molecular tech. rdt 11 50-2020

1. Dr. Vishnu Kumar
Professor, Department of
Biochemistry, SRMS IMS, Bareilly
vkawasthi@hotmail.com
madhwapur1976@gmail.com
Recombinant DNA Technology
and its Applications

2. Learning Objectives
After completion of this lecture learner
should be able to define:
 Recombinant DNA Technology
 Restriction Endonucleases
Sticky Ends and Blunt ends
 DNA Cloning
 Vectors (plasmids, Bacteriophases and cosmids)
 Gene Therapy
 DNA Library
 Applications of RDT

3. Recombinant DNA technology
This is a novel technology in which DNA
sequence is manipulated and chimeric DNA
molecule is produced by a method called
genetic engineering.
Next the chimeric DNA molecule is allowed
to multiply.
This is a biological method of DNA
amplification.

4. • This technology has revolutionized
Biology and is having an ever
increasing impact on clinical medicine.

7. Enzyme used in RDT
Enzyme(s) Function
Type II restrictions
endonucleases
Cleave DNAs at specific base sequences
DNA ligase Joints two DNA molecules or fragments
DNA polymerase I (E.coli) Fills gaps in duplexes by stepwise
addition of nucleotides to 3’ ends
Reverse transcriptase Makes a DNA copy of an RNA molecule
Polynucleotide kinase Adds a phosphate to the 5’-OH end of a
polynucleotide to label it or permit ligation
Terminal transferase Adds homopolymer tails to the 3’-OH end
of a linear duplex
Exonuclease III Removes nucleotides residues from the 3’
ends of a DNA strand
Bacteriophage λ Exonuclease Removes nucleotides from the 5’ ends of a
duplex to expose single –stranded 3’ ends
Alkaline phosphatase Removes terminal phosphates from either
the 5’ or 3’ end (or both)

9. Sticky ends & Blunt ends
• Some restriction endonucleases make
staggered cuts on the two DNA strands,
leaving two to four nucleotides of one strand
unpaired at each resulting end.
• These unpaired strands are referred to as
sticky ends, because they can base-pair
with each other or with complementary sticky
ends of other DNA fragments.
• Other restriction endonucleases cleave both
strands of DNA at the opposing
phosphodiester bonds, leaving no unpaired
bases on the ends, often called blunt ends.

10. Sticky & Blunt ends
haeIII
G G C C
C C G G
EcoRI
G AA T T C
C T T AA G

14. Few related definitions
• Clone  population of cells having the
same genetic composition and derived
from a single cell by repeated mitosis.
• DNA cloning  Involves isolation of
DNA fragments and their insertion into
the DNA from another biological source
( vector ) for manipulation and
multiplication.
• To clone means to make identical
copies.

15. Some other terms.
• Vector  they are small molecules to which the
target DNA is attached. There are three types of
vectors : plasmids, bacteriophages and cosmids.
• Polylinker is a shortsegment of DNA which
contains up to 20 restriction sites.
• Restriction Endonucleases ( R. E.)  Enzymes
that cleave double stranded DNA at specific sites
called restriction sites.

17. Application of R. D. T.
1) Diagnosis of diseases like sickle
cell anemia, thalassemia.
2) Production of proteins for
treatment e.g. insulin, growth
hormone.
3) Production of vaccines e.g.
hepatitis B.
4) Forensic help e.g. paternity test,
prenatal diagnosis and detection of
criminal.

18. Application of R. D. T.
• 5) Gene therapy for potentially curing
diseases caused by a single gene
deficiency, e. g. sickle cell disease,
thalassemia.
• 6) offers rational approach to
understand the molecular basis of a
disease e.g. sickle cell anemia.

19. Procedure.
• 1) Isolation of gene gene is selected
and cut at precise location by R.E.
which act as molecular scissors.
• A vector is selected and cut by the
same R.E.
• The segment of DNA to be cloned is
joined to the vector. This composite
DNA is called recombinant DNA.

20. Procedure
• The recombinant DNA is introduced
into bacterial cell. This step is called
transfection. Now each transformed
cell would multiply and amplify the
target DNA in huge amount.
• The cells are grown under conditions
that are favorable for protein synthesis.
The proteins are isolated from these
cells by usual method. Example of
clinically important protein is INSULIN.

21. vector
• The vector is a self replicating entity
which is capable of carrying target DNA
into the host cell. Essential properties
of a cloning vector are as follows:
• 1) It must be capable of autonomous
replication.
• 2) It must have at least one specific region
for restriction endonuclease.

22. vector
• A cloning vector may be a plasmid,
bactriophage, or a cosmid. An essential
technique in genetic engineering is to
introduce the human gene into a
vector. Covalent binding of the two
results in production of recombinant
DNA, which is then carried to a suitable
host cell. The inserted gene is then
replicated, transcribed and translated.

23. Plasmid.
• Plasmids are circular, extra
chromosomal, DNA molecules of
2000-200,000 bps present in some
prokaryotic cells.
• A single cell may contain 10- 20
copies of plasmids. They undergo
replication with each cell cycle.

24. Plasmid.
• Plasmids carry genes that impart
antibiotic resistance.
• Their function is to deliver a desired
DNA segment into a host cell, the
process is called transfection.
• Properties1) they can pass from one
cell to another. 2) foreign genes can be
inserted into plasmids quite easily.

26. Bacteriophage.
• Lambda phage can carry DNA inserts
much larger than that carried by
plasmids.
• It is a linear double stranded DNA.
• Foreign can be inserted easily.

28. Cosmids.
• A cosmid is an artificially constructed
circular DNA molecule.
• It combines useful features of both the
plasmid and phage.
• It has an antibiotic resistant gene and
several restriction sites.
• Permits insertion of large fragments of
DNA.

30. DNA Library.
• 1) Genomic library Refers to a large
collection of bacterial cells, each containing
a random piece of human genomic DNA
attached to a vector.
• Entire DNA of a cell is cut into pieces by
using different R.E. these cut fragments are
introduced into appropriate vector.
• These recombinant DNAs are introduced into
bacteria to form a genomic library.

32. DNA Library.
• cDNA Library It is a collection of all the
expressed DNA of a particular cell type.
• For building cDNA library the mRNA is
extracted from a tissue. Taking the mRNA as
a template cDNA is synthesized by the
enzyme reverse transcriptase. From the
cDNA d/s DNA is obtained by using DNA
polymerase ,which is attached to a vector
and introduced into bacteria.

34. Gene Therapy.
• Gene therapy is a rapidly growing field
of medicine in which defective genes
causing diseases are substituted by
correct genes in the patient to cure a
disease.
• Diseases caused by deficiency of a
single gene product can be treated by
gene therapy e.g. thalassemia, sickle
cell anemia, phenyl ketonuria, Lesh-
Nyhan syndrome.

35. LONG ANSWER QUESTIONS
1. Describe the RDT and its application in
detail with suitable figures.

36. SHORT ANSWER QUESTIONS
1.Sticky ends and Blunt ends Restriction
Endonucleases with suitable figures.
2.Gene Therapy
3.DNA Library
4.Vector
5.Plasmids, Bacteriophases & Cosmids

37. MCQ RDT & its Applicatipon
1. Which enzyme is used for preparing
Recombinant DNA molecule
a. Restriction Endo-nuclease
b. RNA Polymerase
c. DNA Polymerase
d. Topoisomerase
Answer: a

38. 2. The cDNA is prepared by using the enzyme:
a.RNA Polymerase
b. DNA Polymerase
c. Reverse transcriptase
d.Restriction Endonuclease
Answer: c