MLS TERM
MOLECULAR BIOLOGY
LABORATORY
[TRANS] ACTIVITY 1: INTRODUCTION TO MOLECULAR BIOLOGY
MOLECULAR BIOLOGY
- combines laboratory testing with the precision of
molecular biology and has revolutionized the way
clinical and public health laboratories investigate - Possibility of false-positive results from DNA
the human, viral, and microbial genomes, their
genes, and the products they encode (CDC).
- a battery of widely applied, powerful, and sensitive
techniques used to identify biologic markers in a
genome and proteome by detecting bacterial
genes (with PCR-based techniques) and measuring
expressed bacterial infection – specific proteins
(with enzyme-linked immunosorbent assay [ELISA]
and proteomics).
HISTORY OF MOLECULAR BIOLOGY:
 Origin - started in the 1930s and 1940s
 The term “molecular biology” was first
used in 1938 by Warren Weaver.
 Transforming Principle - (1944)
 Oswald Avery, Colin MacLeod, and
Maclyn McCarty reported that dna rather
than protein served as the carrier of
hereditary material
 DNA as the genetic Material (1952)
 Alfred Hershey and Martha Chase
confirmed that DNA is the genetic
material using bacteriophages or viruses
that infect and replicate in bacteria
 Watson and Crick Model of the DNA (1953)
 Watson and Crick described the dna as
having two helical chains coiled in the
same axis, with the sugar and phosphates
outside.
 Semi-Conservative DNA Replication (1958)
 Matthew Michaelson and Franklin Stahl
provided evidence on the semi
conservative nature of DNA replication
(old DNA strands separate and become
templates for the synthesis of a new DNA
strand).
BASIC LABORATORY PROCEDURES:
- Pipetting
- Specimen collection
- Sterilization
ADVANTAGES:
- Rapid-real time generation of results
- Highly specific
- Highly sensitive
- High throughput
DISADVANTAGES:
- Costly
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- Short shelf life of consumables
- Long time to analyze (depending on method)
- Varying stability
contamination
- Possibility of false-negative results from PCR
inhibitors
- Skilled Personnel Training
- Must have prior knowledge of identity of pathogen
- Does not discriminate between viable and non-
viable organisms
- Processing RNA genomes require special
precautions
CLINICAL APPLICATIONS:
- Non-culturable agents (HBV, HPV)
- Fastidious, slow growing agents (Mycobacterium
tuberculosis, Legionella pneumophilia)
- Dangerous to culture infectious pathogens
(Francisella tularensis, Brucella species,
Coccidioidis immitis)
- In situ detection of pathogens (Helicobacter
pylori, Toxoplasma gondii)
- Pathogens with low numbers (HIV, CMV)
- small volumes of specimen
- Genotype determination of antigenically similar
infectious agents
- drug susceptibility (detect genes responsible for
resistance, determine genome alterations
associated with resistance)
- organisms associated with immune complexes
- distribution of an infectious disease across
populations
- Identify genetic and environmental risk factors
WORKFLOW:
 PREANALYTICAL
 Reduction of identification errors
 Sample receipt handling
 Minimization of cross-contamination
 Quality Control Metrics
 Specimen requirements
 DNA requirements
 ANALYTICAL
 DNA and RNA Extraction
 Storage of DNA and RNA
 Standard Devices for Sample Quality
Assessment
 Validation of Comprehensive Molecular
Assay
 Performance of Molecular Tests
 POST-ANALYTICAL
 Data analysis
 Understanding of test results
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  Decision-making process
 Molecular diagnostic report
MOLECULAR METHODS
- Nucleic Acid Hybridization Techniques
- Nucleic Acid Amplification Techniques
- Microarrays
NUCLEIC ACID HYBRIDIZATION TECHNIQUES:
 FISH (FLUORESCENCE IN SITU HYBRIDIZATION)
 allows the detection of microbial nucleic
acid directly from the sample (or cultured
sample).
 without prior nucleic acid amplification.
 specimen fixation on a microscope slide,
hybridizing the prepared sample with a
specific fluorescent-labelled probe, and
visual detection of the hybridization with
a fluorescent microscope.
 LINE PROBE ASSAY
 Specific oligonucleotide probes are
attached at known locations on a
nitrocellulose strip as parallel lines and
hybridized with biotin-labelled PCR
products.
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NUCLEIC ACID AMPLIFICATION TECHNIQUES:
- CYCLING AMPLIFICATION
- ISOTHERMAL AMPLIFICATION
- MICROARRAYS
CYCLING AMPLIFICATION:
 POLYMERASE CHAIN REACTION
 most frequently used molecular
diagnostic techniques in the diagnosis of
infectious pathogens
 Very high sensitivity &specificity
 allows the amplification of millions of
identical DNA copies from an originally
small amount of pathogen genome in a
clinical sample
 Specific oligonucleotide primers are
annealed to the DNA in a lower
temperature, followed by an extension
phase during which the DNA polymerase
enzyme copies the template strand
 cycle is repeated, usually 30–40 times,
resulting in millions of identical DNA
copies.
 LIGASE CHAIN REACTION
 a modification of PCR
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 used for the production of additional DNA
templates.
 TRANSCRIPTION-MEDIATED AMPLIFICATION
(TMA)
 similar to NASBA, except that it uses the
RNAse H activity of reverse transcriptase,
whereas NASBA uses a separate enzyme
for that

 STRAND DISPLACEMENT AMPLIFICATION (SDA)

 first set of primers containing a restriction
site is annealed to DNA template
 second primers are then annealed
adjacent to the first ones and start
amplification
 after which restriction enzyme HincII is
introduced in order to nick the
synthesized DNA
 an exonuclease-deficient form of the
Klenow fragment of Escherichia coli DNA
polymerase I starts the amplification
again, displacing the newly synthesized
strands
 MICROARRAYS
 consist of a two-dimensional matrix of
 two adjacent probes hybridize to one biomolecules which are printed or
strand of the target DNA
 the small gap between the two adjacent
primers is recognized by a highly specific
thermostable DNA ligase, and ligated to
form a single probe
 the ligated products then serve as
templates for the amplification process
 LCR allows the detection of only single
base pair mutations, and it is very
specific

ISOTHERMAL AMPLIFICATION:
 NUCLEIC ACID SEQUENCE-BASED
AMPLIFICATION (NASBA)
 the target RNA is converted to double-
stranded DNA by using T7 RNA
polymerase, RNAse H, and a primer with
a T7 promoter
 DNA acts as a template to produce
multiple copies of RNA with a polarity
opposite that of the target, which can be

synthesized on a glass, silicon, plastic or

nylon membrane
 detect both nucleic acids and antibodies,
which attach to the immobilized
biomolecule, which can be, e.g., an
oligonucleotide or a protein

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  Positive reaction can be detected with  Reagent preparation room
highly advanced scanners by use of target  Sample preparation room
labelling with fluorescent probes or  PCR room
antibodies

POTENTIAL SOURCES OF CONTAMINATION:

REQUIREMENTS FOR A MOLECULAR LABORATORY:

1. Specific area for the following:
 Specimen reception area
 Virus inactivation and nucleic acid
extraction (Pre-PCR)
 Reagent storage and handling
 PCR
 Clerical activity area
2. Unidirectional workflow
3. Prototype floor plan should be followed.
4. Controlled and adequate ventilation with
the prescribed air changes per hour shall
be maintained for each specific area.
5. Adequate lighting shall be provided in all
areas.

- Amplification product contamination

- Cross contamination between specimens
- Laboratory surfaces
- Ventilation ducts
- Reagents/supplies
- Hair, skin, saliva, and clothes of lab personnel

HOW TO CONTROL CONTAMINATION?

- Laboratory design
- Laboratory practices
- Chemical and enzymatic controls

ENVIRONMENTAL MANAGEMENT:
1. There shall be a written plan and program of
proper disinfection and preventive maintenance of
the facility.
2. There shall be appropriate signage, and that only
authorized personnel shall be allowed entry.
3. The use of Personal Protective Equipment and
adherence to Infection Control Policies shall be
strictly observed.
4. There shall be procedures for the proper disposal
of infectious wastes and toxic and hazardous
THE MOLECULAR LABORATORY SET-UP
substances in accordance with R.A. No. 6969
- A Molecular Biology Lab must be separate from
known as “Toxic and Hazardous Substances and
other usual labs in the school.
Nuclear Wastes Act” and other related policy
- Needs adequate space, which ideally consists of
three separate areas, namely:

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 guidelines and/or issuance (e.g. DOH Healthcare
Waste Management Manual).
5. There shall be a Memorandum of Agreement with
infectious waste and toxic and hazardous
substances hauler.

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