Recombinant DNA technology allows for the isolation, cloning, and manipulation of genes. Two key advances enabled this field: genetic engineering using restriction enzymes to isolate and modify genes in vitro, and DNA sequencing to determine the order of nucleotides. Recombinant DNA is generated by joining DNA from different sources, and molecular cloning produces large quantities of a particular DNA fragment through construction of a recombinant vector, introduction into a host cell, selective propagation of cells containing the vector, and extraction of the cloned DNA.

1. Recombinant DNA technology
and Cloning
Mai Masri
Dep. of Zoology
University of Khartoum

2. Recombinant DNA technology
Two primary advances helped in the new era of molecular
genetics in the late 1970s to the early 1980s:
(1) The use of recombinant DNA technology (genetic
engineering) used to isolate and manipulate genes in
vitro in order to endow cells with new synthetic
capabilities (restriction enzymes)
(2) The ability to synthesize and determine the linear
order of nucleotides of DNA molecules (DNA
sequencing).

3. Advances of DNA technology
Recombinant DNA technology has made it possible
to:
• Clone (isolate and make copies of individual
genes).
• Transfer genes between bacterial species and strains
or from eukaryotes into bacteria (or vice versa).
• Causing the engineered cells to produce, sometimes
in relatively large quantities, proteins of great
economic importance such as enzymes (e. g. ,
amylases, proteases), hormones (e. g. , insulin,
growth hormone).

4. Recombinant DNA
• Recombinant DNA is
generated by covalently
joining DNA molecules
from different sources.
• The technology associated
with the construction
application of recombinant
DNA is referred to as
genetic engineering.

6. Molecular coloning
• Molecular cloning is an in vivo technique for the
production of large quantities of a particular DNA
fragment.
• It contains four major steps:
1. Construction of recombinant vector.
2. Induction of the recombinant vector into suitable
host cell.
3. Selective propagation of cells containing the
vector(cloning).
4. Extraction and purification of the cloned DNA

7. Construction of recombinant
vector, which involve cutting,
modifying and joining donor
and vector DNA in vitro.
Induction of the recombinant
vector into suitable host cell.
Extraction and purification of
the cloned DNA
Selective propagation of cells
containing the vector(cloning).

8. Restriction Endonucleases
Using restriction enzymes II to create recombinant
DNA
• Cutting DNA by using restriction enzymes is
one of the most common Molecular Biology
techniques. The cut ends can be joined using
DNA ligase.
• The availability of pure restriction enzymes was
one of the first major advances in the new
science of Molecular Biology.

9. Restriction Endonucleases
restriction enzymes
• These enzymes occur naturally in bacteria.
• Enzymes that “cut” DNA in a sequence-specific
manner. recognition sequence
• Serve as a natural defense mechanism for bacteria
against viral infection (bacteriophage).
• Bacteria protect their DNA from cutting by their own
enzymes through methylation.

10. • The simplest way of cloning is to cut the donor
and the vector DNA with the same enzyme and
then join them with ligase.

11. Examples
Enzyme Recognition sequence
• EcoRI GAATTC
• HindIII AAGCTT
• BamHI GGATCC
• EcoRV GATATC
• Recognition sequences are usually 4-8 base
pairs in length and are usually palindromic

12. Restriction Map

13. Cloning vectors
• DNA fragment does not contain origin of
replication (or a replicon).
• It should be joined to a replicon (vector).
• Those include : Plasmids, Viruses and
chromosomes.

14. An ideal cloning vector should be:
1. Episomal (do not integrate to the host genome,
can be separated easily).
2. Replicate autonomously giving high copy
number.
3. Allow the identification of DNA-carrying
vector those lacking them.
4. Allow the identification of cells (host) crying
DNA-carrying vector.
5. Should maintain characters that enable them to
be used in applications after cloning.

15. • Plasmids and bacteriophages are considered as
naturally poor vectors. So they are
manipulated by entering restriction sites
without affecting the sequence of the plasmid
or bacteriophages.
1. Plasmids
• Naturally occurring in bacteria.
• Used to copy already present copy of a gene
in the genomic library (sub-cloning).
• Can be used in post transcriptional activities
(small and easy to deal with).

16. 2- Bacteriophage λ:
• Used in DNA libraries.
• Can contain larger inserts
• Can be stored for long periods.
3- Cosmids
• Vectors constructed from both plasmids and bacteriophages
• Can contain large inserts thats why used in genomic libraries.
4-Phagemids:
• Plasmids that contain the OriC of the M13 Bacteriophage.
Thus can produce single stranded DNA ( can be used for
sequencing, probe synthesis and mutagenesis)
5-phasmids:
Used in post-transcriptional applications.
6-Artificail chromosomes
Can carry large DNA sequences
YACs
BACs

17. Types of Cloning Vector
Types size of cloned DNA (kb)
Plasmid 20
Lambda Phage 25
Cosmid 45
P1 phage 100
BAC 300
YAC 1000

19. ex. pBR322
• Is a plasmid and was the first
widely-used E. coli cloning vectors.
• Created in 1977 in the laboratory
of Herbert Boyer (University of
California San Francisco).
• It was named after the Mexican
postdoctoral researchers who
constructed it (p stands for
"plasmid," and BR for "Bolivar"
and "Rodriguez.“
• pBR322 is 4361 base pairs in
length and contains the replicon of
plasmid pMB1.

22. DNA ligases
• DNA ligases catalyze formation of a phosphodiester bond
between nucleotides.
• This enzyme is used to covalently link or ligate fragments of
DNA together.
• Most commonly, the reaction involves ligating a fragment of
DNA into a plasmid vector, which is a fundamental technique
in recombinant DNA work.
• One of the most used enzymes to ligate DNA fragments is T4
DNA ligase, which originates from the T4 bacteriophage

23. DNA transfer to the cloning host
• Once recombinant vector has been constructed in
vitro it should be introduce into a host cell.
• E.coli is the major host cells in general, however
it can not introduce the DNA naturally into the
cell. To over come that problem:
• 1- Electroporation (electron transformation
through high voltatge).
• 2-Heat shock
• 3-Treatment with calcium ions Ca++.

25. Vector and recombinant selection
Construction of recombinant DNA and transfection
are not always 100% successful.
Vector selection (test transfection):
• Markers: Antibiotic resistance marker.
Only the colonies that containing the plasmid can
grow in a medium containing that antibiotic.
Selection of recombinant DNA(test recombination):
• Blue- white selection:
Insert disrupts a marker (gene) that turns the cell into
blue if not distrusted hence has white color if
disrupted.

26. Selection of colonies

27. Copyright © 2010 Academic
Press Inc.
27
Figure 22.16
Copyright © 2010 Academic Press Inc.

28. Recovery of cloned DNA
• The cells are cultured in solid media.
• The colonies that contain the insert are selected by
the previous methods.
• Transferred into a liquid media to generate large
quantities from it.
• Collection of host cells to re-extract the plasmids:
• Using cloning we can generate libraries for genes or
parts of genes that contain thousands of copies

29. DNA Library
1. Genomic libraries:
Collection of DNA sequences from a living
organism that has been copied into a vector
so as to be used and stored and analyzed.
2. Complementary DNA (cDNA) libraries.
Only coding parts of the genome

30. How to construct a genomic library
• The construction of a genomic library begins with cleaving
the genome into small pieces by a restriction endonuclease.
• These genomic fragments are then either cloned into
vectors & introduced into a microbe or packed into phage
particles that are used to infect the host.
• At the end many thousands of different clones each with a
different genomic DNA insert –are created.
• Therefore each clone will act as a “book” in this “library”
of DNA fragments.
• If the genomic library has been inserted into a microbe that
expresses the foreign gene, it may be possible to assay each
clone for a specific protein or phenotype

31. Digestion of the chromosomal DNA
with restriction endonuclease
Production of cDNA
(complimentary DNA)
Genomic Library
Insertion of each DNA fragment into
vector (recombinant DNA)
Transformation of Bacteria
using the recombinant vectors
Cloning of
the bacterial cells
Each clone produced is a “book”
in the “Library” of DNA fragments
cDNA Library
Extraction of mRNA
Extraction of chromosomal
DNA
Insertion of each cDNA into vector
(recombinant DNA)
Transformation of bacteria
Cloning of each recombinant
Production of Bacterial cell (clones)
containing the gene of interest