MODX 311: Molecular Biology and Diagnostics│Laboratory

2022-2023 3RD YEAR, 2 ND SEMESTER OUR LADY OF FATIMA UNIVERSITY | COLLEGE OF MEDICAL LABORATORY SCIENCE

a. Molecular testing costs a lot

DNA Isolation
(Preliminary Term, 2nd Topic) SPECIMEN COLLECTION
Trans Outline:
I: DNA Isolation VI: Nucleic Acid SOURCES OF DNA FOR MOLECULAR BIOLOGY
II: Considerations Isolation WORK:
III: Specimen VII: Reagents, ❖ Bacteria: mid-log to late-log phase
Collection Enzymes, Chemicals o This is because the bacterial growth is
IV: Specimen Collection needed within an optimal amount, they are alive
and Storage VIII: Extraction and can isolate pure and enough DNA for
V: Basic Steps for DNA Methods testing.
Extraction IX: Sample Assessment o Not plateau (many dead bacteria) or lag
X: NucleoSpin® Blood (low bacterial count) phase.
Method ❖ Fungi: luxuriant growth of mold phase or yeast phase
XI: NucleoSpin® ❖ Worms: frozen: cracked or partially grounded
Microbial DNA ❖ Blood: fresh or from stained material
❖ Tissue: fresh or preserved
❖ Human Cells: from swabs, sediments, etc.
DNA ISOLATION o Most of the time human cells are mostly
❖ The process of separating nucleic acid material received in a molecular laboratory.
from its surroundings: ❖ Plant materials: roots, meristem
o Surrounding can include tissues, debris, cells,
proteins, lipids, or carbohydrates SPECIMEN COLLECTION AND STORAGE
❖ DNA is fairly stable over a wide range of [table at last page]
temperatures; hydrolysis poses a major threat to
nucleic acids remaining intact.
COLLECTION AND TRANSPORT
• Common procedure used in the molecular laboratory, • Whole blood or bone marrow: WBC
in isolating the DNA from the nucleus of the cells. should be isolated, due to the nucleus
• Its purpose is to obtain the DNA in a relatively present. RBC can only isolate RNAs.
purified form. Separating it from other cell • Yellow-top-tube: contains Acid Citrate
components. Dextrose (ACD)
• Using it for further investigation (e.g., PCR, • Tissues are on ice to prevent further
sequencing techniques) metabolism of cells in the tissue and
• Hydrolysis is when intact DNA is separated from each prevent tissue necrosis
other due to the loss of hydrogen bond and this may be • Buccal swabs: mostly used for DNA testing or
due to contaminating enzymes; nucleases or exposed
parental or parentage analysis
to a very high temperature.
o Proteins would start to denature at 90oC. • Microorganisms should be collected in a correct
collection system depending on the organism
CONSIDERATIONS FOR CHOOSING AN detected.
ISOLATION METHOD • Forensic: small samples are only given
1. Specimen type
a. Either genomic DNA, plasmid DNA, RNA, or STORAGE TEMPERATURE (SHORT/ LONG)
from a virus • Short storage: refrigerator temperature only
2. Amount of sample and desired yield • Long storage: -70oC (ultralow freezer)
a. In forensic pathology only small amount of
• Buccal swab: only placed in freezer temperature
sample is acquired.
b. More sample more nucleic acid isolated
3. Purity and size of isolate BASIC STEPS FOR DNA EXTRACTION
a. Either pure or if there are other interferences 1. Cell lysis: aka Cell disruption
4. Ease of operation and throughput a. Breaking the cell open to expose the DNA
5. Costs and hazards inside the nucleus

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MODX 311 | BSMLS 2024 MOLECULAR BIOLOGY AND DIAGNOSTICS STUDENT NOTES ONLY| PREPARED BY: GARCIA, K.M.L.B.
2022-2023 3RD YEAR, 2 ND SEMESTER OUR LADY OF FATIMA UNIVERSITY | COLLEGE OF MEDICAL LABORATORY SCIENCE

b. DNA: nuclear and mitochondrial (small Table 1: Main characteristics of chemical and
amount only) mechanical to extract nucleic acid (adapted from
c. Cytoplasm: mRNA is seen Harrison 2003) [*table at last page*]
2. Removal of membrane lipids: using detergent Additional notes:
3. Removal of proteins: using proteinase K • Alkali treatment: no longer performed because
4. Precipitation with alcohol: ethanol or isopropanol smaller DNA are destroyed.
• DNA is insoluble in alcohol; precipitating the DNA o Applicable only for genomic DNA or longer
forming a pellet after centrifugation. DNA
o Ex: sodium hydroxide
NUCLEIC ACID ISOLATION • Homogenization: done on tissue specimen
DISRUPTION: • Mechanical: are mostly harsh on the mode of lysis
1. Mechanical Disruption: physically grinding the
sample using a mortar and pestle MOST COMMONLY USED DNA EXTRACTION
2. Enzymatic disruption: use of Proteinase K PROCEDURES
a. Cell membrane is mostly made up of protein ➢ Organic (Phenol-Chloroform) Extraction
3. Chemical disruption: solubilize cell membrane o Uses organic solvent (PCL)
lipids o Phenol-Chloroform: used to separate nucleic
a. SDS: Sodium dodecyl sulfate: detergent; acid from proteins and lipids
removes membrane lipids o Organic and Aqueous phase is formed when
b. CTAB: cetyltrimethylammonium PCL are mixed together
bromide: used for fungal specimens due to o Organic phase: proteins and lipids are seen
its thick cell wall. o Aqueous phase: DNA can be seen
➢ Inorganic (Proteinase K and Salting out [high salt
REAGENTS, ENZYMES, CHEMICALS NEEDED solution])
o Developed due to the hazard formed by Phenol-
Reagent/ Enzyme/ Chemical Function Chloroform
RNAse Degrades single stranded o Amount and quality of DNA being extracted is
RNA
not that good compared to the PCL method.
Buffer I Dissolve RNAse
➢ Solid-Phase Isolation method: easiest, fastest, and
Lysozyme Lyse Gram-negative
safest method to isolate pure DNA.
bacterial cell wall
Achromopeptidase Lyse Gram-positive o No need to use organic solvent. Buffer solutions
bacterial cell wall are what is used alongside a column.
Sodium dodecyl sulfate Solubilize cell membrane o In the column, there are active beads, and
lipids when centrifuged the other components
Protein K Digest proteins present will be washed out, in the lower part of
PCL solution (Phenol- Separates DNA from other the column, there are silica particles where the
Chloroform) cellular components DNA attaches.
Ethanol Precipitates DNA from o Lysis buffer contains Guanidinium
the solution isothiocyanate (GITC); hazardous chemical.
TE buffer (Tris EDTA) Dissolved precipitated and
dried DNA EXTRACTION METHODS
• RNAse: only used when pure DNA is needed; DNAse ➢ LIQUID PHASE
when RNA is needed.
❖ Used for large sample volumes
• PCL: organic extraction of DNA. ❖ Phenol-chloroform is a biphasic organic
• TE buffer: dissolves the precipitated DNA, used for extraction method
storage; considered as the water of the stored DNA. o Hydrophobic portion: Lipids and debris on
the bottom (organic)
o Hydrophilic portion: Aqueous phase
contains the DNA on top

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MODX 311 | BSMLS 2024 MOLECULAR BIOLOGY AND DIAGNOSTICS STUDENT NOTES ONLY| PREPARED BY: GARCIA, K.M.L.B.
2022-2023 3RD YEAR, 2 ND SEMESTER OUR LADY OF FATIMA UNIVERSITY | COLLEGE OF MEDICAL LABORATORY SCIENCE

o
The DNA is collected from the upper ▪ Bands = presence of nucleic acid
phase and then precipitated with o Polyacrylamide gel: Smaller sequences (1
isopropyl alcohol bp - 2 kb)
❖ Inorganic method substitutes the harsh chemicals of ▪ Vertical position
organic extraction with high salt conditions at a low ▪ Neurotoxin
pH. ▪ Used for sequencing
o To precipitate DNA and remove proteins, • Positive charge particle: cation; migrates
other interferences can’t be fully removed. toward cathode
Lipids or cell debris can still be seen in the • Negative charge particle: anion; migrates
upper phase. toward anode
o The DNA is collected from the upper
phase and then precipitated (pellet) with B. SPECTROPHOTOMETER
isopropyl alcohol ❖ Wavelength: 260 nm (highest absorbance of nucleic
acid; DNA and RNA)
• Aqueous phase: alcohol ratio: 1:1 or 2 (alcohol):1 o Determination of DNA
(aq. phase) Concentration/ Purity
o Through estimation of the
➢ SOLIDS PHASE A260/A280 ratio
❖ More commonly used because of ease of use, fewer ▪ A260 ÷ A280
safety concerns, ability for high throughput, and ❖ Ratio of 1.8 to 2.0 = high quality/ pure
automation: DNA
o Column filter o <1.8: CHON contamination
o Magnetic beads: automated o >2.0: contaminated with chloroform,
▪ Samples would be separated by a phenol, or RNA (solvent contamination)
machine, and DNA would be attached to • Proteins peak absorbance: 280nm
magnets. • 1.8 to 2.0 ratio is hard to achieve
• In an actual laboratory, 1.2 to 2.0 is still considered
SAMPLE ASSESSMENT and acceptable.
❖ Quantification of nucleic
acids is important so that NUCLEOSPIN® BLOOD METHOD
there is consistency of ❖ NucleoSpin® Blood kits are designed for the rapid
amount used in DNA or isolation of highly pure genomic DNA from whole
RNA work. blood, serum, plasma, or other body fluids.
A. Electrophoresis ❖ DNA can be purified successfully from blood
B. Spectrophotometer samples treated with EDTA, citrate, or heparin.
• To know whether there is DNA isolated. As long as leukocytes are not destroyed.
❖ If leukocyte rich materials like buffy coat are used,
A. ELECTROPHORESIS apply smaller volumes and dilute the samples with
❖ Applying electricity in gel sterile PBS (Phosphate Buffered Saline)
❖ Both DNA and RNA (nucleic acids) carry a ❖ The kits allow purification of highly pure genomic
negative charge and will migrate toward the DNA with an A260 / A280 ratio between 1.60 and
positive-charge electrode (anode) 1.90 and a typical concentration of 40–60 ng per
o Negative pole: black; sample wells are μL
placed; cathode
o Positive pole: red Storage conditions and Preparation of Working
❖ Gels act like a sieve for nucleic acid molecules Solutions
o Slab gel (vertical or horizontal) or using gel Before starting any NucleoSpin® Blood protocol prepare
polymer inside a capillary the following:
o Agarose gel: Larger fragments (20 bp - 10 ❖ Wash Buffer B5 (NucleoSpin® Blood): Add the
Mb) indicated volume of ethanol (96–100 %) to Wash
▪ Horizontal position Buffer B5 Concentrate.

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MODX 311 | BSMLS 2024 MOLECULAR BIOLOGY AND DIAGNOSTICS STUDENT NOTES ONLY| PREPARED BY: GARCIA, K.M.L.B.
2022-2023 3RD YEAR, 2 ND SEMESTER OUR LADY OF FATIMA UNIVERSITY | COLLEGE OF MEDICAL LABORATORY SCIENCE

o
Mark the label of the bottle to indicate that • Microbial cells are hard to lyse because it has a thick
ethanol was added. membrane compared to human cells.
o Store Wash Buffer B5 at room temperature (18– • NucleoSpin® Bead Tube is used; contains glass
25 °C) for up to one year. beads to help the lysis of the bacterial cell.
❖ Proteinase K: Add the indicated volume of • EB + DNA = testing
Proteinase Buffer PB to dissolve lyophilized
Proteinase K.
o Proteinase K solution is stable at -20 °C for up
to 6 months.
• Wash Buffer B5: only one that needs preparation

PROCEDURE
[table at last page]
• B3: used for lysis
• Residual after 2nd wash = dry silica membrane;
contains the DNA
• BE: elution buffer; remove the attachment of DNA
from silica particles
• Step 6: uses microcentrifuge tube
o Washed out elution buffer already contains
the DNA; which can be used for testing.

NUCLEOSPIN® MICROBIAL DNA

❖ The NucleoSpin® Microbial DNA kit is designed
for efficient isolation of genomic DNA from
microbial samples.
❖ DNA can be isolated from a wide variety of
microorganisms such as gram-negative, and gram-
positive bacteria as well as yeast, e.g., Escherichia
coli, Bacillus subtilis, Corynebacterium glutamicum,
Saccharomyces cerevisiae.
Storage conditions and Preparation of Working
Solutions
Before starting any NucleoSpin® Blood protocol prepare
the following:
❖ Wash Buffer B5: Add the indicated volume of
ethanol (96–100 %) to Wash Buffer B5 Concentrate.
o Mark the label of the bottle to indicate that
ethanol was added.
o Wash Buffer B5 can be stored at room
temperature (18–25 °C) for at least one year.
o 6mL Wash Buffer B5 (concentrate) + 24mL
ethanol
❖ Liquid Proteinase K is ready to use.
o After first time use, store Liquid Proteinase K at
4 °C or -20°C.
• In the bottles of the reagents, it is already indicated
whether it needs to be diluted or resuspended.

PROCEDURE
[table at last page]

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MODX 311 | BSMLS 2024 MOLECULAR BIOLOGY AND DIAGNOSTICS STUDENT NOTES ONLY| PREPARED BY: GARCIA, K.M.L.B.
2022-2023 3RD YEAR, 2 ND SEMESTER OUR LADY OF FATIMA UNIVERSITY | COLLEGE OF MEDICAL LABORATORY SCIENCE

SPECIMEN COLLECTION AND STORAGE

SPECIMEN COLLECTION AND STORAGE CONSIDERATIONS
TRANSPORT TEMPERATURE (oC)
(Short/Long)
Whole blood or bone Lavender or yellow-top 4 / -70 Avoid heparin tube, remove red
marrow tube for whole blood blood cells before storage to avoid
hemolysis
Tissue Freeze solid tissues or on 4 / -70 May be paraffine-embedded
ice
Buccal swabs Rinse or swab oral cavity, 4 / -20 Less invasive collection procedure
collect in buffer or
transport medium
Microorganism Special collection systems 4 / -70 Viral RNA should be stored at
for various target 24oC, avoid contamination that
organisms may result in false-positive
findings
Forensic: Blood, hair, Evidence labeled air dry 4 / -70 Chain of custody for evidence,
nails, secretions, etc. blood-stained clothes, 24oC for blood stored on avoid heat and contamination,
separate paper bags for special filter paper aliquot to avoid repeated heating
each item and thawing

Method Technique Principle Mode of Cost Most usual application

lysis
Osmotic shock Osmotic rupture of Gentle Cheap Spheroplasts and
membrane Protoplasts
Enzymatic Digestion of cell wall Gentle Cheap Gram-positive and Grame-
digestion negative bacteria
Chemical Detergents Solubilization of Gentle Moderate General use
membranes
Alkali treatment Solubilization of Harsh Cheap Plasmid DNA
membrane
Homogenization Shredding of cells Moderate Moderate Animal tissues
(blade or pestle) (method of
choice for large
scale)
Ultrasonication or Disruption of cells Harsh Moderate to Good for spheroplasts but
Mechanical cavitation by pressure expensive not primary cells
Pressure cell Disruption of cells Harsh Moderate Used for Gram-negative
(“French press”) by shear force and some Gram-positive
bacteria
Ball mill Cells crushed Harsh Cheap Used for bacteria, yeast,
between glass/ steel microalgae, unicellular
balls/ beads animal cells

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MODX 311 | BSMLS 2024 MOLECULAR BIOLOGY AND DIAGNOSTICS STUDENT NOTES ONLY| PREPARED BY: GARCIA, K.M.L.B.
2022-2023 3RD YEAR, 2 ND SEMESTER OUR LADY OF FATIMA UNIVERSITY | COLLEGE OF MEDICAL LABORATORY SCIENCE

PROCEDURE FOR NUCLEOSPIN® BLOOD METHOD

1 Lyse blood samples 200 uL blood
25 uL Pro.K
200 uL B3

Mix

70oC, 10-15 mins (incubate)

2 Adjust DNA binding conditions 210 uL ethanol

3 Binds DNA Load all to the column and receiving

vessel/ collection tube

11,000 x g, 1 min (centrifuge)

4 Wash silica membrane 1st wash: add 500 uL BW and 11,000 x g,

1 min

2nd wash: add 600 uL B5 and 11,000 x g,

1 min

5 Dry silica membrane 11,000 x g, 1 min

6 Elute highly pure DNA 100 uL BE, (70oC)

RT, 1 min (incubate)

11,000 x g, 1 min

PROCEDURE FOR NUCLEOSPIN® MICROBIAL DNA

1 Prepare sample <40 mg microbial pellet (wet weight)

100 uL BE

2 Lyse sample Transfer sample in NucleoSpin® Bead Tube Type

B
40uL Buffer MG
10uL Liquid Proteinase K

Agitate on a swing mill or similar device, 4-12 min

11,000 x g, 30 s
3 Adjusting binding conditions 600 uL Buffer MG

Vortex 3 s (mix)
11,000 x g, 30 s

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MODX 311 | BSMLS 2024 MOLECULAR BIOLOGY AND DIAGNOSTICS STUDENT NOTES ONLY| PREPARED BY: GARCIA, K.M.L.B.
2022-2023 3RD YEAR, 2 ND SEMESTER OUR LADY OF FATIMA UNIVERSITY | COLLEGE OF MEDICAL LABORATORY SCIENCE

4 Bind DNA Load 500-600 uL sample on NucleoSpin®

Microbial DNA Column

11,000 x g, 30 s

5 Wash silica membrane 1st wash: 500 uL BW

11,000 x g, 30 s
2nd wash: 500 uL B5
11,000 x g, 30 s
6 Dry silica membrane 11,000 x g, 30 s

7 Elute DNA 100 uL BE

RT, 1 min (incubate)
11,000 x g, 30 s

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MODX 311 | BSMLS 2024 MOLECULAR BIOLOGY AND DIAGNOSTICS STUDENT NOTES ONLY| PREPARED BY: GARCIA, K.M.L.B.