This document describes the process of constructing a recombinant DNA molecule: 1. DNA/RNA is isolated and the gene of interest is amplified via PCR or RT-PCR. 2. Restriction enzymes are used to cut the DNA molecule into fragments that are inserted into a plasmid vector. 3. The recombinant molecule is transformed into E. coli host cells which then produce many copies of the molecule that can be isolated.

1. How to construct a recombinant
DNA molecule?
• DNA/RNA isolation
• Gene isolation by PCR/RT-PCR
• Or Cutting of DNA molecule with the help of
restriction enzymes
• Transfer of DNA molecule into a suitable vector
with the help of DNA ligases
Transformation of recombinant molecule into
suitable host like E. coli
• Production of large number of copies of the
recombinant molecule in the host
• Checking the gene expression

2. By Prof Amer Jamil

3. Overexpression of proteins

5. • TYPE I: Restrict away from recognition
site (~1000 bp away)
•TYPE II: Restrict within recognition site
•TYPE III: Restrict away from recognition
site (~26 bp away)
Restriction endonucleases

6. • Also called restriction enzymes
• Occur naturally in bacteria
• Hundreds are purified and available commercially
• Named for bacterial genus, species, strain, and type
Example: EcoRI
Genus: Escherichia
Species: coli
Strain: R
Type II Restriction endonucleases

7. 4-7
Restriction Endonuclease Specificity
Restriction endonucleases
recognize a specific DNA
sequence, cutting ONLY at
that sequence
– They recognize 4-bp, 6-bp,
8-bp palindromic sequences
– The frequency of cuts
lessens as the recognition
sequence is longer
– They cut DNA reproducibly
in the same place

8. 4-8
Restriction-Modification System
• What prevents these
enzymes from cutting up
the host DNA?
– They are paired with
methylases (modification
methylase)
– Theses enzymes recognize,
methylate the same site
• Together they are called a
restriction-modification
system, R-M system
• Methylation protects DNA,
after replication the parental
strand is already
methylated

9. Restriction enzymes conditions
• Buffer systems (4 buffer system): pH and ionic
strength
• Optimal temperature
• Star activity (non-specific cutting)
• Enzyme activity (IU; umoles of substrate
transformed into product in one minute).
For restriction enzymes: Enzyme required to
digest 1 ug double stranded DNA in 60 minutes)
• Isoschizomers (two different enzymes
recognizing the same sequence

11. Gene is transferred to a suitable vector
• Plasmids:
– pUC18 (Expression vector)
– pBR322 (cloning vector)
• COSMID
• Lambda vector
• Agrobacterium tumefaciencs (used for plants)
• YAC (yeast artificial chromosome)
» And many more
Commonly used vectors
Vectors have selectable markers, origin of replication
and increased copy numbers

12. Plasmids pBR322 and pUC
Three most important features of a plasmid:
1. Origin of replication
2. Selectable marker
3. MCS (multiple cloning site)

13. By Prof Amer Jamil

14. Plasmid preparation for DNA ligation
• The plasmid after digestion by
restriction enzymes is
dephosphorylated prevents
recircularization and re-ligation
of linearized cloning vehicle
DNA by removing phosphate
groups from both 5´-termini
• Dephosphorylation enzyme:
• CIAP: calf-intestinal alkaline
phosphatase
• BAP: Bacterial alkaline
phosphatase
By Prof Amer Jamil

15. Blunting incompatible sticky ends of DNA
By Prof Amer Jamil

16. Blunting incompatible sticky ends of DNA
By Prof Amer Jamil
May remove overhangs:
Exonuclease III removes nucleotide residues from the 3’-end of a DNA
strand
Bacteriophage ƛ exonuclease removes nucleotide residues from the 5’-end
of a DNA strand

17. DNA ligation
• T4 DNA ligase is used
• It makes phosphodiester linkage (sealing
the nick)
By Prof Amer Jamil
Component Conc./amount in 20 uL
T4 DNA ligase buffer 10X 2 uL
Vector DNA (e.g. 4 kb) 50 ng (0.02 picomol)*
Insert DNA (e.g. 1 kb) 37.5 ng (0.06 picomol)*
T4 DNA ligase 1 uL
Nuclease free water to 20 uL
*molar ratio of 1:3 vector to insert

18. •Heat shock or Electroporation
•Competent cells: E. coli
By Prof Amer Jamil
DNA Transformation

19. Ref. NEB
Competent cells

20. DNA Transformation

21. DNA Transformation
Cell recovery period

22. DNA Transformation
Cell plating (with antibiotic)
IPTG/X-gal in case of blue-white screening

23. Growth on Agar plates
 Blue colonies represent Ampicillin-resistant
bacteria that contain Vector and express a
functional fragment, (beta galactosidase) from
an intact LacZ coding sequence.
betagalactosidaze+x-gal= blue coloured product
White colonies represent Ampicillin-resistant
bacteria that contain Insert and do not produce
LacZ, fragment.
• IPTG: Inducer of LacZ
• X-gal: Analogue of Lactose

24. Selection of recombinant clones
(blue-white screening)
lac operon control
A part of lacZ gene is in
vector, and other part in
chromosomal DNA
Substrate: X-gal (dye)
(5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside)
Inducer: IPTG
(Isopropyl β-D-1-thiogalactopyranoside)
By Prof Amer Jamil

25. Killed by antibiotic
such as ampicillin
No vector in cell
By Prof Amer Jamil

26. By Prof Amer Jamil
IPTG + antibiotic
By Prof Amer Jamil