MOLECULAR BIOLOGY AND

DIAGNOSTICS
Course Delivery
DR. MAGDALENA F. NATIVIDAD
PASMETH Past President
FEU – NRMF

28 JULY 2020 2:00PM VIA ZOOM

CHED - FEU-NRMF WORKSHOP 2018
CHED - FEU-NRMF WORKSHOP 2018
MBD Training
at UST
 Outline
• Course Description
• Course Intended Learning Outcomes
• Course Content
• Resources
• Lab Set Up
• Lab Equipment, Reagents, other materials
• Storage of Reagents
• Sterilization of Buffers and Solutions
• Assessment
Molecular Biology and Diagnostics

• 3-unit course:
• 1 unit Lecture; 2 units Lab

• Prerequisite Course:
• Biochemistry
Importance of the Course
• One of the new courses in the CMO 13, S. 2017.
• A competency expected of a medical
technologist/medical scientists.
• Molecular diagnostics is slowly evolving in our
laboratories and we must keep up to this new
technology, otherwise we might find non-MT/MLS
doing diagnostic work in these laboratories.
• This is now very evident with the appearance of SARS-Cov-2
 Course Description
• This course applies the basic concepts of structure and function of nucleic acids and
proteins to molecular diagnostic techniques such as DNA and RNA isolation, gel
electrophoresis, hybridization, nucleic acid amplification, restriction enzyme
applications, microarrays, DNA sequencing, protein analytic techniques.
• It also covers the application of the techniques in the diagnosis of infectious and
non-infectious diseases, in forensics, and in genetic testing.
• Bioinformatics and its applications will also be covered.
• The laboratory exercises will introduce the students to common laboratory
techniques in molecular biology, allow them to gain hands-on experience in these
techniques and to apply biosafety practices.
SUGGESTED: Course Intended Learning Outcomes
(CILOs)
At the end of the course, the student shall :
1. Apply the concepts of the structure and functions of DNA, RNA, and proteins,
and of mutation to understanding of molecular diagnostic techniques.

2. Apply good laboratory practices, biosafety and biorisk principles.

3. Discuss the principles and applications of molecular techniques in the analysis
of DNA, RNA, and proteins.

4. Perform laboratory techniques in the analysis of DNA, RNA, and proteins.

5. Discuss the applications of molecular methods in the diagnosis of infectious and
non-infectious diseases, in forensic science, and in genetic testing.

6. Apply bioinformatics tools in the analysis of genes and proteins and the design
of primers.
7. Analyze current molecular biology literature.
Course Schedule of Activities
WEEK LECTURE (1 hour/week) LAB (6 hours/week)
1 Orientation Basic Lab Practices
2 Biosafety and Waste Management Lab Math, Getting acquainted with molecular Biology
lab equipment
3 Review of DNA, RNA, and Protein structure and function, Pipetting exercise, Reagent Preparation, Use of pH
Mutation meter
4 DNA and RNA Isolation Techniques DNA, RNA structure, Mutation (Pen and Paper)

5 DNA Hybridization Techniques: Dot Blot, Southern blot, DNA isolation, Spectrophotometry
Northern blot, ISH, FISH
6 PRELIM EXAM
7 Fundamentals of PCR, PCR reagents Gel Electrophoresis
8 Other Amplification Techniques: RT-PCR, qPCR, Multiplex PCR: Pre-analytic (planning)
PCR, Digital PCR Optimization, Master mix preparation
9 DNA sequencing techniques, Microarray PCR: Analytical Phase
10 Restriction Endonucleases, RFLP PCR product visualization
11 Mutation Detection Techniques: allele-specific Restriction Endonuclease digestion, Restriction mapping
oligonucleotide hybridization, allele-specific PCR,

12 MIDTERM EXAM
13 Techniques in Protein analysis: Western blot, SDS-PAGE, DNA Sequencing (Pen and paper)
ELISA, MALDI-TOF, others
14 Bioinformatics 1: The Basics ELISA technique, SDS-PAGE
15 Bioinformatics 2 (Primer Design) Bioinformatics: Sequence Alignment
16 Applications of Molecular technique in Disease Bioinformatics: Primer design
17 Applications of Molecular Techniques in Forensics, Wrap up
Genetic testing
18 FINAL EXAMINATION
 Suggested Textbooks
• Campbell, Farrel, McDougal. (2018) Biochemistry. Cengage Learning
• Hofmann A and Clockie S. Wilson and Walker’s Principles and Techniques of Biochemistry and Molecular
Biology.(2018). Cambridge University Press, united Kingdom.
• Grenn MR, J Sambrook. Molecular Cloning: A Laboratory Manual (2012) Cold Spring Harbor Laboratory
Press
• Miller H, Witherow DS and Carson S. (2019) Molecular Biology Techniques: A Classroom Laboratory Manual
4th Edition Elsevier Academic Press
• Nolan T., Bustin SA. (2013) PCR Technology: Current Innovation CRC Press
• Clark DP, Pazdernick NJ. Molecular Biology. (2013) 2nd ed. Elsevier Academic Press.
• Buckingham L, Flaws ML. (2007) Molecular Diagnostics: Fundamentals, Methods and Clinical Applications
F.A. Davis Company Philadelphia
• Coleman WB, Tsongalis GJ. Diagnostic Molecular Pathology. A Guide to Molecular Testing. (2016).
Academic Press.
 Laboratory
Resources
Biosafety and Waste Management
• https://www.who.int/hiv/pub/drugresistance/
HIVDR_Mod_16_Biosafety_Waste_Mgt.ppt?ua
=1

• WHO Lab Biosafety Manual

 The Molecular Laboratory Set-Up

• A Molecular Biology Lab must

be separate from other usual
labs in the school.
• Needs adequate space,
which ideally consists of three
separate areas, namely
üReagent preparation room
üSample preparation room
üPCR room.
Why separate areas? Contamination

• Introduction of unwanted nucleic acids into

specimen
• the sensitivity of PCR techniques makes them vulnerable
to contamination
• Repeated amplification of the same target
sequence leads to accumulation of amplification
products in the laboratory environment
•Buildup of aerosolized
•A typical PCR generates as •Aerosols from pipettes will amplification products will
many as 109 copies of target contain as many as 106 contaminate laboratory
sequence amplification products reagents, equipment, and
ventilation systems
Potential Sources of Contamination

• Amplification product contamination

• Cross contamination between specimens
• Laboratory surfaces
• Ventilation ducts
• Reagents/supplies
• Hair, skin, saliva, and clothes of lab personnel
What happens if • Incorrect results
there is lack • Require extensive
contamination
cleanup
control?
• Loss of credibility
• Impact on economy
and performance
How to Control Contamination

• Laboratory design
• Laboratory practices
• Chemical and enzymatic controls
Laboratory Equipment
-20oC Freezer

Autoclave

Biosafety Cabinet
Refrigerator 4oC
freezer: -18oC
 Laboratory Equipment

Analytical Balance pH Meter

Vortex mixer

Spectrophotometer
Laboratory Equipment

A dry bath or a
water bath

Electrophoresis apparatus
Thermocycler

UV Transilluminator
and
Gel Documentation
(optional)
microcentrifuge
Laboratory Equipment

Refrigerated centrifuge

Ice Shaver Microwave Oven

microcentrifuge
 Pipette tips

Pipettors Pipette type Volumes (µL) Tip color

P10 0.5–10 white

P20 2–20 yellow

P200 20–200 yellow

P1000 200–1000 blue

P5000 1000–5000 white

 Microcentrifuge tubes
0.2-ml for PCR
Tip racks
0.5-ml
Different sizes
1.5-ml Spray bottles
Other Materials

Spatulas Baker’s cups Graduated conical tubes

Different sizes
Other materials

Microcentrifuge tube racks Freezer boxes Ice buckets

 Reagents
Agarose Powder Gel loading dye or buffer
Tris Base Gel Red (for gel electrophoresis)
NaCl Molecular weight markers
MgCl2 PCR kit (dNTPs, MgCl2, Taq polymerase, buffer)
EDTA powder Restriction enzymes
Absolute ethanol DNA isolation kit
Nuclease free water/Nanopure water ELISA kit (Biorad)
Distilled Water (e.g., Wilkins) Proteinase K
Glacial acetic acid Sodium hypochlorite (bleach)
HCl Autoclave tapes
NaOH
Boric acid
Na Acetate
Sterilization of Commonly used Reagents and
Materials
Even if you require the
• Autoclave: buffer for a non-sterile
application, it should
1. Most buffers be sterilized, because
microbial growth can
cause pH changes

if a heat-labile or otherwise non-autoclavable ingredient must be added to

an autoclavable buffer, autoclave the buffer first.
When the buffer has cooled to room temperature, add the filter-sterilized
(non-autoclavable) ingredient.

2. Undefined bacterial and yeast media

3. Tips, microcentriguge tubes
Sterilization of Commonly used Reagents
• Do Not Autoclave:
• Corrosives (acids, bases, phenol), solvents or volatiles (ethanol,
methanol, chloroform)
• Liquids containing bleach, formalin, or glutaraldehyde
• Buffers with detergents, such as 10%SDS, since they can boil over
• Heat-labile ingredients such as serum and vitamins, antibiotics, and
proteins (BSA)
• Dithioethreitol (DTT) or β-mercaptoethanol (BME)-containing solutions
Sterilization of Commonly used Reagents

• Alternative: Filter-sterilization (0.2 Um)

Storage of Buffers and Solutions
Room Temperature 4o C -20oC -70oC

Detergents Bacterial cultures Enzymes Frozen bacterial cultures

Ethanol Buffers* RNA Lipids

Buffers* DNA

Concentrated solutions Media

Acids and bases PCR products

Serum
*Buffers: 4oC or at room temperature. Some, especially concentrated ones, precipitate in the cold; heat at 37 C for a few
minutes dissolves the ppt.

Hazardous Chemicals: refer to MSDS (Materials Safety Data Sheet) which describes
the composition and properties, toxicology, and instructions for handling, spill control,
and waste disposal.
Assessment
• First Prelim Exam 15%
• Midterm Exam 15%
• Final Exam 15%
• Weekly Shifting Exams 15%
• Laboratory Performance 40%
• Lab Worksheet/Report
• Other graded activities

• TOTAL 100%
Reference:
• Barker K. 2005. A the Bench. A Laboratory Navigator.
Cold Spring harbor Laboratory Press