Pusat Penyelidikan Halalan Thayyiban

Halalan Thayyiban Research Centre

DNA-based
Analysis [Part I]
HS4322 Halal Food Analysis
Instructors
Hamzah Mohd. Salleh (Prof. Dr.)
hamzah.salleh@unissa.edu.bn

Siti Nur Idayu Haji Matusin (Dr.)

idayu.matusin@unissa.edu.bn
Fundamentals
Learning Objectives

• Recall the structure of

nucleic acids
• Recall the biochemical
structure of DNA and RNA
What are
nucleic acids?
Long polymers of
repeating subunits
called nucleotides.

The elements of
nucleotides:
C, H, O, N, P

There are TWO classes

of nucleic acids – DNA
and RNA.
Nucleotides
• A nucleotide is composed of
THREE subunits:
• A pentose (or 5-carbon) sugar
– either deoxyribose or ribose;
• A phosphate group;
• A nitrogenous base (adenine,
thymine, guanine, cytosine or
uracil).
Formation of
nucleic acids
• Nucleotides can form
polymers by joining the
phosphate of one
nucleotide to a hydroxyl
group on the sugar of
another nucleotide by a
dehydration reaction.

• This forms a
phosphodiester bond.
Structure of DNA and RNA
Pentose Sugar
• Carbon #2
Structure of DNA and RNA
Nitrogenous Base

DNA RNA

Adenine (A) Adenine (A)

Thymine (T) Uracil (U)
Guanine (G) Guanine (G)
Cytosine (C) Cytosine (C)
Structure
of RNA
• Single-stranded.

• Uses Uracil (U),

instead of
Thymine (T).
Structure of DNA
• Made up of 2 strands
that ‘complement’ each
other.
• Strands run in opposite
direction (anti-parallel).
• Thus, creating a double-
stranded molecule that
looks like a long, twisted
ladder.
Structure of DNA
• Held together in double-helix
structure by the hydrogen bond
between the bases (base pair):
• A with T
• G with C
• The particular sequence of bases
along the DNA molecule determines
the genetic code.
• The order of bases from one strand
can be inferred from the
complementary strand

5’ – CCAGTAG – 3’
3’ – GGTCATC – 5’
Function

DNA RNA
• Contains nucleotides • Has ribose-containing
that store genetic nucleotides that help
information. manifest the genetic
code as a protein.
Fundamentals
Learning Outcomes

• Remember the structure of

nucleic acids
• Remember the biochemical
structure of DNA and RNA
ELECTROPHORESIS DNA-BASED RELATED
ANALYSIS
 Electrophoresis
Learning Objectives
• What is electrophoresis?
• Describe the principle of electrophoresis.
• Describe the components of
electrophoresis.
• Outline the process of electrophoresis.
• Describe the analysis or interpretation of
electrophoresis results.
• Describe the application of
electrophoresis in HFA.
What is electrophoresis?

• A separation technique based on

movement of charged particles in an
electric field according to the size of the
molecules.
• A standard method for separation,
identification, analysis, and purification
of molecules:
• DNA
• Protein
Electrophoresis
Principle No electric
Any charged ion or field
molecule migrates when With electric
field
placed in an electric
field.

The rate of migration

depend upon its net
charge size, shape, and
the applied electric
current.
 Movement depend on
particles’ net charge
What is the charge of DNA and RNA?
• DNA and RNA are
negatively charged:
• The phosphate
backbone of DNA is
negatively charged,
which is due to the
presence of bonds
created between the
phosphorus and oxygen
atoms.
Electrophoresis
Components of the system [1]
Power supply and Buffer
chamber • A fluid mixture of water and ions.
• Electrophoresis buffer allow current to be carried
through the sample while resisting pH changes in the
overall solution.
• Common buffer used: Tris-acetate-EDTA (TAE) or Tris-
borate-EDTA (TBE) buffer at pH 8.3
• Tris acetic acid or Tris boric acid provide ions to support
conductivity and maintain pH.
• EDTA prevents breakdown of molecules.
• Concentration of the buffer affects molecules migration
• Use of water will produce no migration.
• High buffer concentration could melt the agarose gel
Electrophoresis
Components of the system [2]
Electrophoresis Gel
• A porous material that molecules migrates
through.
• Small molecules can pass through the pores
of the gel more easily than large
molecules.
• Small molecules migrate farther in the gel
than large molecules.
Electrophoresis Gel
Types
AGAROSE POLYACRYLAMIDE
A complex sugar chain from red seaweed Chain of acrylamide molecules
Has large pore size Has small pore size
Best for separating large molecules e.g., Best for separating smaller molecules e.g.,
DNA >100bp proteins, DNA <100bp
Unable to tell small differences between Able to tell small differences between sizes
sizes of bands of bands
Gel usually runs horizontally Gel usually run vertically
Factors in choosing agarose gel
concentration
• Concentration affects DNA molecules migration
• Low concentration = larger pores → better resolution of larger
DNA fragments
• High concentration = smaller pores → better resolution of smaller
DNA fragments

1% agarose 2% agarose
Electrophoresis
Components of the system [3]

Sample and Marker

• Loaded onto the gel for analysis

Electrophoresis
Process
1. Sample and marker preparation
2. Gel preparation
3. Load sample and marker onto gel
4. Run electrophoresis
5. Analyse results
Agarose gel preparation
Agarose gel concentration depends on the size
of the DNA fragments to be analysed
Concentration (% w/v) DNA fragment range (kb)
0.3 5-60
0.5 1-30
0.7 0.8-12
1.0 0.5-10
1.2 0.4-7
1.5 0.2-3
2.0 0.1-2
 Once gel is prepared and loaded onto the
Electrophoresis electrophoresis chamber, running buffer is loaded into

Process
the chamber before sample and marker is loaded
into the wells. Run started and once finished, gel is
analysed.
Interpreting gel
electrophoresis
 HOW DO YOU KNOW THE
Agarose and polyacrylamide RESULTED BAND IS YOUR
gel electrophoresis – DNA INTENDED DNA?

• Results appears as bands

on the gel
• Stains are used to make
the bands visible
• Ethidium bromide /
GelRed / GelGreen:
visible under UV light
Electrophoresis
Marker / Ladder

• DNA ladder or marker is a

standard that contains
fragments of known lengths
and concentrations.
• It is a must for it to be
included in electrophoresis
and run next to your sample.
• It can be used to determine
the size of DNA in your
samples.
Example

• PCR product of several

species are listed:
• Fish – 200bp
• Pork – 800bp
• Duck – 300bp
Electrophoresis
Application in HFA
• Application often in
combination with other
equipment such as PCR.
Hence, DNA fragments are
separated according to their
size.
• When the PCR technique
was used, DNA from the
samples was extracted
and amplified for
electrophoresis.
 Electrophoresis
Learning Outcomes
• What is electrophoresis?
• Describe the principle of electrophoresis.
• Describe the components of
electrophoresis.
• Outline the process of electrophoresis.
• Describe the analysis or interpretation of
electrophoresis results.
• Describe the application of
electrophoresis in HFA.