticky ends into blunt ends

 DNA proofreading

 Fill gaps in DNA

26. Why Blunt Ends Are Needed

 Different enzymes produce incompatible ends

 No suitable restriction sites available

 Universal ligation possible

27. Alkaline Phosphatase

Definition

Enzyme that removes 5′ phosphate groups from DNA.

Importance

Prevents vector self-ligation.

Without phosphate:
Ligase cannot join vector ends.
Types

 CIAP

 SAP

 BAP

28. Reverse Transcriptase

Definition

Reverse transcriptase is RNA-dependent DNA polymerase.

It converts RNA into cDNA.

Importance

Bacteria cannot process introns.

Therefore:
mRNA → cDNA → cloning.

29. cDNA

Definition

Complementary DNA produced from mRNA.

Features

 No introns

 Used for cloning eukaryotic genes

30. Applications of Reverse Transcriptase

1. cDNA Library Construction

Used to prepare cDNA libraries.

2. RT-PCR
RNA converted to cDNA before PCR.

3. Gene Cloning

Cloning of expressed genes.

4. Viral Research

Used in HIV studies.

Important PYQ Topics

1. Restriction enzymes and types

2. Star activity

3. Homopolymer tailing

4. Linkers vs Adaptors

5. DNA ligase role

6. T4 DNA polymerase role

7. Reverse transcriptase in cloning

8. Gene cloning steps

9. Restriction digestion

10. Isoschizomers and heterohypokomers