Chapter 6

Molecular
Diagnostics
Objectives
• Explain what molecular diagnostics is and why it is important.
• Know the basics of DNA sequencing
• Understand the principle and procedure of gel electrophoresis
• Understand the principle and procedure for PCR
Molecular Diagnostics
• What is it?
• The process of identifying a disease by studying molecules, such as proteins, DNA,
and RNA in a tissue or fluid.

• Why is it important?
• To detect and measure the presence of genetic material or proteins associated with
a specific health condition or disease.

• Examples of how it is used?

• HLA genotyping, disease risk management, genetic diseases due to mutations, aid in
diagnosis of various cancers, identify infectious diseases
Two different
nucleic acids
1.Deoxyribonucleic acid
(DNA)

2. Ribonucleic acid (RNA)

Flow of Genetic
Information
1. Replication: DNA makes a copy of
itself during cell division.

2. Transcription: process during

which DNA is converted into
complementary sequence of
nucleotide bases in mRNA.

3. Translation: process of
converting nucleotide sequence in
mRNA to linear sequence of amino
acids in proteins.
Polymerase Chain Reaction (PCR)
• Target amplification method
• Used to rapidly make many copies of a specific DNA sample
• Goal is to make enough of the target DNA region that it can be analyzed.
• Visualized by gel electrophoresis
• Mimics the steps of DNA replication in vivo
Molecular Methods
• Hybridization Assays
• Blotting Techniques
• DNA Microassays
• Amplification Techniques
• Cycles conducted in a
thermocycler

• Requires multiple cycles

• Automated
• Quick

• Advantage/Disadvantage
• Extremely sensitive
• Highly prone to
contamination
One Cycle Multiple Cycles
Gel Electrophoresis
Gel = suspension of tiny particles in a medium, occurring in a solid form. Typically, an
agarose gel but also polyacrylamide gel is used.
Electro = flow of electricity
-phoresis = to carry across

Process that uses electricity to separate charged molecules (DNA, RNA, proteins) on a gel
slab.
Gel Electrophoresis
What is needed?
• Solidified agarose gel
• Power supply gel box
• Buffer
• Micropipette
• Loading dye
• Molecular weight markers
• UV light box
Gel & Buffer
• Agarose Gel
• Agarose is most used
• Polysaccharide, generally extracted
from certain red seaweed.
• Requires a specific concentration
• Buffer
• TAE or TBE most common
• pH 7-9 range
• Prevents evaporation
Power Supply
Box
• Power supply sends the
electrical force through the
electrophoresis system.
• Maintains constant power,
current voltage.
• Recommended voltage does
not exceed 5 volts/cm
Loading Samples

•Proper technique
•Loading dye
•Marker (control)
•Samples
Gel Electrophoresis
How it work?
• DNA has an overall negative charge
due to the negative charge on the
phosphate groups.

• DNA is exposed to an electrical field

and the particles migrate toward
the positive electrode.

*Picture to right briefly shows

how to set up, load samples, and
run the test.
 Gel Electrophoresis
• DNA fragments migrate toward the positive
electrode at a rate that depends on their size.
Large DNA fragment = moves slower and stays
closer to the wells.
Small DNA fragments = move faster and move
further from the wells.

• The length of base pairs is determined by

comparison to a commercially available ladder.
Gel Electrophoresis
Factors that affect gel electrophoresis
• Dimension of the gel pores
• pH change
• Size of the DNA
• Voltage used
• Concentration of dye