LESSON 4 BASIC METHODS & TOOLS IN MOLECULAR BIOLOGY & DIAGNOSTICS Flashcards
type of specimen collected when testing for current or past infection with SARS-CoV-2 is based on the
test being performed and its manufacturer’s instructions
• CDC recommendation for initial dx test:
upper respiratory specimen
– easier to do
• Nasopharyngeal swab and Oropharyngeal swab
• Specimen with the highest viral load is nasopharyngeal wash/aspirate
• Nasopharyngeal swab and Oropharyngeal swab
Work within [?] of patients
6ft
• Maintain proper
infection control
• Follow
standard precautions
• Ppe with [?] or higher level of respirator
n95
The respirator must be put on [?] and worn during the exposure.
correctly
The respirator filter must capture more than [?] of the particles from the air that passes through it.
95%
The respirator must [?] against the user’s face to ensure that there are no gaps between the user’s skin and respirator seal.
fit snugly
is a critical component to a respiratory protection program.
Fit testing
OSHA requires an initial respirator fit test to identify the right [?] respirator for each worker.
model, style, and size
ensure that users continue to receive the expected level of protection.
• Annual fit tests
Collect as soon as possible after symptoms begin (ideally within?)
7 days
PCR - can detect virus from day [?] (virus is already shedding and multiplying; there are already remnants of RNA)
1 to day 7
- Antibody production against SARs-Cov 2 genes takes[?]
17 days/2 weeks
- Antigen can be detected during the
first 4 days
Ideally before [?] are administered
V antiviral medications
Collect multiple specimens on
multiple days
Use only [?] that have been designed for sampling the nasopharyngeal mucosa
synthetic fiber swabs with thin plastic or wire shafts
Do not use [?], as they may contain substances that inactivate some viruses and may inhibit molecular tests
calcium alginate swabs or swabs with wooden shafts
If both NP and OP specimens are collected, combine them in a single tube to maximize [?] and limit use of [?]
test sensitivity
testing resources.
Should be [?]
drayon, rayon, or polyester fiber swabs
Select swabs with [?]
serrated shafts
Do not use [?] nor ones with [?]; they may inhibit PCR
calcium alginate or cotton swabs; wooden sticks
- Tilt patient’s head back [?]
- Insert [?] through nares parallel to palate (not upwards) until:
a. Resistance is met, OR - Completely insert until there is resistance (about [?])
b. Distance is equivalent to half the distance from the [?]. - Gently [?] the swab.
- Leave swab in place for [?] to absorb secretions.
- Slowly remove the swab while rotating it and immediately place in sterile tube containing [?].
70°
flexible shaft mini-tip swab
2 inches
patient’s ear to their nostril
rub and roll
several seconds
transport medium
Insert swab into the
posterior pharynx and tonsillar areas
Rub swab over both tonsillar pillars and posterior oropharynx and avoid touching the
tongue, teeth, and gums
Place swab, tip first, into the [?] provided.
transport tube
Store respiratory specimens at [?] after collection
2-8°C for up to 72 hours
If a delay in testing or shipping is expected, store specimens at
70°C or below
Store extracted nucleic acid samples at
-70°C or lower
allows the safe transfer of
viruses, chlamydia and mycoplasma, conventional cell culture methods, diagnostic tests, and molecular biology techniques
One of the standardized components of VTM by CDC and WHO
to prevent microbial growth:
o Calcium
o Magnesium
o heat-inactivated Fetal Bovine Serum (FBS)
o Antibiotics (Gentamycin and Amphotericin B)
and contains heat-inactivated Fetal Bovine Serum (FBS)
Calcium and Magnesium
: growth supplement for in vitro cell culture
Fetal Bovine Serum (FBS)
UNACCEPTABLE SPECIMENS
[?] specimens (specimen container info and form do not match)
No identification on [?]
Specimens with [?] for testing
Improper specimen type sent
Spillage or possibility of [?]
Specimens shipped at [?]
Excessive specimen [?]
Improperly identified
specimen container or form
insufficient Quantity
cross contamination
improper temperature
transport time
One of the most critical part in molecular techniques because downstream procedures and analysis relies on the quality of the DNA used for the assay
DNA ISOLATION
Refers to the process of separating DNA from other cellular materials such as proteins and membranes.
DNA ISOLATION
Removes potential inhibitors to the polymerase chain reaction PCR) amplification and produces a stable solution of highquality DNA that can be stored for prolonged durations without degrading
DNA ISOLATION
SOURCES OF DNA
Plasmid DNA
Viral Nucleic Acids
Genomic DNA from Blood and Biological Fluids
Genomic DNA from Tissue and Cells
Genomic DNA from Forensic Samples
Genomic DNA from Plant and Fungi
Genomic DNA from Food and Feed
Ancient DNA (archeologic)
3 BASIC STEPS
01 LYSIS
02 PRECIPITATION
03 PURIFICATION
The cell and the nucleus are broken open to release the DNA inside.
01 LYSIS
separates DNA from the cellular debris.
02 PRECIPITATION
Extracted DNA can be rinsed with alcohol to remove any remaining unwanted material and cellular debris.
03 PURIFICATION
At this point the purified DNA is usually redissolved in water for easy handling and storage.
03 PURIFICATION
LYSIS METHODS
- Mechanical disruption
- Using the following detergents and enzymes:
- can be a manual method using mortar and pestle, homogenizer
- Mechanical disruption
- Using the following detergents and enzymes:
Organic: NaOH, Sodium Dodecyl Sulfate
Inorganic: Tris-EDTA (TE), EDTA, SDS
PRECIPITATION METHODS
- Na+ ions
- Ethanol/ Isopropanol
is added and causes DNA to precipitate out of the aqueous solution because it is not soluble in alcohol
- Ethanol/ Isopropanol
neutralize the negative charges on the DNA molecules which makes them more stable and less water soluble.
Na+ ions
- normally, DNA has a
(-) charge
: to precipitate and prevent dissolving
- less water soluble
- dehydrants
Ethanol/ Isopropanol
- also causes DNA to precipitate out of the aqueous solution
Ethanol/ Isopropanol
- to separate DNA and precipitate
Ethanol/ Isopropanol
- to purify or clean further with an alcohol or buffer
PURIFICATION
- redissolve and resuspend in water
PURIFICATION
REAGENTS USED FOR IDENTIFICATION
designed to lyse outer cell membrane, but will not break down nuclear membrane
CELL LYSIS BUFFER
Nucleus is intact
CELL LYSIS BUFFER
remove contaminating protein
PHENOL
allows protein to settle at the bottom
PHENOL
prevents cutting of DNA during isolation.
CHLOROFORM
It solubilizes lipids and a lot of protein to remove them from the DNA
CHLOROFORM
Comes with phenol
CHLOROFORM
Contaminating proteins and lipids also settles at the bottom
CHLOROFORM
DNA floats at the top of the solution
CHLOROFORM
remove most of the protein by digesting with proteolytic enzymes
PROTEINASE K
Active against a broad spectrum of native proteins and denatured proteins
PROTEINASE K
Alternative for phenol and chloroform
PROTEINASE K
An enzyme method
PROTEINASE K
chelating agent of d Mg2+.
EDTA
Mg2+is a cofactor for DNase if the Mg2+is bound up by
EDTA
DNAses are inactivated.
EDTA
Protects the DNA from damage
EDTA
Inactivates DNAses that destroys the DNA
EDTA
: important cofactor of DNAse; once chelated, DNAse will not work anymore
Magnesium
is an anionic detergent which helps cell membrane and nuclear envelope to break open
SODIUM DODECYL SULFATE (SDS)
Inhibit RNAse and DNAse.
SODIUM DODECYL SULFATE (SDS)
A lysis regant
SODIUM DODECYL SULFATE (SDS)
Inactivates nucleases
SODIUM DODECYL SULFATE (SDS)
Prevent DNA and RNA degradation
TRIS-EDTA (TE) BUFFER
Used for final resuspension and storage because it is a buffer that preserves any changes in pH
TRIS-EDTA (TE) BUFFER
Preserves DNA and RNA
TRIS-EDTA (TE) BUFFER
Precipitation of DNA
95% ETHANOL OR ISOPROPANOL
: organic procedure
Ethanol
: inorganic procedure
Isopropanol and phenol
DIFFERENT TYPES OF DNA EXTRACTION METHODS
1.Chemical based DNA extraction method
2.Solid phase DNA extraction method
A.Organic solvent-based DNA extraction
- Phenol-chloroform Method
Commonly used in the laboratory
Phenol-chloroform Method
Used in the RNA isolation of covid 19 testing
Phenol-chloroform Method
Advantage: Rapid denaturation of nucleases (destroys RNA and DNA) and stabilization of RNA (phenol and chloroform can be used)
Phenol-chloroform Method
Drawbacks: Laborious and manually intensive processing
Phenol-chloroform Method
Silica Column-based DNA Extraction Method
Phenol-chloroform Method Steps:
(open it up to free the nucleus; addition of buffer/detergent/enzyme)
Lyse/Homogenize cells
: addition of phenol and chloroform then centrifuge
Extraction
: organic phase separated
Precipitation
: aqueous soln with precipitated DNA
Top
: Protein contaminants and cellular debris
Bottom
: Proteins (separate by placing to another container → add water → clean it further by washing with buffer or alcohol → resuspend with water)
Middle
Precipitated DNA will be purified or rehydrated or resuspended with water or buffer
Phenol-chloroform Method
Phenol + chloroform
Phenol-chloroform Method
is good for stabilization
Chloroform
Lipids and cell membrane lipids will dissolve in [?] since it is an organic compound
phenol
After release of DNA from the cell, further purification requires removal of contaminating proteins, lipids, carbohydrates, and cell debris through
high salt, low pH and phenol & chloroform
: extracting reagents
phenol & chloroform
: increases the yield and purity of DNA
high salt, low pH
: DNA will be pipeted for precipitation with ethanol
Supernatant
: Phenol and Chloroform will extract the proteins
Bottom
: added to prevent RNA contamination
RNAse
B. Inorganic solvent-based DNA extraction
- Salting out Method
- Proteinase K Method/ Enzymatic Method
Salting out Method Steps:
Precipitation
Salting out Method
Precipitation: [?] helps in the DNA extraction
Isopropanol and salts ( sodium acetate, sodium chloride, potassium acetate and ammonium acetate)
facilitates high DNA yield but it is time-consuming.
Proteinase K Method/ Enzymatic Method
Good in research, experimentation, forensic
Proteinase K Method/ Enzymatic Method
Extract as much DNA
Proteinase K Method/ Enzymatic Method
Also if not maintained well in a cold chain, the (?) cannot be utilized for a longer period of time
Proteinase K Method/ Enzymatic Method
The lower stability of the enzyme is another major issue in this method.
Proteinase K Method/ Enzymatic Method
Disadvantage: Labile
Proteinase K Method/ Enzymatic Method
Solid phase DNA extraction method
A. Silica column based DNA extraction method
: (+) charged; has affinity for DNA to isolate it
Silica column/Silica beads
: Rapid denaturation of nucleases and stabilization of RNA
Silica column based DNA extraction method
: Laborious and manually intensive processing
Silica column based DNA extraction method
Difficult method
Silica column based DNA extraction method
Principle: works on the unique chemistry interaction between silica and DNA. A positively charged particles (column/bids) bind/ adsorbed with the negatively charged DNA during centrifugation
Silica column based DNA extraction method
Silica column based DNA extraction method
Blue:
Black:
Droplet:
Blue: silica
Black: DNA
Droplet: elite DNA
Silica column based DNA extraction method
1. Lyse cells and [?] to remove large cell debris
2. Apply [?] to silica column (spin column)/ beads
3. Addition of [?] (extraction rgt) and acidification to remove contaminating
4. [?]/ Nucleic acid extractor machine
5. Adsorbed DNA will be washed using [?]
6. Final product [?] (purified DNA; extraction of adsorbed DNA)
spin
lysate reagent
alcohol
Microcentrifugation
buffers
Elute DNA
Widely accepted because of its good quality DNA yield and
minimal simple operating system.
Silica column based DNA extraction method
The method is most rapid, reliable, accurate and consumes less time.
Silica column based DNA extraction method
This extraction lasts up to 30 mins
Silica column based DNA extraction method
RNA Extractor Machine
Silica column based DNA extraction method
is a cation-chelating resin (positively-charged) binds to the negatively charged phosphate of DNA and helps in the extraction of DNA
Chelex
rapid and used in forensic
DNA extraction using the anionic resins
Positively charged magnetic beads attract the negatively charged DNA.
DND extraction by magnetic beads
The DNA is separated under the magnetic field.
DND extraction by magnetic beads
is used to fractionate DNA based on density
CsCI Density gradient method
A procedure for separating particles (such as viruses or ribosomes or molecules such as DNA ) in which the sample is placed on a preformed gradient such as sucrose or caesium chloride.
Density gradient Centrifugation
Upon centrifugation either by rate zonal or equilibrium procedures, the macromolecules are ‘banded” in the gradient and can be collected as a pure fraction.
Density gradient Centrifugation
Blood sample + sucrose or caesium chloride (ficoll: rgt)
Ficoll Density Gradient Centrifugation
Source of DNA: WBC
Ficoll Density Gradient Centrifugation
Nuclear DNA and Mitochondrial DNA stays
Ficoll Density Gradient Centrifugation
DNA can be stored for [?] in a refrigerator or stored frozen for [?]
months
years
Extracted DNA is typically stored at [?] for long-term storage, to prevent nuclease activity - To prevent DNAse from degrading the DNA
-20°C and up to -80°C
are protein enzymes found in cells that degrade DNA to allow the cells to recycle nucleotide components
Nucleases
Nucleases need [?] to work properly so one of the measures to prevent them from digesting DNA in blood is the use of EDTA
magnesium
The [?] chelates, or binds up, most of the free magnesium and thus helps prevent the nucleases from destroying the DNA in the collected blood sample
EDTA
: good for inactivating nucleases
EDTA
is usually more stable over time.
Highly concentrated DNA
DNA can gradually hydrolyze in
acidic conditions
As such, storage buffers are slightly basic such as
Tris buffer
of total RNA is ribosomal RNA
80-90%
is messenger RNA
2.5-5%
SARs-COV 2: [?] is extracted
RNA
Difficult and very crucial since [?] are major contaminant
RNAse
One of the most critical part in molecular techniques because downstream procedures and analysis relies on the quality of the DNA used for the assay
RNA ISOLATION
Refers to the process of separating DNA from other cellular materials such as proteins and membranes.
RNA ISOLATION
Removes potential inhibitors to the polymerase chain reaction PCR) amplification and produces a stable solution of highquality DNA that can be stored for prolonged durations without degrading
RNA ISOLATION
Requires STRICT precautions to avoid sample degradation.
RNA ISOLATION
RNA especially
labile
Goal: Prevent RNA degradation by
RNAses
is a common contaminant
RNAse
- naturally occurring
RNAse
- bacterial and human source
RNAse
- could be difficult to inactivate because it can reactivate even after autoclaving
RNAse
Must be eliminated or inactivated before isolation of RNA
RNAse
There must be a
RNAse-free
Protecting Specimen Against RNAse:
• Wear [?] at all times
• Use [?]
• Use [?], chemicals
• Pre-treat materials with extended heat (180 C for several hours), wash with [?]
• Supplement reactions with [?]
• Reusable [?] is seldom used
gloves
RNase-free tubes and pipet tips
dedicated, RNase-free
DEPC-treated water, NaOH orH202
RNase inhibitors
glassware
Common: Phenol-Chloroform Extract
- Cell lysis step for RNA isolation is performed in detergent or phenol in the presence of [?].
- RNA is precipitated by addition of two volumes of ethanol or one volume of isopropanol
a. Top:
b. Bottom: - RNA precipitate is then washed in [?] and resuspended in RNF buffer or water.
high salt or RNase inhibitors
aqueous solution with total RNA ; Protein contaminants solvents
70% ethanol
is added at the lysis step or purification step to eliminate contamination of DNA
DNase
is a strong denaturant of RNases and can be used instead of high-salt buffers
Guanidine isothiocyanate (GIC)
A positively charged silica particles bind with the negatively charged RNA during centrifugation
Silica column based RNA extraction method
mRNA: only [?] of the total RNA
2.5% to 5%
Principle: All mRNAs have a [?] (many adenine)
poly (A) tail
could be used to specifically capture mRNA with magnetic beads/ columns with poly T chain
Poly(A tail)
[?] columns or beads bind the polyA tail of mRNA
Oligo polythymine (or polyuracil)
The bead/column has a
3’ T 5’
mRNA has
5’ A 3’
mRN(A) is attracted to
T
3 Adenine will form hydrogen bonds with
3 Thymine
After washing away residual RNA, [?] is eluted by washing the column with warmed, low-salt buffer containing detergent to break the hydrogen bonds between the mRNA and the column.
polyA RNA
WHY DO WE WANT TO EXTRACT ONLY THE MRNA?
Because mRNA represents the genes that are expressed and will be translated into
proteins
mRNA with instructions for making the [?] is developed in a lab
spike protein (s-gene)
(: lacks s-gene)
omicron
mRNA enters the cell (extracted using ?)
poly-T bead
COVID-19 virus [?] created
spike protein
are recognized by the immune system, which produces specific antibodies against the COVID-19 virus
Spike proteins
If you’re infected with the COVID-19 virus, [?] bind to virus & stop it from replacing
antibodies
Proteinase K Method/ Enzymatic Method
[?] of blood + [?] TE buffer
Mix well and centrifuge at [?]
To pellet add [?] TE buffer
Mix well and centrifuge at [?]
To pellet add [?] Proteinase K
+ (?) of DNA extraction buffer
Mix well and incubate it At [?]
Add [?] + Salt: NaCI / sodium acetate
Mix well and centrifuge at [?]
Wash with [?]
Mix well and centrifuge at [?]]
Dissolving DNA in [?]
2ml; 20uL
2500pm, 20min
20uL
2500pm, 15min
20uL
2 ml
56°C to 60°C for 2hrs
Isoamyl alcohol
9000 rpm, 2 min
ethanol
9000pm, 2 min
TE Buffer
