5/22/2016

RECOMBINANT DNA
TECHNOLOGY

BIO20 Intro. to
Biomimetics Prof. UREAH THEA A. SEVILLA

Recombinant DNA Technology

The intentional alteration of DNA sequence

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Importance of rDNA Technology

 Agriculture
Pest-
resistant
plants

Frost-resistant
plants

Importance of rDNA Technology

 Medicine

recombinant blood
clotting factor Recombinant
recombinant human
vaccine
insulin

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Importance of rDNA Technology

 Environment

rDNA Technology: DNA Cloning

1. Creation of a recombinant DNA
2. Cell Transformation
3. Cloning of Transformed cells
4. Identification of cells containing rDNA
5. Growing of pure culture containing rDNA

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DNA Cloning
 Amplification of rDNA using host organism’s
replication mechanism

DNA Cloning

The recombinant DNA is returned to the host

cell. This process is called Cell Transformation.
This cell is then grown in culture, forming a clone
of cells.
The foreign DNA spliced into the plasmid is
replicated with the rest of the plasmid as the host
cell multiplies. In this way, the gene of interest is
“cloned”.
Identification of the bacterial clone carrying the
gene of interest.

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DNA Cloning

Cloned genes and proteins:

 For pest resistance inserted into plants
 Gene used to alter bacteria for cleaning up toxic
waste
 Basic research on genes
 Protein dissolves blood clots in heart attack
therapy
 Human growth hormone treat stunted growth
 Basic research on proteins

Procedure in Recombination

1. isolation of desired DNA fragments

- gene of interest and host DNA (DNA vector)

2. joining them in new combinations

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DNAs in Recombinant DNA Technology

 DNA VECTORS (host DNA)  GENE OF INTEREST

Small, circular, double-stranded Refers to the gene that is
DNA molecule within a bacteria genetically engineered with
cell than can replicate improved features e.g. pest-
independently. It often carry resistant gene.
genes that may benefit the
survival of the organism e.g.
Antibiotic resistance

DNA Vector (Cloning Vector)

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Recombination
Gene of
interest

+


Enzymes involved
 RESTRICTION ENDONUCLEASES / RESTRICTION
ENZYMES

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Restriction Endonucleases
Blunt ends – when both strands of the
molecule are cut at the same position that
leaves no unpaired nucleotides.
Staggered or “Sticky” ends – when each
strand of the molecules is cut at different
position so that one strand (either 5’ or 3’)
overhangs by several nucleotides, these single-
stranded ends can be spontaneously base pair
with each other –that is, they are sticky or
cohesive.

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Cleavage done by endonucleases

Restriction Enzymes

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Enzymes involved
 DNA LIGASE

DNA Ligase

is the “glue’ that joins two DNA molecules

joins DNA fragments that have complimentary sticky

ends or blunt ends; catalyzes formation of covalent
bonds between the sugar and phosphate of the
adjacent nucleotides, requiring only that one
nucleotide have a free 5’-phosphate and adjacent one
have a 3’-hydroxyl group

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Restriction enzyme
recognition sequence

5’ GAAT TC 3’
3’ CT TAAG 5’

Restriction enzyme
cuts DNA

Sticky ends
5’ G AA T T C 3’
3’ CT TAA G 5’

Addition of DNA fragment from DNA fragment

another source; fragments stick produced by same
together by base pairing enzyme
AA T T C G
G CT TAA
DNA ligase seals
the strands

5’ G A AT T C G A A T T C 3’
3’ C T T AA G C T T AA G 5’

Recombinant DNA

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Recombinant DNA
The enzyme that cuts both the vector and DNA of
interest internally is called
a) DNA ligase
b) DNA nuclease
c) Restriction endonuclease
d) DNA exonuclease

Determine what type of cut is

made on the DNA:
A.

B.

C.

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Cloning Vectors

Cell Transformation
 introduction of new DNAs into organisms or to a host for gene cloning.

Electroporation – protoplasts of host organism are exposed to a

brief electrical pulse, which is thought to introduce transient
openings in the cell membrane through which DNA molecule
enters.

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Electroporation

Cell Transformation

 introduction of new DNAs into organisms or to a host for gene cloning.

Microprojectile bombardment or biolistics – very small (4μm)

microprojectiles made of gold or tungsten are coated with DNA and
is shot at high velocity from a particle gun into cells or tissues.

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Cell Transformation

 introduction of new DNAs into organisms or to a host for gene cloning.

Microinjection – for multicellular animals, DNA is injected directly

into the nucleus of animal with an extremely fine pipette. After the
DNA is transferred into the cell, it is integrated into the
chromosome, and the transformed fertilized egg is implanted into
an animal for completion of development.

Microinjection

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Cloning of Transformed Cells

Identification of cell clones carrying gene of

interest

Nucleic acid hybridization

-process in which complimentary single strands of
desired gene form hydrogen bond with nucleic acid
probe that is labeled by radioactive isotope or
fluorescent tag.

Denaturation of the cells

-separation of the two strands by heat or
chemicals.

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Nucleic Acid Hybridization

Cloning of Transformed Cells

Once cell clones are

identified, they are
grown in liquid culture
and then can easily
isolate large amounts of
the gene.

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rDNA
Technology

rDNA and Cloning

Arrange the steps in DNA Cloning (1–5)
__Cloning of transformed cells
__Growing of pure culture containing rDNA
__Creation of a recombinant DNA
__Identification of cells containing rDNA
__Cell Transformation

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rDNA and Cloning

It is the host DNA
a. Plasmid c. Vector
b. Cosmid d. All of the given

Step where the rDNA is returned back into the host

cell is called
a. Recombination c. Cell Cloning
b. Cell Transformation d. Cell propagation

DNA Techniques
•Polymerase Chain Reaction
•Gel Electrophoresis

•Southern Blot Hybridization

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Polymerase Chain Reaction

Polymerase Chain Reaction (PCR)

-a technique by which any piece of DNA can be quickly
amplified without using cells.
PCR is used to:
 Rapidly isolate specific sequences for further analysis
or for cloning
 Identify specific genetic loci for diagnostic or medical
purposes
 Generate DNA fingerprints to determine genetic
relationships or to
establish identity in forensics
 Rapidly sequence DNA

PCR Components

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Polymerase Chain Reaction

1. denaturation 2. annealing 3. polymerization

Steps in PCR
DENATURATION
Double-stranded DNA sample is unwound by heating to 92-94ºC. The
single strands will serve as templates for reaction.

ANNEALING WITH PRIMERS

Short nucleotide sequences are mixed with the DNA sample to base-
pair.

The mixture is cooled 42-55ºC and the short nucleotide sequences

base-pair with the ends of the template strands. These short
nucleotides serve as primers.

ELONGATION
DNA molecules in the mixture become doubled.
The mixture is heated again which cause unwinding of the double-
strands and the cycle repeats producing new DNA strands.

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Polymerase Chain Reaction

Polymerase Chain Reaction

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Polymerase Chain Reaction

After cycle 30, >1B copies generated (230= 1.07 X 109)

Gel Electrophoresis
 A technique used to separate macromolecules
(nucleic acids or proteins) on the basis of size,
electrical charge, and other physical properties. It
sorts a mixture of DNA molecules into bands, each
consisting of DNA molecules of the same length.

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Gel Electrophoresis Set-Up

Agarose Gel Electrophoresis of DNA

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Agarose Gel Electrophoresis of DNA

Southern Blot Hybridization

 Southern blot is a method used for detection of a
specific in DNA samples. Southern blotting combines
transfer of electrophoresis-separated DNA
fragments to a filter membrane and subsequent
fragment detection by probe hybridization.

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Southern Blot Hybridization

Southern Blot Hybridization

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Southern Blot
Hybridization

Other Hybridization Techniques

Similar blotting and hybridization methods:
 Northern Blotting – used to probe RNA molecules
separated by denaturing agarose gel electrophoresis.
It is useful for identifying isolated RNAs and for
studying the expression of specific genes.
 Western Blotting – method to transfer
electrophoretically separated proteins to a
membrane for antibody binding to detect specific
proteins.

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RFLPs = DNA Fingerprinting

 Restriction Fragment Length Polymorphism is a
technique that exploits variations in homologous
DNA sequences. It refers to a difference between
samples of homologous DNA molecules from
differing locations of restriction enzyme sites.

 In RFLP analysis, the DNA sample is broken into

pieces and (digested) by restriction enzymes and
the resulting restriction fragments are separated
according to their lengths by gel electrophoresis.

RFLPs = DNA Fingerprinting

 RFLPs separated by gel electrophoresis can be
further analyzed using Southern Blot Hybridization.

 RFLP analysis was the first DNA profiling technique

or aka DNA fingerprinting

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DNA Fingerprinting

Amplified DNAs

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DNA Fingerprinting

Which is the father?

DNA Fingerprinting

Based on the results

shown for a single locus
probe, which of the
children could not be the
progeny of these
parents?

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DNA Fingerprinting
Based on the results
shown for a single locus
probe, which of the
children could not be the
progeny of these
parents?

DNA Fingerprinting

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References:
 Voet, Voet and Pratt, Principles of Biochemistry 3rd
ed. Wiley Publication (2008).
 Lodish H, Berk A, Zipursky SL, et al. Molecular Cell
Biology, 5th edition. New York: W. H. Freeman;
(2004).
 Barnum, S., Biotechnology, Wadsworth – Thomson
Publishing Co. (1998).

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