DNA Recombinant Technology

• Assoc. Prof. Dr. Atif Amin Baig ,

• Faculty of Medical Sciences,
• University Sultan Zainal Abidin.
• atifamin@unisza.edu.my
 Recalls
• Designing of Primers

• Hands-on
 Learning Outcomes
• What are Omics technologies
• Branches of Omics technologies
• Cloning
• Types of cloning
• Somatic cell nuclear transfer
• Genetic recombination
• Restriction analysis and Ligases
• Transformation, Conjugation and Transduction
• Holiday Junction
 Omics
Genome  Ome
Chromosome  Ome

The term omics refers to the comprehensive analysis of biological systems

The complete sequencing of the human genome has ushered in a new era of
systems biology referred to as Omics technology

a term invented by Hans Winkler in 1920

 Omics
The word genomics is said to be appeared in the 1980s and
became widely used in the 1990s

The first genome was completely sequenced by Sanger in

Cambridge, UK, in the 1970s

Chromosome  Color and body

Genome  Collection of group
 Branches of Omics
• Antibodyome & Antibodyomics • Metabolome & Metabolomics
• Bacteriome • Neurogenome
• Cardiogenomics • Nucleome
• Cellome & Cellomics • Nutrigenomics
• Diseaseome & Diseaseomics • Proteome & Proteomics
• Epigenome & Epigenomics • Sequenceome
• Transcriptomics
• Foodome &Foodomics
• Virusomics
• Genome & Genomics
• Glycome & Glycomics
• Healthome & Healthomics
• Herbome & Herbomics
• Hygienome &Hygienomics
• Immunolome & Immunolomics
• Lipidome & Lipidomics
• Lipoproteome & Lipoproteomics
 BIOINFOMATICS

MOLECULAR GENETICS
MEDICINE

NUTRIGENOMICS

PHARMOCO- MOLECULAR
-GENOMICS NUTRITION

MOLECULAR
BIOLOGY
 The Omic Technologies
Omics needs throughput molecular biology
techniques including sequencing and
genotyping (genomics), Transcriptomics,
proteomics and metabolomics.
Genome Transcriptome Proteome

? Metabolome
 DNA cloning or Genetic
recombination are same?

Molecular Cloning?

Or Cloning

Production of genetically identical individuals which have

identical nuclear DNA
Types of Cloning Technology

1. Recombinant DNA technology

• DNA cloning
• Molecular cloning
• Gene Cloning

2. Reproductive cloning

3. Therapeutic cloning
• Embryo cloning
 DNA Cloning
Transfer of DNA of interest from one organism to a self replicating genetic element such as
bacterial plasmid

Plasmids: self replicating extra chromosomal circular DNA molecules,

distinct from normal bacterial genome

Uses of DNA cloning

Gene therapy
Genetic engineering of organisms
Genome sequencing
 Reproductive cloning

A technology to generate an animal that has some nuclear DNA as

another currently or previously existing animal has

Dolly is an example

How Reproductive cloning is done ?

Somatic cell nuclear transfer (SCNT)
 Somatic cell nuclear transfer (SCNT)

Starts from removal of polar body from and chromosome from an oocyte
(enucleated oocytes)

Donor cell then inserted into the perivitelline space of the enucleated oocyte

Oocytes and donor cells are fused and activated by an electric impulse
to start cell division

Developed embryos transfer to surrogate females

Birth of an organism
 Pipette injecting the
This is human oocyte A pipette penetrating an egg somatic cell into the egg

Nucleus removed from an egg The enucleated egg Egg with the somatic cell

Source of cells?

Cells from individuals

Culture
Pipette penetrating the egg Frozen sections
The human oocyte with the somatic cell

Process of Enucleation
 Therapeutic cloning

Production of human embryos to be used in research

(To harvest stem cells that can be used to study human development
and to treat disease)

Stem cells: are the cells which have ability to divide and give rise to both
“specialized cells and more stem cells”.

Either adult stem cells or embryonic stem cells (preimplantation embryo)

Heart disease, Alzheimer's diseases, Cancer, Diabetes,

Parkinson's disease, spinal cord injury
 Molecular cloning
Inserting a piece of DNA molecule (of interest))into a
DNA carrier (vector) to generate multiple copies in a
host cell such as bacteria

Purposes
Separate a gene from others
Amplification of modified forms of genetic materials
Manipulation of DNA for further experiments

Transfer of DNA of interest from one organism

to a self replicating genetic element such as
bacterial plasmid
 How it Works?
We need:
• Method of amplification of a specific gene or region of DNA
• Molecular enzymes (DNA amplifiers or modifiers)
• Host
• Vectors
Plasmids
Cosmids
YAC
Bateriophage
Virus

Amplification Get clones of

Insert into a vector Transfer to a host
of a gene of interest the DNA
Enzymes that can cut DNA

Enzymes that can join DNA

 Types/Groups of Plasmids (addressing plasmids in E. coli, mainly)

Conjugative vs. Non-ConjugativeTypes

F plasmids: Sex plasmids

Host cell with an F plasmid is able to transfer F and chromosomal genes to a cell lacking F

R plasmids: Drug resistance plasmids

Host cells are resistant to one or more antibiotics and can often transfer resistance to Rcells

Col plasmids: Colicinogenic factor plasmids

Host cells synthesize colicins which kill closely related bacterial strains lacking Col plasmids

Stringent vs. relaxed; Low vs. high copy number

 In each of the cells there are about 20-30,000 genes

250,000,000 base pairs

Learning about genes have required scientists to develop tools to manipulate the genome

DNA Scissors DNA Glue

Method of amplification of
DNA or separation
Cut double stranded DNA or Derived from bacteria or archea
single stranded DNA Physiologically provided for defense against

Restriction modification system

First isolated
HindIII
DNA Scissors

Restriction endonucleases

500 + restriction
enzymes are known yet Blunt end
Recognition sequence
Restriction site

Sticky end
 Blunt end restriction
Recognition sites will not be in one plane at both incisions
palindromic
Usually Type II

ATGC CGAT
TAGC GCTA
PO3-
No 5’ or 3’ overhang

5’ ATGC 3’ 5’ CGAT 3’
3’ TACG 5’ 3’ GCTA 5’

PO3-
S -- deoxyribose P -- phosphate groups

3’ 5’

5’ 3’
 Sticky end restriction
Recognition sequences are at different planes at both incisions
Palindromic or non palindromic
Type II or Type III restriction enzymes

G AATTC
CTT AA G
PO3-

G AATTC
CTTAA G

PO3-
 Sticky end

Sticky end
Double stranded DNA
DNA Glue
DNA Ligase

V(D)J Junction
Non homologous end joining
T cell receptors modification
RNA editing in immune system

XRCC4

DNA repair genetic recombination DNA replication

Base seiling mutation
Type III Type IV Type I
 Some possible ligation reaction products:

Recombinant No insert Fragments No ligation

 Conjugation and Gene replacement

Donor Recipient
aphA
oriR6K

Kanamycin, Ampicillin and

Polymyxin resistant
 Merodiploid

sacB gene codes for levan sucrase, which metabolize

Sucrose sucrose and produces toxic by-product leavan.
selection The host carrying the sacB gene will be killed if it is
grown in the presence of sucrose.
 Bacteriophages as DNA carriers

- Natural vectors that transduce DNA from one bacterial cell to

another.

- A virus for a bacterial cell

- Cannot “live” or reproduce without getting inside a bacterial cell

Transfection
 A single phage can infect and clear out many
bacterial cells and creat a “plaque” on a bacterial
lawn

A plaque contains a homogeneous population of a

phage
Holiday’s Junction
Q and A

Thank you