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PTT 104
Introduction to
Biotechnology
Lecture 3
Techniques in Biotechnology
Recombinant DNA & PCR
Madam Noorulnajwa Diyana Yaacob
Course Outcome
CO 2: Ability to demonstrate
important recent advances in
methods and applications of
biotechnology with regards to
microorganisms and plants.
Biotechnology today
• Focuses on DNA
Deoxyribonucleic Acida double-stranded
helical molecule that
stores genetic
information for the
production of all the
organism’s proteins
What Is the Structure of DNA?
• DNA Is Composed of Four Nucleotides
• DNA Is a Double Helix of Two Nucleotide
Strands
• Many people contributed to the discovery,
but Francis Crick and James Watson (and
Maurice Wilkins) got the Noble prizes.
Hydrogen Bonds
• Hydrogen bonds hold certain
nitrogenous base pairs together
– A bonds with T, G bonds with C
– Bonding bases called complementary
base pairs
• Ladder-like structure of the two DNA
strands are twisted into a double helix
What Is the Structure of DNA?
• Hydrogen Bonds Between
Complementary Bases Hold the
Two DNA Strands Together
• The Order of Nucleotides in DNA
Can Encode Vast Amounts of
Information
5 end
3 end
3 end
Figure E9-6 Biology: Life on Earth 8/e ©2008 Pearson Prentice Hall, Inc.
5 end
Recombinant DNA
• DNA produced by joining
segments of DNA from different
sources
• eg. To produce human insulin,
scientists have combined bacterial
plasmid DNA + human DNA
Tools for Producing
Recombinant DNA
Restriction enzymes: enzymes that
cleave the DNA double helix at
specific nucleotide sequences
Use of the Restriction Enzyme
Bam H1
5’— G G A T C C — 3’
3’— C C T A G G — 5’
Results in
sticky end
5’— G
3’— C C T A G
sticky end
G A T C C — 3’
G — 5’
Tools for Producing
Recombinant DNA
Vector: carrier of DNA; can be virus or plasmid
Plasmid: extrachromosomal, independently
replicating, small circular DNA molecule
Producing Recombinant DNA
Mix together
Add DNA Ligase
restriction
enzyme
Treat source
DNA with
restriction
enzyme
Treat plasmid
DNA with
same enzyme
Many recombinant DNA
molecules are produced,
each with a different
piece of source DNA
restriction
enzyme
Transform
bacterial cells
Each bacterial cell
carries a different
recombinant plasmid
Tools for Producing
Recombinant DNA
Probe: sequence of DNA that is
complementary to the gene of interest;
Used to locate a copy of the gene by
hybridization
AGCTTAGCGAT
TCGAATCGCTA
Denature DNA by heating
Add Probe
Probe Binds to gene
AGCTTAGCGAT
AATCGC
TCGAATCGCTA
Building
a
DNA
Library
Applying Your Knowledge
1.
2.
3.
4.
Probe
Clone
Plasmid
Restriction Enzyme
A. An enzyme that cleaves DNA at specific
sequences is a __________ .
B. A sequence of DNA that is complementary to
the gene of interest is a _________.
C. A small, independently replicating DNA
molecule is a ___________.
Polymerase Chain Reaction
• PCR is a technique that is
used to amplify a sample of
DNA from miniscule
amount of DNA (ex., DNA
from a crime scene,
archaeological samples,
organisms that can’t be
cultured).
Who developed PCR?
• PCR was developed by Kary Mullis.
• Kary Mullis is a scientist and surfer
from Newport Beach, California.
• He won a Nobel Prize in Chemistry in
1993 for the development of PCR.
• He was working for Cetus Corporation
in the 70’s and received $10,000 bonus
for the idea.
How is PCR used?
• Medical Diagnosis: To detect and identify the causes of
infectious diseases from bacteria and viruses.
• Genetic testing: To determine whether a genetic mutation
has been passed on (ex. cystic fibrosis).
• Evolutionary study: To gather archaeological samples
and analyzed for similarities/differences.
• DNA fingerprinting: To profile DNA from blood, hair, and
skin cells for criminal identification and forensics
Stages of PCR
PCR is divided into 3 stages:
1. Denaturation
2. Anneal
3. Extension
What is a primer?
• A primer is a short
oligonucleotide which is
the reverse complement
of a region of a DNA
template.
• It would anneal to a DNA
strand to facilitate the
amplification of the
targeted DNA sequence.
oligonucleotide
Primer Selection variables
•
•
•
•
•
•
Primer length
Melting Temperature
GC content
Hair-pin loop
Self-dimerization
Cross-dimerization
Primer Length
• Should be between 18 – 25
bases.
• The longer the primer, the more
inefficient the annealing.
• If primers are too short, they will
cause non-specific annealing
and end up amplifying nonspecific sequences.
Melting Temperature
• Formula (18-25 bp range):
– Tm = 2(A+T) + 4(G+C)
• The forward and reverse primers
should be having similar Tm, or else
amplification will be less efficient.
• Melting Temperature should be
between 55ºC and 65ºC.
GC Content
• GC% = (G + C) / length of seq * 100%
• The base composition should be in the range of 45% to
55%.
• Poly G’s or C’s can result in non-specific annealing.
Hairpin Loop
• Primers with hairpin loop may interfere
with annealing to the template by forming
partially double-stranded structure.
Self-dimerization
• Primers may form inter-primer homology
with its own copies.
Cross Dimerization
• Forward and Reverse primers may
hybridize to form primer-dimer.
Algorithm for primer design
Input DNA
sequence
Input the
start and
end of
central
region
Input the
length of
primers
N
55-65oC
Y
GC content
45-55%
Y
Hairpin and selfdimerization
List of
acceptable
primers
N
Y
N
N
Cross Dimerization
Y
Excluded primers
Tm:
Polymerase Chain Reaction (PCR)
 Amplifies a specific region in the DNA
 Used for identification, especially
if the amount of DNA is small
 Uses repeated cycles of heating to
denature DNA and cooling to synthesize
new DNA
Involves the use of
---Taq polymerase (withstands heat)
---primers to begin synthesis
Polymerase Chain Reaction:
One PCR Cycle
90 °C
50 °C
Taq Polymerase
Original
Doublehelix
DNA
Separate
DNA
Strands
72 °C
Primer
Primers &
Taq
polymerase
bind
DNA
synthesized