PPE Donning and Doffing MOLECULAR BIOLOGY

Dr. Andro Garcia | May 16, 2021 TRANS 01

OUTLINE II. SECOND VIDEO

I. First Video
A. Donning A. DONNING
B. Doffing 1. Foot cover
II. Second Video 2. Perform hand hygiene using alcohol-based sanitizer.
A. Donning 3. 1st glove- inner gloves
B. Doffing 4. Gown
III. For breaches in PPE 5. Mask
6. Outer gown/apron
7. 2nd glove- outer gloves
LEGEND 8. Goggles
Remember Lecturer Book Previous Presentation
Trans *Remove watch,jewelry and content bof the pockets
Tie your hair

I. FIRST VIDEO B. DOFFING

A. DONNING
1. Perform hand hygiene using alcohol-based sanitizer. 1. In case of visible contamination, use 1:10 bleach
2. Gown disinfectant to wipe it off
3. Mask 2. Disinfect outer gloves but do not remove it yet
4. Goggles 3. Remove outer gown/apron
5. Gloves 4. Disinfect outer gloves but do not remove it yet
5. Remove foot cover
*Remove watch,jewelry and content bof the pockets 6. Disinfect outer gloves but do not remove it yet
Tie your hair\ 7. Remove outer gloves
8. Disinfect inner gloves but do not remove it yet
B. DOFFING 9. Remove goggles, avoid touching its front surface
1. Remove gown- remove by rolling it away from your body, 10. Disinfect inner gloves but do not remove it yet
taking care to not contaminate your uniform with the outside 11. Remove the hood of the gown from your head
of the gown 12. Disinfect inner gloves but do not remove it yet
2. Remove gloves 13. Remove gown (lift chin) and discard (touch only the inside
3. Perform hand hygiene of the gown prior to disposal)
4. Put a new pair of gloves 14. Disinfect inner gloves but do not remove it yet
5. Remove and disinfect goggles 15. Remove inner glove
6. Remove 2nd pair of gloves 16. Perform hand hygiene
7. Perform hand hygiene 17. Put a new pair of gloves
8. Remove mask 18. Remove mask by tilting the head slightly forward and w/o
9. Perform hand hygiene touching the front surface of the mask
19. Disinfect gloves
*Put all PPE in a leak-proof bag. 20. Disinfect your boots
21. Disinfect and remove gloves
22. Perform hand hygiene

*Put all PPE in a leak-proof bag.

III. FOR BREACHES IN PPE

WASH skin with soap and water.

IRRIGATE mucous membrane with copious amount of water or

an eyewash solution.

REPORT the incident immediately.

END OF TRANSCRIPTION

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 Introduction to Mol Bio and Diagnostics MOLECULAR BIOLOGY

Dr. Andro Garcia | May 16, 2021 TRANS 02

OUTLINE
I. Molecular Biology
A. Tools of Mol Bio B. Terminologies
B. Terminologies • Functional Genomics- study of expression of large number of genes.
C. Application of Mol Bio • Transcriptomics- study of transcriptomes (all the transcripts of an
organism makes at any given time)
• Proteomics- study of proteomes (set of expressed proteins in a given
LEGEND type cells or an organism at a given time under defined conditions).
Remember Lecturer Book Previous Presentation • Transcription- copying of DNA to produce RNA
Trans • Splicing- cutting of DNA
• Translation- formation of protein
• Reverse Transcription- enzyme-mediated synthesis of a DNA
I. MOLECULAR BIOLOGY molecule from an RNA template

- a branch of biology that study gene structure and function at the C. Application of Molecular Biology
molecular level (chromosomes, RNA, DNA) 1. Research
- this field overlaps with other areas (Genetics & Biology) 2. Diagnosis
- It allows the laboratory to be predictive in nature; events that occur in 3. Transplantation
the future. Trend in medicine, for example; babies are being screen to 4. Paternity Testing
know if they will have future diseases because gene components are 5. Forensic Analysis
already being examined. Ex. thalassemia, down syndrome. 6. Gene Therapy- the movie One more try “pahiram ng asawa
- Used to diagnose viral diseases mo”. Yung gene sa BM ng magiging anak ni angel gagamitin
para masave anak nya
7. Drug Design
A. Tools of Molecular Biology
• PCR- gold standard for coronavirus detection END OF TRANSCRIPTION
• Vector Molecular Cloning- cloning of causative agent

• Electrophoretic Separation of Nucleic acid- part of PCR

Electrophoretic
Nucleic acid Detection of
Separation of
Fractionation Genes
Nucleic acid

Polymerase DNA: Southern

DNA sequencing
Chain Reaction Blotting

Probes RNA: Northern

Microassay
Hybridization Blotting

Vector,
Nucleic acid Protein:
Molecular
Enzyme Western Blotting
Cloning

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 Biosafety & Biosecurity MOLECULAR BIOLOGY

Dr. Andro Garcia | May 16, 2021 TRANS 03

OUTLINE

I. Biosafety II. Biosecurity - BSL-2 lab is expected to have more controls than BSL-1
A. Biosafety Level • In addition to the Standard Laboratory Practices of
B. PPE BSL-1 these practices must be followed in BSL-2
C. Microbes labs:
D. Factors Affecting • Using biosafety cabinet
Disinfectant
E. Biological Safety • Trained personnel
Cabinet • Availability of eye wash station
• Vaccination for the workers if applicable
LEGEND 3. BIOSAFTEY LEVEL 3 (BSL-3)
Remember Lecturer Book Previous Presentation - This level includes the microbes or agents that cause
Trans serious or fatal diseases thru inhalation
- Requires autoclave
- BSL-3 facilities are usually under the control on
I. BIOSAFETY government agencies and the laboratory staff are medical
- Prevention of large-scale loss of biological integrity, focusing on both surveillance.
ecology and human health.
- EXAMPLE: Yellow fever & Tuberculosis bacteria
- For protection from harmful incidents. Keeping contained bacteria and
viruses well contained. - BSL-3 facility should maintain unidirectional air flow from
clean air to the infectious air.
A. Biosafety Level • The recirculation of air happens thru the HEPA
-Set of bio containment controls that requires to separate the biological filter.
agents based on the risk they cause on the environment and the human - Additional laboratory practices must include the following:
beings. • Periodic medical testing for workers
-These levels are rank from 1 to 4. Each level has specific controls for
• Full body garment with respiratory protection
containment or biological agents and microbes.
-They control based on the: • Restricted access at all times
• Infectivity of the disease
• Severity of the disease 4. BIOSAFTEY LEVEL 4 (BSL-4)
• Source of the agent - BSl-4 facilities are rare in the world being the highest level
• Route of invasion into the human body of biological safety. Requires the strictest protocols
-These biosafety levels play an important role in:
- Requires autoclave
• Designing the facility
- BSL-4 microbes cause fatal infections.
• Safety environment
• Laboratory practices • The diseases caused by these agents usually do
-Each biosafety level has its own standard laboratory practices one must not have vaccines or treatment.
follow. - EXAMPLE: Ebola and Marburgvirus
-However, each microbiology laboratory in respective on their biosafety level - BSL-4 facility must be isolated and have dedicated supply
must follow the basic good laboratory practices.
air and exhaust air.
1. BIOSAFTEY LEVEL 1 (BSL-1) - In addition to the other BSL laboratories, these practices
- Lowest biosafety level must be followed in BSL-4 labs:
- Includes the microbes that are non-pathogenic. • Decontamination of materials before exiting
- Laboratory personnel can work with minimum risk in these • Full body garment
facilities • Taking shower after leaving the facility
- BSL labs do not need special containments and equipment. •
• People can work on open bench tops Regardless of the level, knowing the basics of biosafety is
• This lab is typically used for students and trainee absolutely imperative in protecting yourself and others.
microbiologists.
WHO BIOSAFETY GUIDELINES FOR COVID-19
- EXAMPLE: Non-pathogenic strain of E.coli
Recommendations are:
- BASIC SAFETY PRACTICES: 1. Initial processing of specimen should take place in a validated
• Hand washing BSC or primary containment device.
• Wearing lab coat, glove and eye protection 2. Non-propagative diagnostic lab work (ex. sequencing nucleic
• Limited access to people acid, amplification test [NAAT]) should be conducted at a facility
using procedures equivalent to Biosafety Level 2 (BSL-2)
• No mouth pipetting
3. Propagative work (ex. culturing virus, neutralization assays)
• Cleaning and decontamination of area should be conducted in a containment lab with inward
• Warning signs directional airflow (BSL-3)

2. BIOSAFETY LEVEL 2 (BSL-2)

- For microbes that are associated with human diseases
• That means, pathogenic or infectious bacteria and
viruses are included in this level
- EXAMPLE: Staphylococcus and Hepatitis virus
RITM Guidance for COVID-19
1. Routine diagnostic test and other non-viral propagative
research can be safely conducted in BSL-2 with
enhancements based on local risk assessment.

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Biosafety & Biosecurity TRANS 03

2. Initial processing &inactivation of respiratory specimens in a

certified Class II biosafety cabinet.
3. In BSC, start working from clean to dirty E. Biological Safety Cabinet
4. In hierarchy of controls , PPE is the last line of defense, not
the first. We should not assume that we are safe by just 1. Wash hand before and after using biological safety cabinet
wearing PPE 2. Never store items in BSC
3. Disinfect all work surface before & after using
Basic Biosafety Rules 4. Discard container must be inside your BSC
Basic Biosafety Rules are very similar to Standard Lab Safety: 5. BSC must be certified to ensure efficiency
5. No eating & drinking inside the lab a. HEPA filter must be tested
6. Never put contacts or apply cosmetics inside lab b. Proper airflow velocities and pattern must be observed
7. Never wear sandals/shorts.
8. In case of emergency, every lab requires a first aid kit CDC & American Biological Safety Association do not support
9. Immediately inform your Lab Biosafety Officer if exposure to installing UV lamps in biological safety cabinets
infectious agents occur.
10. Read the info of the reagents from its MSDS sheet to be
guided when handling reagents. Biological Safety Cabinet
Class II Type A2- air is recirculated within the cabinet to provide a
B. PPE sterile working area and discharge into the lab through a HEPA filter to
-design to protect your body ensure that lab is not compromised either.
-should be chosen based on the work
-only worn and stored in the lab
-all employees should be trained properly Validating the autoclave procedure:
Mechanical Indicators- detects cycle error
C. Microbes Autoclave Tape- turns color when exposed to 121 C
Pathogen- microbes that cause diseases. Chemical Indicator Strips- for monitoring sterilization
Biological Indicator- means of validation that sterilization has been
achieved.
How to keep you safe
• Store & transport carefully Emergency Procedures
• Incorrect pipetting can produce aerosol which can be harmful for the 1. Conduct regular drills of mock spills and other potential
worker. emergency scenarios
• Choose Disinfectants Wisely (EPA Registered Disinfectant)
• Adhere to all biosafety Guidelines
II. BIOSECURITY
D-value: time required for control to kill 90% mmicroorganism 1. Increase biosecurity awareness among front-liners
2. Restrict access to patient areas and laboratories
D. Factors affecting Disinfectants 3. Moving sample to lab ASAP
1. Concentration of anti-microbial agents a. Ensure that there is a chain of custody sign off
2. Composition of the microbial population b. Knowing who you are handling the sample
3. Contact time 4. Decontaminate all potentially contaminated sample that are
no longer use.

SARS COV-2 is susceptible to commonly used disinfectatnt in

laboratory:
1. 70% ethanol
Biosafety and biosecurity are also a key part of the COVID-19 response
2. Sodium hypochlorite
efforts

Sterilization & Disinfection differ

END OF TRANSCRIPTION

MOLECULAR BIOLOGY 2 of 2
 Molecular Biology: Basic Science TRANS 04

LEGEND
Remember Lecturer Book Previous Presentation Basic differences between eukaryotes and prokaryotes
Trans Attribute Eukaryotes Prokaryotes
Organisms Plants, animals and Bacteria and
fungi cyanobacteria
Three Domain of Life Cell wall No (animals); Yes yes
• Eukaryotic (plants)
• Prokaryotic Chromosome Mitotic spindle Cell membrane
• Archaea segregation
Meiosis + -
Eukaryotic Cell Ribosome size 80s 70s
• Eukaryotes are generally more advanced than prokaryotes. Cell organelle
“eu” means “true” – a cell having a true nucleus. Nuclear membrane + Absent
Endoplasmic + -
reticulum
Golgi apparatus + -
Mitochondria + -
Chloroplasts + -

Archaea
• Archaea is prokaryotes; organisms without nucleus but
some aspects of their molecular biology are more similar to
those of eukaryotes.

• Eukaryotic cells are found in animals, plants, fungi and

protists cell;
• Cell with a true nucleus, where the genetic material is
surrounded by a membrane
• Eukaryotic genome is more complex than that of
prokaryotes and distributed among multiple chromosomes.
• Eukaryotic DNA is linear arrangement
• Eukaryotic DNA is complexed with proteins called The Genome
histones. Prokaryotes does not have histones. • Totality of genetic information of an organism.
• Numerous membrane-bound organelles; • Encoded in the DNA (for some viruses, RNA).
• Complex internal structure; Remember each cells have DNA and individual cells with
• Cell division by mitosis DNA have its own genetic information that is why they can live
independently. Except for RNA viruses like the SARS-Cov 2
Prokaryotic Cell wherein its genetic information is encoded in the RNA.
• Unicellular organisms, found in all environments. These
include bacteria and archaea.
• Without a nucleus; no nuclear membrane (genetic material
dispersed throughout cytoplasms;
• No membrane-bound organelles;
• Cell contains only one circular DNA molecule contained in
the cytoplasm;
• DNA is naked (because no histone);
• Simple internal structure; and
• Cell division by simple binary fission

Unit bp stands for base pair.

MOLECULAR BIOLOGY 1 of 6
 Molecular Biology: Basic Science TRANS 04

Species/Number of Chromosome

Species Number of Chromosome

Human 46
Mouse 40
Rat 42
Fruit flies 8
Bacteria 1

Human Genome
Human Genome: Arranged on multiple chromosomes; twenty
three pairs of chromosomes;
• Twenty two pairs (autosomes)
• One pair (sex chromosome) (xx) (female) or (xy) (male).
Humans have 23 pairs of chromosomes in every cell (except
mature red blood cells.); Gametes or sex cells (sperm and egg)
have half the normal complement of chromosomes.
Nucleotides
Nucleotides; ring shaped structures composed of:
1. Nitrogenous base; these bases are classified based
on their chemical structures into two groups:
✓ Purine; double ringed structure (Adenine and
Guanine).
✓ Pyrimidine; single ring structures (cytosine and
thymine).
2. Sugar
3. Phosphate group

• DNA: Four different types of nucleotides differ in nitrogenous

base:
✓ A is for adenine;
✓ G is for guanine;
✓ C is for cytosine and
✓ T is for thymine.

• RNA: thymine base replaced by uracil base.

Included in the list of probable questions in the board exam;
the number of percent of each genes/functionality of DNA on a
person.
Nuclear genome is composed of 3.2 Giga base pair and
the Mitochondrial genome is 16.5.
Nuclear<Mitochondrial.
Under Nuclear genome, Extragenic DNA (75%) >
Genes and gene related DNA (25%)
Out of the genes and gene related DNA, Coding and Bases
regulatory DNA (10%) < Non-coding DNA (90%) • Types: - adenine and guanine (fused five- and six-
Out of the 90% Non-coding DNA, it includes the less membered heterocyclic compounds) – Purines
functional. (Ex. Pseudogenes, Gene fragments, Introns,
Untranslated sequences)
For the Mitochondrial genome; it includes the rRNA,
tRNA and polypeptide encoding genes. These are
the functional units needed by an individual to maintain
the integrity or life of a cell. That is why Mitochondrial
genome is greater in number.

General Structure of Nucleic Acid

• DNA and RNA are long chain polymers of small chemical
compound called nucleotides.

• Cytosine & thymine (six-membered rings)-Pyrimidines.

• A fifth pyrimidine base, called uracil (U), usually takes the
place of thymine in RNA and differs from thymine by lacking a
methyl group on its ring.
• PAIRING: A =T and A=U

MOLECULAR BIOLOGY 2 of 6
 Molecular Biology: Basic Science TRANS 04

Sugar + Base = nucleoside

Phosphate + sugar + Base = nucleotide

THE DNA Dominant and Recessive

• Deoxyribonucleic Acid (DNA); the genetic material of all Dominant
cellular organisms and most viruses. • The one pair of allele that masks the effect of the other when
• DNA; the gigantic molecule which is used to encode genetic present in the same cell.
information for all life on Earth. Exception is when paired with another dominant allele in
• A human cell contains about 2 meters of DNA. DNA in the the complementary cell, it cannot be fully expressed.
body could stretch to the sun and back almost 100 times. So Recessive
it is tightly packed. • The one pair of allele that is masked by the other when
• DNA responsible for preserving, copying and transmitting present in the same cell and capable of producing its
information within cells and from generation to generation. characteristics phenotype in the organism only when two
alleles are present and identical.
DNA Double Helix
• Linked as a twisted ladder.
• The curving sides of the ladder represent the sugar-
phosphate backbone of the two DNA strands; the rungs are
the base pairs.
• Possess antiparallel polarity.
• Stabilized by hydrogen bonds between the bases.

Gene Structure
• Most of the genes consist of; short coding sequences or
exons are interrupted by a longer intervening noncoding
sequence or introns; although a few genes in the human
genome have no introns.

The Gene
DNA Organization
• The gene; it is a segment within a very long strand of DNA.
• DNA molecules complexed with other
• Genes are the basic units of hereditary.
proteins, especially basic proteins
• Genes located on chromosome on its place or locus.
called histones to form a substance
• Allele; a variant of the DNA sequence at a given locus. Each
known as chromatin.
allele inherited from a different parent.
Each part of our DNA has its own loci, and each loci
Chromatin=DNA + Histones
retains the characteristic of that cell which is called the
allele – the variant of the DNA where information is
collected that came from the parent cell. Mawala man
ang parent cell pero the allele is still present sa portion
ng DNA na yun, nandun parin and genetic information.
In each gene, a portion of Exon and Intron is present.
Intron – non-coding portion, usually mas mahaba
Exon - coding region;short

MOLECULAR BIOLOGY 3 of 6
 Molecular Biology: Basic Science TRANS 04

• Ribosome; factory for protein synthesis; composed of

ribosomal RNA and ribosomal proteins (known as a
Ribonucleoproteinor RNP).
• rRNA provides a mechanism for decoding mRNA into amino
acids. Need ng decoding para alam kung anong amino acid
ang ibibigay ni tRNA.

Ribosomes
• Factory for protein synthesis.
• Composed of ribosomal RNA and ribosomal proteins
(known as a Ribonucleoprotein or RNP).
• Translate (mRNA) to build polypeptide chains using amino
acids delivered by (tRNA)
THE RNA The Protein
• Three major classes of RNA: • Proteins are chain like polymers of a few or many thousands
✓ messenger (mRNA), of amino acids. •
✓ transfer (tRNA)
• Amino acids: (3-nucleotide RNA sequences) (codon)
✓ ribosomal (rRNA).
Four Levels of Protein Structure
• Minor classes of RNA include 1. Primary protein structure: basic sequence of a chain of
✓ small nuclear RNA; amino acid.
✓ small nucleolar RNA;
2. Secondary protein structure: A chain of amino acids linked
by hydrogen bonds.
• RNA is a single stranded; the pyrimidine base uracil (U)
replaces thymine and ribose sugar replaces deoxyribose.
RNA=ribose sugar ; DNA=deoxyribose
3. Tertiary protein structure: It occurs when certain attraction
occurs between alpha helices and pleated sheets.
Messenger RNA / mRNA
There is interaction
• Transcripts of structural genes.
• Encode all the information necessary for the synthesis of a 4. Quaternary protein structure: Protein containing more than
polypeptide of protein. one amino acid chains. Ex. Hemoglobin structure
• The 5' terminus is capped by 7 methyguanosine Changes in the shape. Modification of the structure of
triphosphate. proteins.
• Synthesis of the poly (A) tail involves cleavage of its 3' end
and then the addition of about 200 adenine residues.
• Intermediate carrier of genetic information; deliver genetic
information to the cytoplasm.

Transfer RNA / tRNA

• All the tRNAs share a common secondary structure
resembles a cloverleaf: They have four base- paired stems
defining three stem-loops (the D loop, anticodon loop, and
T loop) and the acceptor stem.
• tRNA carry correct amino acids to their position along the
mRNA template to be added to the growing polypeptide
chain. Correct amino acid para macopy ng tamang protein.

Genetic Mutation
• A mutation is a change in the
DNA sequence or arrangement
of DNA.

Ribosomal RNA / tRNA

• The central component of the ribosome.

MOLECULAR BIOLOGY 4 of 6
 Molecular Biology: Basic Science
COMMON TOOLS OF MOLECULAR BIOLOGY
Gel Electrophoresis
• The basic principle is that DNA, RNA, and proteins can all be
separated by means of an electric field.
• In agarose gel electrophoresis, DNA and RNA can be
separated on the basis of size by running the DNA through
an agarose gel.
• Proteins can be separated on the basis of size by using an
SDSPAGE gel, or on the basis of size and their electric
charge by using what is known as a 2D gel electrophoresis.
Polymerase Chain Reaction (PCR)
• The polymerase chain reaction is an extremely versatile
technique for copying DNA.
• PCR allows a single DNA sequence to be copied (millions of
times), or altered in predetermined ways.
For example swab sa nose and the present virus is
sars-cov 2 RNA is dalawa lang. The process of PCR is
to extract the RNA, there are series of steps para
maiwan na lang is the RNA strands. For example meron
ka na pure RNA, it will then be transferred sa PCR
machine para macopy. Yung RNA gagawan ng
complementary copy na gagawing DNA since RNA siya.
Then the copied DNA will go through amplification
hanggang dumami siya and will be detected by the PCR
machine as present kaya magppositive ka.
Kahit isa or dalawa or konti lang ang RNA/DNA na
present, kaya madetect ng PCR.
• PCR has many variations, like reverse transcription PCR
(RT-PCR) for amplification of RNA, and real-time PCR
(QPCR) which allow for quantitative measurement of DNA or
RNA molecules
QPCR – RNA/DNA being multiplied
RT-PCR – RNA convert to DNA then multiplied.
Example: Sars-CoV2. Since it is RNA virus
PCR Analysis
The process follows the principle of DNA replication.
Exponential amplification
CT value/Cycle number – lower value means mas
infected.
• If 1000 ang nacollect na DNA/RNA sa nose
mo, then niprocess sa PCR, then at the early
cycle nadetect na agad na positive ka, this
indicates na mababa ang CT value mo
Primer
• A primer is a strand of nucleic acid that serves as a starting
point for DNA synthesis.
• These primers are usually short, chemically synthesized
oligonucleotides, with a length of about twenty bases. They
MOLECULAR BIOLOGY
TRANS 04
are hybredized to a target DNA, which is then copied by the
polymerase.
• Minimum primer length used in most applications is 18
nucleotides.
• Replication starts at the 3'-end of the primer, and copies the
opposite strand.
• In most cases of natural DNA replication, the primer for DNA
synthesis and replication is a short strand of RNA.
Southern Blotting
• Southern blot is a method for probing for the presence of a
specific DNA sequence within a DNA sample.
• DNA samples are separated by gel electrophoresis and then
transferred to a membrane by blotting via capillary action.
• The membrane is then exposed to a labeled DNA probe that
has a complement base sequence to the sequence on the
DNA of interest.
• Less commonly used due to the capacity of other techniques,
such as PCR.
• Southern blotting is still used for some applications such as
measuring transgene copy number in transgenic mice, or in
the engineering of gene knockout embryonic stem cell lines.
DNA mismo mula sa DNA samples
Northern Blotting
• The northern blot is used to study the expression patterns of
a specific type of RNA molecule as relative comparison
among a set of different samples of RNA.
• RNA is separated based on size and is then transferred to a
membrane then probed with a labeled complement of a
sequence of interest.
• The results may be visualized through a variety of ways
depending on the label used. Most result in the revelation of
bands representing the sizes of the RNA detected in sample.
• The intensity of these bands is related to the amount of the
target RNA in the samples analyzed.
• It is used to study when and how much gene expression is
occurring by measuring how much of that RNA is present in
different samples.
• One of the most basic tools for determining at what time, and
under what conditions, certain genes are expressed in living
tissues.
Western Blotting
• In western blotting, proteins are first separated by size, in a
thin gel sandwiched between two glass plates in a technique
known as SDS-PAGE sodium dodecyl sulphate
polyacrylamide gel electrophoresis.
• The proteins in the gel are then transferred to a nitrocellulose,
nylon or other support membrane.
• This membrane probed with solutions of antibodies.
Antibodies specifically bind to the protein of interest &
visualized by a variety of techniques, including colored
products, chemiluminescence, or autoradiography.
• Antibodies are labeled with enzymes. When a
chemiluminescent substrate is exposed to the enzyme it
allows detection.
• Using western blotting techniques allows not only detection
but also quantitative analysis.
Confirmatory test for HIV wherein the antibody against
HIV is being detected
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 Molecular Biology: Basic Science
Molecular Markers
• Molecular markers are based on naturally occurring
polymorphism in DNA sequence (i.e. base pair deletion,
substitution, addition or patterns).
• Genetic markers are sequences of DNA which have been
traced to specific locations on the chromosomes and
associated with particular traits. It can be described as a
variation that can be observed.
• A genetic marker may be a short DNA sequence, such as a
sequence surrounding a single base-pair change (single
nucleotide polymorphism, SNP), or a long one, like mini
satellites.
Common sa cell na pwede madetect kung meron silang
abnormality
Some commonly used types of genetic markers are:
• RFLP (or Restriction fragment length polymorphism)
• AFLP (or Amplified fragment length polymorphism)
• RAPD (or Random amplification of polymorphic DNA)
• VNTR (or Variable number tandem repeat)
• Micro satellite polymorphism, SSR (or Simple sequence
repeat)
• SNP (or Single nucleotide polymorphism) STR (or Short
tandem repeat)
• SFP (or Single feature polymorphism)
• DArT (or Diversity Arrays Technology)
• RAD markers (or Restriction site associated DNA markers)
Principles of DNA Isolation & Purification
DNA can be isolated from any nucleated cell.
DNA is a giant anion in solution.
Sources of DNA include
• Blood
• Buccal cells
• Cultured cells (plant and animal)
• Bacteria
• Biopsies
• Forensic samples i.e. body fluids, hair follicles, bone & teeth
roots.
DNA isolation is a routine procedure to collect DNA for
subsequent molecular analysis. There are three basic steps in a
DNA extraction:
• Cell disruption: - This is commonly achieved by grinding or
sonicating the sample. Removing membrane lipids by adding
a detergent.
• Isolation of DNA: - Removing proteins by adding a
protease (optional but almost always done). REMEMBER
THIS!
• Precipitating the DNA:-usually ice-cold ethanol or
isopropanol is used. Since DNA is insoluble in these alcohols,
it will aggregate together, giving a pellet upon centrifugation.
This step also removes alcohol soluble salt.
MOLECULAR BIOLOGY
TRANS 04
Basic rules (REMEMBER THIS!!)
• Blood – first lyse (explode) the red blood cells with a gentle
detergent such as Triton-X-100.
• Wash cells – hemoglobin (and other pigments) inhibits
restriction enzymes and TAQ polymerase.
• Work on ice to slow down enzymatic processes.
• Wear gloves to protect your samples from you!!
• Autoclave all solutions and store in fridge (except SDS and
organic solvents!)
• Keep all pellets & supernatants until you have the DNA you
want.
Getting to the DNA
• Cells – lyse all cells in presence of :
• NaCl so that DNA is stabilised and remains as a double helix,
• EDTA which chelates Mg++ and is a co-factor of DNAse
which chews up DNA rapidly.
• Anionic detergent SDS which disrupts the lipid layers helps
to dissolve membranes & binds positive charges of
chromosomal proteins (histones) to release the DNA into the
solution. •
• Include a protease (proteinase K) to digest the proteins
• Incubate the solution at an elevated temperature (56oC to
inhibit degradation by DNAses) for 4-24 hrs.
Getting rid of the protein
• Organic solvent extraction using equal volume
phenol:chloroform (24:1)
• Protein at the interface after centrifugation (10000 rpm at 10o
c for 10 min.)
Precipitating the DNA
• Add 2.5 - 3 volumes ice-cold 95% ethanol to the DNA &
leave at -20oC overnight.
• Centrifuge sample at 10000 rpm, 10 min., 40C.
• Wash DNA pellet to remove excess salt in 70% EtOH and air-
dry.
• Resuspend in sterile distilled water(pH7.4)
• Store at 4oC or frozen at -20oC long term.
Quantifying the DNA
• The amount of DNA can be quantified using the formula:
• DNA concentration (µg/ml) = OD260 x 100 (dilution factor) x
50 µg/ml • 1000
• Nucleic acids have a peak absorbance in the ultraviolet range
at about 260 nm •
• 1 A260 O.D. unit for dsDNA = 50 µg/ml
• 1 A260 O.D. unit for ssDNA = 33 µg/ml
• 1 A260 O.D. unit for RNA = 40 µg/ml
DNA Purity
• The purity of the DNA is reflected in the OD260: OD 280 ratio
and must be between 1.6 and 2.00.
< 1.6 – protein contaminated
> 2.0 – chloroform / phenol contaminated
• Repurify sample.
END OF TRANSCRIPTION
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 Process in Molecular Biology Lab MOLECULAR BIOLOGY

Dr. Andro Garcia | May 16, 2021 TRANS 06

Physical separation:
OUTLINE • 3 areas (ideal)
I. Molecular Laboratory B. Air flow 1. Reagent prep - master mix, primers, controls
A. Contamination C. Decontamination 2. Sample prep - Pt samples, add to master mix
II. Preventing Contamination III. Other consideration in 3. PCR - pre-PCR & post-PCR
A. Unidirectional Work setting-up Molecular
Flow Laboratory • 2 areas
1. Reagent prep
LEGEND Specimen prep use laminar flow hoods
Remember Lecturer Book Previous Presentation Target loading
Trans 2. PCR/Product detection

Features of Work Areas

I. MOLECULAR LABORATORY • Separate equipment & supplies in each area. If not possible,
• Prevent Contamination- main goal of molecular lab disinfection should be performed often and strictly
• Reagents prep- single entrance, reagents should stay in this are
A. Contamination • Sample prep- samples should be kept separate from post-PCR areas
- Introduction of unwanted nucleic acid into a specimen
- Sensitivity of PCR makes it vulnerable to contamination Alternatives to Physical Separation
• Aerosol= most important source of contamination. • Class II BSC
-Amplification products can build up in lab (reagents, equipment, • Dead air box- no ventilation system unlike BSC
ventilation system) • Dedicated, labeled work areas (unidirectional flow crucial)
• Automated specimen processing
Source of Contamination
• Closed-tubed amplification and detection system
1. Cross contamination between specimens- aerosols
2. Contamination of environment by amplification products
B. Airflow
→ Laboratory surface
→ Ventilation ducts 1. Positive Pressure
→ Reagents/supplies - Amount of supply air is greater
3. Contamination of personnel than the exhaust
→ Hair - Prevent the reversal of the
→ Skin direction of air movement
→ Clothes - Reagent prep area
→ Saliva

Stop pushing out the last drop in the pipette as it can produce
aerosols
2. Negative Pressure
II. PREVENTING CONTAMINATION - Exhaust removes air faster than
Strategies to prevent Contamination is supplied
1. Unidirectional Work Flow - Prevents air from migrating from
2. Air flow room
3. Decontamination - Doors must remain closed to
maintain negative air pressure
A. Unidirectional Work Flow - Sample prep area & PCR area
-Working in one direction (personnel & specimen should flow in one
direction without going back to previous area) Rule of thumbs: positive pressure used to keep contaminants
- Work flow should start from: out, negative pressure used to keep contaminants from
- Highly recommended to have a separate equipment and PPE in each escaping
testing area. And to have physical separation between each area.
C. Decontamination

1. BLEACH
- 10%sodium hypochlorite sol’n (used in surfaces & equipment)
- Follow with ethanol or diH20 (rinses the bleach off)

2. UV Light
- Renders nucleic acid inactive. (make sure no specimen placed
in the surface before using uv light)
- Can also be used to decontaminate surfaces especially BSC
and dead air box

3. UNG
Pre-amplification Post-amplification - Uracil-N-Glycosylase enzymatic inactivation
- Used to degrade product that has already been through PCR
(Pre-PCR) (Post-PCR)
process
- UNG used commonly in quantitative PCR testing areas
- Substitute uracil for thymine during PCR

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 Process in Molecular Biology Lab TRANS 06
III. OTHER CONSIDERATION IN SETTING-UP MOLECULAR
Decontamination Consideration LABORATORY
• Clean all work areas & equipment routinely
• Clean PCR workstation at the start and end of each shift Other consideration for setting up molecular testing lab
• Clean exterior & interior of all pipettes • Use cleanroom floor mats
• Clean doorknobs, freezer handles, exterior of instruments, BSC, • Use nuclease-free water
etc. • Conduct wipe test regularly
▪ Monthly: swabbing of surfaces and testing it can detect
environmental contamination
• Temperature & humidity
• Backup power system

END OF TRANSCRIPTION

Figure: Molecular Biology Laboratory Design

This shows a diagram of a molecular testing lab with three physically separated spaces.
• ROOM1 – for specimen receiving & DNA instruction
• ROOM2- for reagent prep and test setup
• ROOM3- for PCR and analysis
• SOLID ARROW- shows the direction of workflow from room one to room three without backtracking.
• PRE-PCR AREA- includes room 1 and 2
• POST-PCR AREA- includes room 3, where your PCR is going to be performed and then any sort of gel electrophoresis, DNA sequencing,
any other manipulation for your post-PCR products are going to occur in room 3

MOLECULAR BIOLOGY 2 of 2