This presentation is prepared to provide a general overview of Recombinant DNA technology. The flow of the presentation is following manner. Slide 1. Introduction Slide 2. The basic principle of Recombinant DNA technology Slide 3. Restriction endonucleases Slide 4. Cloning Slide 5. Vectors Slide 6. Transformation Slide 7. Screening of clones Slide 8. Sequencing and polymerase chain reaction Slide 9. Case Study: Disease identification and therapy discovery

1. RECOMBINANT
DNA
TECHNOLOGY
ABHISHEK GAIKAR

2. FLOW OF PRESENTATION
I. Introduction
II. Basic principle of Recombinant DNA technology
III. Restriction endonucleases
IV. Cloning
V. Vectors
VI. Transformation
VII. Screening of clones
VIII. Sequencing and polymerase chain reaction
IX. Case Study: Disease identification and therapy discovery

3. INTRODUCTION
◼ Recombinant DNA technology involves Isolation and manipulation of DNA to make a chimeric molecules
◼ End to end joining of DNA sequences from different sources (It can be human DNA sequence or Bacterial
DNA sequence)
◼ Importance of rDNA technology is
I. Molecular basis of disease (E.g. Huntington's Disease)
II. Human protein for therapy (E.g. Insulin)
III. Vaccine productions (E.g. Hepatitis B)
IV. Disease Diagnosis (E.g. HIV)
V. Designing personalised medicines
VI. Gene therapy
Source: Weil, V. R. (2018). Harper's Illustrated Biochemistry. McGraw Hill Lange.

4. BASIC PRINCIPLE OF RECOMBINANT DNA TECHNOLOGY
◼ Generation of small DNA fragments of genome and selecting the desired gene of interest
◼ Insertion of gene of interest into vectors ( e.g. Plasmid, cosmid) to create chimeric DNA
◼ Introduction of recombinant DNA to host cells
◼ Multiplication and selection of clone containing recombinant molecule
◼ Expression of selected recombinant clone to generate desired product
Gene of interest
Source: Weil, V. R. (2018). Harper's Illustrated Biochemistry. McGraw Hill Lange.
Figure 1: Insulin production scheme using rDNA technology

5. PROCESS OVERVIEW
Clones
Desired
Clone
selection
Blotting and
Probing techniques
helps to identify the
desired product.
Sequencing
of selected
clones
PCR Amplification
of gene of Interest
Source: Weil, V. R. (2018). Harper's Illustrated Biochemistry. McGraw Hill Lange.
Figure 2: General process flow for rDNA technology

6. RESTRICTION ENDONUCLEASES
◼ Restriction enzymes (RE), are bacterial enzymes that cuts/ splits target DNA at specific site
◼ RE’s are defensive system of bacteria
◼ Enzyme activity are seen at unique recognition sequence to generate cohesive ends or blunt ends
◼ Enzymes are named after the bacterium from which they have isolated (Eco-RI)
◼ Same restriction sites on vector and gene of interest is essential to carry out recombination
Source: Weil, V. R. (2018). Harper's Illustrated Biochemistry. McGraw Hill Lange.
Figure 3: Recognition sequence and restriction reaction of BamHI

7. CLONING
◼ Cloning allows for the production of large number of identical DNA molecules
◼ Chimeric molecules are constructed using cloning vectors like Plasmid, Cosmid and phages
◼ Gene insert size influences the vector selection
◼ Complementary cohesive end resulted from REs are joined together
◼ DNA Ligase helps in Phosphodiester bond formation
Circular plasmid
DNA
EcoRI restriction
endonucleases
Linear plasmid DNA with sticky ends
EcoRI restriction
endonucleases
cleavage
Human DNA
Place of human DNA cut with EcoRI
restriction nucleases contains the
same sticky ends as the EcoRI-
digested plasmid
Plasmid DNA Molecules with
human insert (Recombinant
DNA molecule)
Anneal
Source: Weil, V. R. (2018). Harper's Illustrated Biochemistry. McGraw Hill Lange.
Figure 4: Cloning scheme

8. VECTORS
Vectors DNA insert Size (kb)
Plasmid 0.01-10
Cosmid 10-20
Lambda Phage 35-50
Bacterial Artificial Chromosomes (BAC) 50-250
Yeast Artificial Chromosomes (YAC) 500-300
PLASMID
Source: 1. Weil, V. R. (2018). Harper's Illustrated Biochemistry. McGraw Hill Lange., 2. Product information sheet , Mo Bi Tech.
Table 1: Different types of vectors and respective gene insert size (kb) [1]
Figure 5: Vector map for pUC19 [2]

9. TRANSFORMATION
◼ Insertion of recombinant plasmid to bacterial cell
◼ Methods of Transformation
I. Chemical method (Calcium chloride -Heat shock method)
II. Physical method (Electroporation)
Source: Weil, V. R. (2018). Harper's Illustrated Biochemistry. McGraw Hill Lange.
Recombinant Plasmid
with gene of Interest
inserted
Transformed Bacterial
cells
Figure 6: Transformation process in bacterial cell

10. SCREENING OF CLONES
◼ Whole genome fragments are packed into numerous plasmid
◼ Distinguish between recombinant and non recombinant plasmids
◼ Few screening methods:
1. Disruption of functional region of plasmid-
a. Double positive/ negative selection technique-Plasmid containing two antibiotic resistance marker
b. Blue white screening- β- Galactosidase mediated colour conversion, X-Gal substrate, white colour
colonies are positive
2. Blotting Techniques
a. Southern Blotting- DNA
b. Northern Blotting-RNA
c. Western Blotting- Proteins
Source: Weil, V. R. (2018). Harper's Illustrated Biochemistry. McGraw Hill Lange.

11. SEQUENCING & POLYMERASE CHAIN REACTION
◼ Sequencing- Target sequence obtained during blotting, can be analyzed using sanger dideoxy
sequencing method
◼ Target gene then can amplified using Polymerase gene reaction using heal stable Taq polymerase
Source: Weil, V. R. (2018). Harper's Illustrated Biochemistry. McGraw Hill Lange.
Figure 7: Polymerase Chain Reaction Cycle

12. CASE STUDY: Disease Identification and Therapy Discovery
◼ Huntington’s disease, mutation in exon 1 of IT15 chromosome 4 leads to increase in polyglutamine
repeats ( 37 Q being normal, mutation causes 40-200 Q repeats)
◼ Increase in ‘CAG’ repeats causes 3-D structure disruption of huntingtin protein leads to aggregation
◼ Developing the molecular medicines which prevents this aggregation will improves the disease condition
◼ Aptamer being molecular medicines can be studied using rDNA technology
With RNA Aptamer
Without RNA Aptamer
Mutated huntingtin
protein
Aggregated Mutated huntingtin protein
RNA-Mutated huntingtin protein
Complex
Source: Ipsita Roy., et al. "Inhibition of aggregation of mutant huntingtin by nucleic acid aptamers in vitro and in a yeast model of Huntington's disease." Molecular
Therapy 23.12 (2015): 1912-1926.
Figure 8: Mutant huntingtin protein and RNA aptamer interaction

13. THANK YOU