Recombinant DNA Technology

❖ Recombinant DNA technology

❖ Genetic engineering

❖ Gene manipulation

❖ Molecular cloning

❖ Gene splicing

❖ Gene cloning
Recombinant DNA cloning procedure
1. Cross species barrier
- mixing of genes

2. Produce in bulk defined small

pieces of DNA
- For analysis, expression, manipulation

3. Expression of heterologous
proteins (molecular biotechnology)
- New microbes for fermentation industry
- Pharmaceuticals
- Vaccines
- Molecular diagnosis
 Basic steps in gene cloning

1. Fragment of DNA with gene of

interest
→ insert into vector
→ recombinant DNA molecule

2. Vector carries gene into bact. host

(usually E. coli)
3. Multiplication in host cells → clone
 Basic principles of gene cloning
1. Construction of a recombinant DNA molecule Bacteria DNA

R-plasmid DNA
Chimaera or
Vector
+ recombinant
DNA molecule
Plasmid DNA

Fragment of DNA

3. Multiplication
+
of recombinant 2. Transport into
DNA molecule the host cell

Bacterium

4. Division
of host cell 5. Numerous cell
divisions resulting
in a clone
Bacterial colonies growing
on solid medium
Prerequisites of gene cloning:

i. Gene of interest (or just a

fragment of DNA)
ii. Transport vehicle
iii. Host cells
Important aspects/processes/tools

Must be able to :

- Isolate DNA (extraction)

- Separate DNA (electrophoresis)
- cut DNA (specific sites) (restriction digest)
- Join DNA (specific sites) (Ligation)
- Amplify DNA (make many copies) (cloning, PCR)
- Put DNA into host (eg. bacteria) Transformation
- Select bacteria that has plasmid (+insert) (Selection)
Vectors: Vehicles for gene cloning

❖ Function: transport DNA sequence of

interest into host cell, and replicate
❖ Important features of cloning vectors:
i. Must be able to replicate within host cell
- numerous copies of the rDNA
molecule can be produced and passed to
daughter cells
ii. Relatively small (<10kb)
- large DNA easily sheared and difficult to
manipulate

Prof Sam CK: R-DNA

Desirable qualities of cloning vectors

❖ With restriction endonuclease sites, esp.

unique sites
❖ Copy number can be significantly increased
❖ Convenient selection markers

Prof Sam CK: R-DNA

 Cloning vectors

i. Plasmids
filaments
ii. Bacteriophages
head and tail

iii. Cosmids

Prof Sam CK: R-DNA

 Plasmids

❖ Replicon, autonomous replication (with

origin of replication)
Bacterial chromosome
❖ Extrachromosomal
Plasmids
❖ Can be inherited
Plasmids
❖ Usually dispensable
❖ Been identified in about
all strains of bacteria
❖ Cryptic or with selectable markers

Prof Sam CK: R-DNA

 Use of antibiotic resistance as a selectable
marker for a plasmid
Ampicillin
resistance

RP4 + Cells with plasmid

Cells without plasmid
Kanamycin Tetracyclin
resistance resistance

+
+
+
E. coli cells, some
containing RP4
+ + + +
+ + +
+
+ + + +
+ + + + +
Normal growth medium - no antibiotic Growth medium + 50 mg/ml tetracyclin

All cells can grow Only cells containing RP4 can grow
Replication strategy for a non-
integrative plasmid

Plasmids

Cell division

Bacterial chromosome

Prof Sam CK: R-DNA

Replication strategy for a an episome

Chromosome carrying integrated plasmid

Plasmid

Bacterial chromosome
Cell division

Prof Sam CK: R-DNA

 Plasmid copy number

❖ Copy number
- number of a particular plasmid present in a single
bacterial cell

❖ Stringent plasmid
- low copy of 1 or 2 per cell

❖ Relaxed plasmid
- present in multiple copies of 10 or more per cell

Prof Sam CK: R-DNA

 Enzymes for cutting DNA:
restriction endonucleases

❖ Endodeoxyribo
- nucleases that recognise specific
nucleotide sequences in double-stranded
DNA and cleave both strands of the duplex
(Arber, Smith & Nathan; Nobel Prize 1978)

Prof Sam CK: R-DNA

 Phosphodiester bond

O- O- O-
І І І 5’
-O – P– O – P– O – P– O – CH
2 O Base
one nucleotide
O O O 3’

5’ triphosphate O
І 5’
phosphodiester bond O= P– O – CH2 O Base
І
O- 3’

OH
3’ OH

Prof Sam CK: R-DNA

Restriction endonucleases (R.E.s)
❖ R.E. : types I, II & III
I – cut far from recognition site
II – cut at recognition site
III – cut near recognition site
❖ Type II R.E.
- recognise & break ds DNA within particular
sequences of tetra, penta, hexa, hepta or
octanucleotides which have an axis of rotational
symmetry (palindromic sequences)*
* Exceptions, eg. HinGuII GGATG

Prof Sam CK: R-DNA

 Restriction endonucleases (R.E.s)
❖ Almost all R.E. recognition sequences are
palindromes
- eg. EcoRI 5’ G A A T T C 3’
3’ C T T A A G 5’
(reads the same from both directions )

Prof Sam CK: R-DNA

 Common R.E.s…
EcoRI Eschericia coli GAATTC sticky
BamHI Bacillus amyloliqnefaciens GGATCC sticky
BglII Bacillus globii AGATCT sticky
PvuI Proteus vulgaris CGATCG sticky
PvuII Proteus vulgaris CAGCTG blunt
HindIII Haemophilus influenza Rd AAGCTT sticky
HinfI Haemophilus influenza Rf GANTC sticky
Sau3A Staphylococcus aureus GATC sticky
AluI Arthrobacter luteus AGTC blunt
TaqI Thermus aquaticus GAATTC sticky
HaeIII Haemophilus aegyptius GGCC blunt
NotI Nocardia otitidis-caviarum GCGGCCGC sticky

Prof Sam CK: R-DNA

Production of blunt/flush ends

-N-N-A-G-C-T-N-N
-N-N-T-C-G-A-N-N
AluI

-N-N-A-G C-T-N-N-
-N-N-T-C G-A-N-N-

Prof Sam CK: R-DNA

Production of sticky/cohesive ends

-N-N-G-A-A-T-T-C-N-N
-N-N-C-T-T-A-A-G-N-N

EcoRI
-N-N-G A-A-T-T-C-N-N
-N-N-C-T-T-A-A -G-N-N

Prof Sam CK: R-DNA

 Same sticky ends produced by
different R.E.s

BamHI -N-N-G G-A-T-C-C-N-N-

-N-N-C-C-T-A-G G-N-N-

BglII -N-N-A- G-A-T-C-T-N-N-

-N-N-T-C-T-A-G A-N-N-

Sau3A - N-N-N G-A-T-C-N-N-N-

-N-N-N-C-T-A-G -N-N-N-

Prof Sam CK: R-DNA

Sticky/cohesive protruding 5’ termini

5’ …..GAATTC….. 3’
3’ …..CTTAAG….. 5’
EcoRI

5’…..G-OH 3’ 5’ P-AATTC…..3’
3’…..CTTAA-P 5’ 3’ HO-G…..5’

Prof Sam CK: R-DNA

Sticky/cohesive protruding 3’ termini

5’ …..CTGCAG….. 3’
3’ …..GACGTC….. 5’
PstI

5’…..CTGCA-OH 3’ 5’ P-G…..3’
3’…..G-P 5’ 3’ HO-ACGTC…..5’

Prof Sam CK: R-DNA

 Analysis of DNA fragments…
❖ Gel electrophoresis: agarose, acrylamide
❖ Composition of gel determines pore size
- sizes of DNA fragments that can be separated
- eg. 0.5 cm thick of 0.3% agarose used for 5-60 kb
0.3 mm (very thin) of 40% acrylamide for 1-300 bp
- buffers: many types

❖ DNA (like most macromolecules) carry an electric

charge (-ve charge)
- migrate towards +ve pole
- viewing: ethidium bromide (>25 ng)

Prof Sam CK: R-DNA

…Analysis of DNA fragments

buffer
o
–ve o
electrophoresis

+ –

o DNA fragments

*Buffer
Gel concentration
Voltage (heat)
Time +ve
Molecular
weight marker
Prof Sam CK: R-DNA
Agarose Gel Electrophoresis (AGE)

SASAR, 2012
…Analysis of DNA fragments

Electrophoretic separation
of DNA fragments generated
by digestion of  DNA with HindIII

Prof Sam CK: R-DNA