Introduction to Genetic engineering & rDNA technology, Methodologies and enzymes involved in r-DNA technology

1. INTRODUCTION
TO RDNA
TECHNOLOGY
Dr. S. Sivaranjani
Assistant Professor
Dept.of Biotechnology
Bon Secours College for Women
Thanjavur

2. RECOMBINANT DNA TECHNOLOGY
● A technique mainly used to change the phenotype of an organism (host) when a genetically altered vector is
introduced and integrated into the genome of the organism. So, basically, this process involves the introduction
of a foreign piece of DNA structure into the genome which contains our gene of interest. This gene which is
introduced is the recombinant gene and the technique is called the recombinant DNA technology.
● The technology used for producing artificial DNA through the combination of different genetic materials (DNA)
from different sources is referred to as Recombinant DNA Technology. Recombinant DNA technology is
popularly known as genetic engineering.
● The recombinant DNA technology emerged with the discovery of restriction enzymes in the year 1968 by Swiss
microbiologist Werner Arber.
● This process involves the introduction of a foreign piece of DNA structure into the genome which contains our
gene of interest. This gene which is introduced is the recombinant gene and the technique is called the
recombinant DNA technology.

3. MINIMALIST AESTHETIC SLIDESHOW INFOGRAPHICS
STEP-1
Isolation Of Genetic
Material
STEP-3
Amplifying The Gene
Copies Through PCR
STEP-5
Insertion Of RDNA Into Host
STEP-2 Cutting The Genes At
The Recognition Sites
STEP-4 Ligation Of The DNA
Molecules

4. Step-1. Isolation of Genetic Material.
The first and the initial step in Recombinant DNA technology is to isolate the desired DNA in its pure form i.e. free
from other macromolecules.
Step-2.Cutting the gene at the recognition sites.
The restriction enzymes play a major role in determining the location at which the desired gene is inserted into the
vector genome. These reactions are called ‘restriction enzyme digestions’.
Step-3. Amplifying the gene copies through Polymerase chain reaction (PCR).
It is a process to amplify a single copy of DNA into thousands to millions of copies once the proper gene of
interest has been cut using restriction enzymes.
Step-4. Ligation of DNA Molecules.
In this step of Ligation, the joining of the two pieces – a cut fragment of DNA and the vector together with the help
of the enzyme DNA ligase.
Step-5. Insertion of Recombinant DNA Into Host.
In this step, the recombinant DNA is introduced into a recipient host cell. This process is termed
as Transformation. Once the recombinant DNA is inserted into the host cell, it gets multiplied and is expressed in
the form of the manufactured protein under optimal conditions.

6. ENZYMES INVOLVED IN RDNA TECHNOLOGY
1.DNA ligase
2.Reverse transcriptase
3.Restriction endonuclease
4.Terminal transcriptase
5.Nuclease
6.DNA polymease
7.Ribonuclease-H
8.Alkaline phosphatase
9.Polynucleotide kinase

7. DNA LIGASE
• DNA ligase is isolated from E.coli and Bacteriophage commercially and used in
recombinant DNA technology.
• The enzyme DNA ligase joins the DNA fragments with cloning vector.

8. REVERSE TRANSCRIPTASE
• It is also called RNA-directed DNA polymerase, an enzyme encoded from the genetic material
of retroviruses that catalyzes the transcription of retrovirus RNA (ribonucleic acid)
into DNA (deoxyribonucleic acid). This catalyzed transcription is the reverse process of normal
cellular transcription of DNA into RNA, hence the names reverse transcriptase and retrovirus.
Reverse transcriptase is central to the infectious nature of retroviruses, several of which cause
disease in humans, including human immunodeficiency virus (HIV).
• Reverse transcriptase is also a fundamental component of a laboratory technology known
as reverse transcription-polymerase chain reaction (RT-PCR), a powerful tool used in research
and in the diagnosis of diseases such as cancer.

9. RESTRICTION ENDONUCLEASE
• Endonucleases can recognize specific base sequence (restriction site) within DNA or RNA molecule and
cleave internal phosphodiester bonds within a DNA molecule. Example: EcoRI, Hind III, BamHI etc.
• These enzyme is isolated from wide variety of microorganisms. Endonuclease enzyme degrades foreign
genome when enter inside microbial cell but the host cell own DNA is protected from its endonuclease by
methylation of bases at restriction site.
There are 3 types of restriction endonuclease:
1. Type I Restriction endonuclease:
It has both methylation and endonuclease activity.
It require ATP to cut the DNA
It cuts DNA about 1000bp away from its restriction site
eg. EcoKI
2. Type II Restriction endonuclease:
It does not require ATP to cut DNA
It cuts DNA at restriction site itself
eg. EcoRI, Hind III
3. Type III Restriction endonuclease:
It requires ATP to cut DNA
It cuts DNA about 25bp away from restriction site.
eg. EcoPI

10. Property Type I RE Type II RE Type III RE
Abundance Less common than
Type II
Most common Rare
Recognition site Cut both strands at
a non- specific
location > 1000 bp
away from
recognition site
Cut both strands at
a specific, usually
palindromic
recognition site (4-
8 bp)
Cleavage of one
strand, only 24-26
bp downstream of
the 3´ recognition
site
Restriction and
modification
Single
multifunctional
enzyme
Separate nuclease
and methylase
Separate enzymes
sharing a common
subunit
DNA cleavage
requirements
Two recognition
sites in any
orientation
Single recognition
site
Two recognition
sites in a head-to-
head orientation

11. TERMINAL TRANSCRIPTASE
• It is the enzyme that converts blunt end of DNA fragments into sticky end.
• If the restriction enzyme cuts DNA forming blunt ends, then efficiency of ligation is very low. So the
enzyme terminal transferase converts bunt end into sticky end.
• Terminal transferase enzyme synthesize short sequence of complementary nucleotide at free ends of
DNA, so that blunt end is converted into sticky end.

12. NUCLEASE
• The enzyme nucleases hydrolyses the phosphodiester bond on DNA
strand creating 3’-OH group and 5’-P group.
• It usually cut DNA on either side of distortion caused by thymine dimers or
intercalating agents
• The gap is filled by DNA polymerase and strand is joined by DNA ligase
• Nucelase are of two types; endonuclease and exonuclease

13. DNA POLYMERASE
• DNA polymerase is a complex enzyme which synthesize nucleotide
complementary to template strand.
• It adds nucleotide to free 3′ OH end and help in elongation of strand
• It also helps to fill gap in double stranded DNA.
• DNA polymerase-I isolated from E. coli is commonly used in gene cloning
• Taq polymerase isolated from Thermus aquaticus is used in PCR

14. RIBONUCLEASE –H [RNASE H]
• Reverse transcriptases are enzymes composed of distinct domains that exhibit different
biochemical activities. RNA-dependent DNA polymerase activity and RNase H activity are the
predominant functions of reverse transcriptases, although depending on the source
organisms there are variations in functions, including, for example, DNA-dependent DNA
polymerase activity.
• RNase H cleaves the RNA template of the RNA:cDNA hybrid concurrently with
polymerization.The RNase H activity is undesirable for synthesis of long cDNAs because the
RNA template may be degraded before completion of full-length reverse transcription. The
RNase H activity may also lower reverse transcription efficiency, presumably due to its
competition with the polymerase activity of the enzyme.

16. ALKALINE PHOSPHATASE
• The enzyme Alkaline phosphatase helps in removal of terminal phosphate group from 5′ end
• It prevents self annealing of vector DNA soon after cut open by restriction endonuclease

17. THANK YOU