BN2301

Biochemistry and Biomaterials for

Bioengineers

Lecture 3

Dr. Cheow Lih Feng [email protected]

 Announcement

• Tutorial (Problem Set 1) will be held after

lecture
• Solutions to Problem Set 1 will also be
uploaded later today
Last time

Biomolecules:
DNA
RNA

Processes in the cell:

Replication
Transcription
Translation
COVID-19 Vaccines
 Mechanism of action of mRNA vaccine

https://www.youtube.com/watch?v=DCk7LyMslxo
How is mRNA vaccine produced
Short Film – GATTACA (1997)

https://www.youtube.com/watch?v=BpzVFdDeWyo
Two main concepts

Genetic Engineering DNA Testing

Outline

DNA Technology
1. Genetic Engineering
Technique: Recombinant DNA Cloning

2. DNA Fingerprinting
Technique: Polymerase Chain Reaction
Gel Electrophoresis

3. DNA Sequencing (next week)

Why do we study this

DNA Technology

Biotechnology Analysis
(Topic 1: Genetic Engineering) (Topic 2: DNA Fingerprinting)
Biotechnology

“use of an organism, or a component of an organism or other

biological system, to make a product or process for a specific use”
Genetically modified organism

Gene Therapy (e.g. Cystic Fibrosis)

Pharmaceuticals (e.g. Penicillin)
Insulin production
Genetic Engineering

“confer useful trait by introduction of foreign DNA”

DNA Cloning

• DNA cloning is the process of making multiple, identical copies of a piece of DNA.
• In a typical DNA cloning procedure, the gene or other DNA fragment of interest is
first inserted into a circular piece of DNA called a plasmid.
• Next, the recombinant plasmid is introduced into bacteria.
• Bacteria carrying the plasmid are selected and grown up. As they reproduce, they
replicate the plasmid and pass it on to their offspring, making copies of the DNA it
contains.
Plasmid

• Plasmids are small,

circular DNA molecules
in bacteria
Bacterial chromosome
– duplicate separately
from chromosome
– can carry almost any Plasmids
gene

Colorized TEM
Remnant of bacterium
Schematic of a Plasmid
Recombinant bacteria

Bacterium Cell containing gene

of interest DNA of chromosome
(“foreign” DNA)
Bacterial Plasmid 1 Gene inserted
chromosome
Recombinant Gene of Into plasmid
DNA (plasmid) interest
2 Plasmid put into
bacterial cell
Recombinant
bacterium
3 Host cell grown
In culture to form
a clone of cells
Useful products
Figure 12.3-2
Clone of cells
Some uses Genes containing the Some uses
of genes inserted gene of interest of proteins
into other Protein for
organisms “stone-washing”
. jeans
4 The gene
and protein Harvested
Genes for of interest proteins may
cleaning up are isolated be used
toxic waste directly
from the Protein for
bacteria. dissolving
clots
Gene for pest
resistance
Cut and Paste DNA
• Cut: Restriction enzymes
– Cuts DNA at a predetermined sequence of nucleotides,
e.g. EcoRI cuts GAATTC
• Question: What is the function of restriction
enzymes in bacteria?
– Bacteria use restriction enzymes to disable unknown DNA
Restriction enzymes

• DNA may be cut by some special naturally-occurring

proteins called restriction enzymes.
• Each restriction enzyme only identifies and “cuts” at a
very specific sequence (recognition site) in the DNA
strand.
• Restriction enzymes typically recognize a symmetrical
sequence of DNA, such as the site GAATTC.
Restriction enzymes
Example 1

"
Read to the left
Read to the right
"
• Palindromic sequence: The top strand is the same as the
bottom strand when you read backwards.
• When the enzyme EcoRI cuts the strand between G and
A, it leaves overhanging chains:

• These are termed "sticky ends" because the base pairs

formed between the two overhanging portions will glue
the two pieces together, even though the backbone is cut.
Example 2

Another symmetrical sequence of DNA, CCCGGG:

When the enzyme SmaI cuts the strand between C and
G, it leaves no overhang. This is known as a “blunt end”
Cut and Paste with DNA

Recognition site for

DNA
restriction enzyme

1 A restriction enzyme cuts the Restriction enzyme

DNA into fragments.
ky end
Sti c
Stick
y en
d
Cut and Paste with DNA

Recognition site for

DNA
restriction enzyme

1 A restriction enzyme cuts the Restriction enzyme

DNA into fragments.
k y end
Sti c
Stick
y en
d

2 A DNA fragment cut with

the same enzyme is added
from another source.
Cut and Paste with DNA

3 Fragments stick together by

base pairing.

4 DNA ligase joins the DNA ligase

fragments into strands.

Recombinant DNA molecule

Restriction enzyme = scissors

DNA ligase = glue
Getting the plasmid into bacteria

During transformation, bacterial cells are given a shock

(such as high temperature) that encourages them to
take up foreign DNA.
Select bacteria that took up plasmid

• A plasmid typically contains an antibiotic resistance gene, which

allows bacteria to survive in the presence of a specific antibiotic.
• Thus, bacteria that took up the plasmid can be selected on nutrient
plates containing the antibiotic. Bacteria without a plasmid will die,
while bacteria carrying a plasmid can live and reproduce.
• Each surviving bacterium will give rise to a small, dot-like group, or
colony, of identical bacteria that all carry the same plasmid.
Protein Production

• We can now grow a large culture of plasmid-bearing bacteria.

• The bacteria serve as miniature “factories," churning out large
amounts of protein. For instance, if our plasmid contained the
human insulin gene, the bacteria would start transcribing the
gene and translating the mRNA to produce many molecules of
human insulin protein.
Summary: Genetic Engineering in Bacteria
A glimpse into the future: Human DNA Editing
CRISPR-Cas9

https://www.youtube.com/watch?v=avM1Yg5oEu0
 Use of DNA Technology for Analysis
• Unless they are identical twins, every individual have unique DNA
sequence
• DNA fingerprinting: Identify individual by taking advantage of
difference in DNA sequence
• The process of DNA fingerprinting begins by extracting DNA
• E.g. blood, skin, body fluid, garments
• If there is only a small amount of DNA, amplify DNA with a
technique called Polymerase Chain Reaction (PCR)
• PCR is doing DNA replication in a test tube (in vitro)
Polymerase Chain Reaction

o Kary Mullis
o Chemist in Cetus Corporation
o Invented PCR in 1983
o 1993 Nobel Prize in Chemistry
o Make millions of pieces of
identical copy of DNA at a
short time
o Revolutionized biotechnology
Basic components of PCR

o Primers
o Template DNA
o dNTP
o Taq Polymerase
(Thermus Aquaticus)
Important Concepts

There are four bases (dNTPs)

Each of the bases can only pair up with its complementary base

A primer is a short stretch of synthetic DNA (oligonucleotide) that can pair up

with the front part of DNA to initiate DNA polymerization
DNA polymerase can copy the
template DNA

Free dNTPs

Polymerase
Polymerase Chain Reaction

**Important: Given a target region to be copied, determine the primer

sequences to be used
Another example of primer design

C
D
(Denaturation 95°C)

(Annealing 55°C- 60°C)

(Extension 72°C)

Why do we need to use Taq polymerase?

https://www.youtube.com/watch?v=oOzsVEuXyfw
Variable Number Tandem Repeat (VNTR)
VNTR is a location in a genome where a short nucleotide sequence
is organized as a tandem repeat, and often show variations in length
(number of repeats) among individuals.
Each variant acts as an inherited allele, allowing them to be used for
personal or parental identification.
Restriction Fragment Length Polymorphism
The difference in VNTR alleles can be identified using a technique
known as Restriction Fragment Length Polymorphism (RFLP)

Due to the variable lengths of VNTR, restriction enzyme digestion of

DNA lead to different fragment length
Gel Electrophoresis
• DNA of different fragment lengths can be separated via gel
electrophoresis
• Electrophoresis involves running a current through a gel
containing the molecules of interest.
• Based on their size and charge, the molecules will travel through
the gel at different speeds, allowing them to be separated from
one another.
slowest
Question: What is the charge of DNA?
−

+ fastest
Gel Electrophoresis

Mixture of DNA
fragments of
different sizes
-

Power
source

+
Gel Electrophoresis

Mixture of DNA
fragments of
different sizes
- -

Power
source

+ +
Gel Electrophoresis

Mixture of DNA
fragments of
Band of longest
different sizes
- - (slowest) fragments

Power
source

+ +
Band of shortest
(fastest) fragments
DNA Fingerprint Analysis

Case 1
Mr. Chan’s family consists of mom, dad and four kids. The parents have one
daughter and one son together, another daughter is from the mother’s
previous marriage, and the other son is adopted. Here are the DNA analysis
results:

1. Which child is adopted? Why?

2. Which child is from the mother’s previous marriage? Why?
3. Who are the own children of Mr and Mrs Chan?
DNA Fingerprint Analysis

Answers:
• Child 4 is adopted.
• Child 2 is the child from the mother’s
previous marriage.
• Child 1 and Child 3 are own children of Mr
and Mrs Chan.
DNA Fingerprint Analysis

Case 2
A blood sample from a crime
scene was collected. DNA
samples of the victim and the
potential suspects (June, Scarlet
and John) were also collected for
DNA analysis. The DNA profile is
shown. Now, you should be able
to identify the potential murderer.

54
DNA Fingerprint Analysis

Answers:
• All of the DNA fragments of Scarlet can be
found in the crime scene sample making her
the most likely suspect.
Lab 1 Preview – DNA Fingerprinting
Lab 1 Preview – DNA Fingerprinting
Thank You