MOLECULAR BIOLOGY – MIDTERM TOPIC 1

Mercado, Ada Audrey Nichole J. BMLS 10-2D

DNA AND RNA ISOLATION

Nucleic Acid Extraction: removal of nucleic acids (DNA and/or

RNA) from the cells in which they normally reside.

• Purpose: release nucleic acid from the cell for:

o Detecting a specific pathogen (bacteria and
viruses)
o Diagnosing disease and genetic disorders
o Other applications: forensics, paternity tests,
ancestry tracking, genetic engineering, vaccines,
hormones

Target Nucleic Acids: free from contamination with

macromolecules such as: proteins, carbohydrates, lipids, or
other nucleic acids
• Later Routine Laboratory Procedures of DNA Isolation
How Nucleic Acid is Removed from the Cell
o Solubility differences among chromosomal DNA,
plasmids, and proteins in alkaline buffers

1. Bust out the cell membrane I. Preparing the Sample:

2. Lysis of cell wall or cell membrane
Specimen Expected Yield
3. Lysis of nuclear membrane
Specimens Adequate for Analysis Without DNA
4. Nucleic acids come out from nucleus
Amplification
5. Purification
Blood¹ (1 mL, 3.5-10×10⁴ WBCs/mL) 50 – 200 µg
6. Determination of the concentration and purity of the sample
Buffy coat¹ (1 mL whole blood)
Isolation of DNA Bone marrow¹ (1 mL) 100 – 500 µg
Cultured cells (10² cells) 30 – 70 µg
• Friedrich Miescher (1869): isolated the nuclein (DNA)
Solid tissue¹ (1 mg) 1 – 10 µg
from the WBCs he obtained from the pus on collected
Lavage fluids (10 mL) 2 – 250 µg
surgical bandages in a nearby hospital. (Precipitation
Mitochondria (10 mg tissue, 10² cells) 1 – 10 µg
process)
Plasmid DNA, bacterial culture (100 mL 350 µg – 1 mg
o pus: composition of dead or alive bacteria,
overnight culture)
offending agent, or pathogen + WBC; product of
Bacterial culture (0.5 mL, 0.7 10 – 35 µg
immune response
absorbance units)
• Early Routine Laboratory Procedures of DNA Isolation
Feces⁵ (1 mg; bacteria, fungi) 2 – 228 µg
o Developed from density-gradient
Specimens Adequate for Analysis With DNA Amplification
centrifugation strategies
Serum, plasma, cerebrospinal fluid¹ 0.3 – 3 µg
▪ Meselson & Stahl (1958): demonstrate the
(0.5 mL)
semi-conservative replication of DNA
Dried blood (0.5 to 1 cm diameter spot) 0.04 – 0.7 µg
Saliva (1 mL) 5 – 15 µg
MOLECULAR BIOLOGY – MIDTERM TOPIC 1
Mercado, Ada Audrey Nichole J. BMLS 10-2D

Buccal cells (500 mg) 3 – 50 µg presence of Tris

Bone, teeth (500 mg) 30 – 50 µg base, ethylene-
Fixed tissue** (5 – 10 × 10-micron 6 – 50 µg diaminetetraaceti
sections; 10 mm²) c acid (EDTA) &
Feces¹¹ (animal cells, 1 mg) 2-100 pg glucose.
* Yields are based on optimal conditions. Assays will vary in B. Viruses
yield and purity of sample DNA.
Viral DNA
* DNA yield will vary with WBC count. • Held within free viruses or integrated into the host
genome along with host DNA.
* DNA yield will depend on type and conditions of tissue.
• Cell-free specimens (plasma) will be used for viral
* DNA yield will depend on types and fungi will yield more or less detection
DNA. • May require concentration of viroids by centrifugation or
other methods
* DNA yield will depend on degree of cellularity.
• virions: basic structure of virus
* Dried blood yield from paper is less than from textiles.

* Mitochondrial DNA are attainable from hair shafts. C. Nucleated Cells in Suspension (Blood & Bone Marrow
Aspirates)
** Isolation of DNA from fixed tissue is affected by the type of
White Blood Cells (WBCs)
fixative used and the age of the preliminary handling of the
• Anticoagulants will be added to prevent clotting and will
original specimen.
also trap WBCs
¹* Cells in fecal specimens are subject to digestion and • Obtained from the blood plasma
degradation. • Purified of red blood cells (RBCs) & other components

A. Bacteria and Fungi: by either differential density-gradient centrifugation or

differential lysis
Gram- Some bacteria & Yeast, • Differential osmotic fragility of RBCs & WBCs
negative fungi with tough cell filamentous • Plasma & WBC: usually recovered for further analyses
bacteria walls fungi, & gram-
• Ficoll & RBC / Erythrocytes / Granulocytes: usually
positive
discarded but can be recovered as rich source of high-
bacteria
quality DNA
• Can be • Can be lysed by • Commercial
lysed by enzyme products reagents
high pH or mechanically designed for
(alkaline) grinding or by isolation of
and vigorously mixing DNA in
detergent with glass beads amplificatio
s (to crack them n
open and to break procedures
down the bonds (PCR)
present) D. Plasma

• Detergent (1% Exosomes

sodium dodecyl • Small vesicles (30 – 100 nm in diameter), which form by

sulfate) & strong imagination & budding from the inside of cellular

base (0.2 M endosome vesicles and are secreted by living cells

NaOH) in the • Contain nucleic acid and can be collected through

centrifugation
MOLECULAR BIOLOGY – MIDTERM TOPIC 1
Mercado, Ada Audrey Nichole J. BMLS 10-2D

• Diagnostic & prognostic analyses purposes (liquid - can be accomplished using an organic mixture
biopsy) (phenol & chloroform)
• Solid-phase collection of nucleic acid - use a combination of high salt, low pH, and an
organic mixture of phenol and chloroform
o readily dissolves
E. Tissue Samples
o hydrophobic contaminants (lipids & lipoproteins)
Fresh Tissue Frozen Tissue Fixed, embedded
o collect cell debris & strips away most DNA-
tissue
associated proteins
• Dissociate • Dissociated • May be
d by by grinding deparaffinized
mincing the and by soaking in
tissue homogenizing xylene and
the tissue tissue is
rehydrated by
soaking it in
decreasing
concentrations
B. Inorganic Isolation Methods:
of ethanol
- does not include organic extraction step
• May be used
- sometimes called “salting out,” makes use of low-pH
directly
& high salt conditions to selectively precipitate
without
proteins, leaving the DNA in solution
dewaxing

Tissue Fixatives Influencing Nucleic Acid Quality

Fixative Relative Quality Average

of Nucleic Acid Fragment
C. Solid-Phase Isolation:
Fixative Size
- uses solid matrices (silica-based products) in the
Range (kB)
form of columns & beads to bind & hold DNA for
10% buffered Good 2.0 – 5.0
washing.
neutral formalin
Acetone Good 2.0 – 5.0
Zamboni’s Not as good 0.2 – 2.0
Clarke’s Not as good 0.8 – 1.0
Paraformaldehyde Not as good 0.2 – 5.0
Methacarn Not as good 0.7 – 1.5
Formalin-alcohol- Not as good 1.0 – 4.0
acetic acid
B-5 Less desirable <0.1
Carnoy’s Less desirable 0.7 – 1.5
Zenker’s Less desirable 0.7 – 1.5 III. Proteolytic Lysis of Fixed Material
Bouin’s Less desirable <0.1 • Paraffin-embedded specimens
o Dewaxed with xylene or other agents
o Rehydrated before nucleic acid isolation
II. DNA Isolation Chemistries
• Some tests (somatic mutation analyses)
A. Organic Isolation Methods:
MOLECULAR BIOLOGY – MIDTERM TOPIC 1
Mercado, Ada Audrey Nichole J. BMLS 10-2D

o Microscopic examination of adjacent stained IV. Proteolytic Lysis of Fixed Material

serial section • Before lysis
Noninvasive Human DNA Isolation o Cells may be washed by suspension &
• Genomic DNA Purification from Hair centrifugation in saline / other isotonic buffers
o Simple alkaline lysis method: Hair with root is • Reagents for cell lysis
incubated (95ºC) for 10 minutes in NaOH buffer & the o Simple screens: detergents (SDS or Triton)
supernatant is subjected to DNA purification after o PCR amplification: mixture of Tris buffer &
centrifugation proteinase K
o Smooth chemical digestion method using
Dithiothreitol (DTT): Hair sample can be incubated
(56ºC for 2 hours with buffer containing Tris-HCl,
EDTA, NaCl, SDS, DTT, & proteinase K, followed by
gentle mixing and incubation (60ºC) for 2 hours or
until the hair has dissolved completely.
• Genomic DNA Purification from Saliva: cells found in
the saliva; exfoliated buccal epithelial cells & other cells
o Buccal swabs (cotton swabs / cytobrushes)
▪ Suspended in lysis buffer which includes Tris,
EDTA, SDS, & proteinase K
▪ Incubated (56ºC) for 1-3 hours until the tissue is
totally dissolved & DNA is extracted from the
solution
V. Rapid Extraction Methods
o Mouthwash Method
• PCR
▪ Samples from saline rinses need to be processed
/ frozen immediately after collection • Rapid lysis methods & DNA extraction / storage cards

▪ Alcohol-containing mouthwash must be used • Chelex

o Genomic DNA Purification from Urine Sample: o Cation-chelating resin that can be used for

▪ Urine Specimen simple extraction of DNA from minimal

• Inverted & swirled in a specimen cup to create samples.

a homogenous suspension of cells followed by o 10% Chelex resin beads is mixed with the
specimen & the cells are lysed by boiling.
the centrifugation
• Supernatant is removed & a dry pellet
containing cells is chilled (-20ºC) for 15
minutes followed by addition of lysis buffer
(Tris, EDTA, SDS, & proteinase K)
• Sample is incubated (56ºC) for 2h & then DNA
is extracted from the solution

VI. Isolation of Mitochondrial DNA

• Approaches
1. First, isolate the mitochondria by centrifugation
2. Isolate total DNA by hybridization or PCR.
MOLECULAR BIOLOGY – MIDTERM TOPIC 1
Mercado, Ada Audrey Nichole J. BMLS 10-2D

• Tissue or cell pellets can be stored and/or

transported in preservative reagents

II. Preparation of Specimen Material

• Reticulocytes in blood & bone marrow samples:
Lysed by osmosis or separated from WBCs by
centrifugation
• Tissue: Kept frozen in liquid nitrogen / immersed in
buffer that will inactivate intracellular RNAses
• Bacterial & fungal RNA: Isolated by chemical lysis by
grinding in liquid nitrogen
Before isolation of RNA:
• Viral DNA: Isolated directly from the serum, plasma
• Strict precautions to avoid sample RNA degradation by culture medium, or other cell-free fluids by means of
ribonucleases (RNAses) specially formulated spin columns or beads
• RNAses must be eliminated or inactivated
o RNAses inhibitors: diethyl pyrocarbonate (DEPC) III. RNA Isolation Chemistries
added to water & buffers; vanadyl-ribonucleoside • Organic Isolation
complexes; and macaloid clays o Cell lysis
• Allocate a separate RNAse-free (RNF) are of the ▪ Detergent or phenol in the presence of high salt
laboratory (0.2 to 0.5 M NaCl) or RNAse inhibitors
▪ Guanidine isothicyanate (GITC) can also be used
Total RNA
▪ Strong reducing agents (2-mercaptoethanol) may
rRNA mRNA tRNA & snRNA also be added
(Ribosomal (Messenger (Transfer RNA & o Acid phenol: chloroform: isoamyl alcohol
RNAs) RNAs) Small Nuclear (25:24:1) solution efficiently extracts RNA
RNA
Most abundant Next most Also present
(80% - 90%) RNA abundant RNA
in all cells fraction (2.5% to
5%)

RNA Isolation

• Solid-phase Isolation
o Begins with similar steps as described for organic
extraction
o The strong denaturing buffer conditions must be
adjusted before application of the lysate to the
column
I. Specimen Collection
• RNA tests (array analysis or quantitative reverse
transcriptase PCR)
o RNA is stabilized from further metabolism or
degradation after collection
• Specialized collection tubes
o To stabilize RNA immediately upon draw
MOLECULAR BIOLOGY – MIDTERM TOPIC 1
Mercado, Ada Audrey Nichole J. BMLS 10-2D

• The quality of RNA from fixed, paraffin-embedded tissue

will depend on the fixation process and handling of the
specimen before fixation
• Commercial reagent sets are available for extraction of
RNA from fixed-tissue specimens
• Specialized reagents or spin columns for removal of
genomic DNA contamination are included in some
systems
• Automated isolation systems have reagent kits and
cartridges

Fixative Relative Quality of Average Fragment

Nucleic Acid Fixative Size Range
IV. Yield of RNA from various specimen sources
(kB)
• 1 million eukaryotic cells or 10-50 mg of tissue = 10 µg of
10% buffered Good 2.0 – 5.0
RNA
neutral formalin
• Yield of RNA from biological fluids will depend on the
Acetone Good 2.0 – 5.0
concentration of microorganisms or other target
Zamboni’s Not as good 0.2 – 2.0
molecules present in the specimen
Clarke’s Not as good 0.8 – 1.0
Specimen Expected
Paraformaldehyde Not as good 0.2 – 5.0
Yield
Methacarn Not as good 0.7 – 1.5
Blood¹ (1 mL, 3.5-10×10⁴ WBCs/mL) 1 – 10 µg
Formalin-alcohol- Not as good 1.0 – 4.0
Buffy coat¹ (1 mL whole blood) 5 – 10 µg
acetic acid
Bone marrow¹ (1 mL) 50 – 200 µg
B-5 Less desirable <0.1
Cultured cells (10² cells) 50 – 150 µg
Carnoy’s Less desirable 0.7 – 1.5
Buccal cells (1 mg) 1 – 10 µg
Zenker’s Less desirable 0.7 – 1.5
Solid tissue¹ (1 mg) 0.5 – 4 µg
Bouin’s Less desirable <0.1
Fixed tissue¹ (1 mm³) 0.2 – 3 µg
Bacterial culture¹ (0.5 mL, 0.7 absorbance 10 – 100 µg
units) VI. Isolation of polyA (Messenger) RNA
*Specimen handling especially affects RNA yield isolation of • RNA often required for analysis: mRNA (2.5% - 5% of
polyA RNA will result in much lower yields. the total RNA yield)
• Single-stranded oligomers of thymine or uracil
*RNA yield will depend on WBC count.
immobilized on a matrix resin are used
*RNA yield will depend on type of cells and the conditions of cell o Approximately 30-40 ng of mRNA can be obtained
culture. from 1 µg of total RNA

*Liver, spleen, and heart tissues yield more RNA than brain,
lung, ovary, kidney, or thymus tissues.

*Isolation of RNA from fixed tissue is especially affected by the

type of fixative used and the age and the preliminary handling of
the original specimen.

*Different bacterial types and fungi will yield more or less RNA.

V. Proteolytic Lysis of Fixed Material

MOLECULAR BIOLOGY – MIDTERM TOPIC 1
Mercado, Ada Audrey Nichole J. BMLS 10-2D

Measurement of Nucleic Acid Quality & Quantity A = Absorbance

∈ = molar absorptivity (L/mol-cm), 50 for dsDNA, 40 for
RNA
b = path length (cm)
c = concentration (mg/L)
• Absorbance is directly proportional to the
concentration of the nucleic acid in the sample
• Beer-Lambert Law: A= ∈bc
o ∈ = molar absorptivity (L/mol-cm), 50 for dsDNA,
40 for RNA
Electrophoresis Spectrophotometry
Separation of particles Technique that uses light Using absorptivity, as a conversion factor from
through a solution or matrix absorption to measure the optical density to concentration:
under the force of an concentration of an analyte in
At 260 nm,
electric current a solution
Fluorometry Microfluidics 1 optical density unit (or absorbance unit)

Measurement of emitted Science & technology of = 50 mg/L (or 50 µg/mL) of dsDNA & 40 µg/mL of RNA
fluorescent light systems that process or To determine concentration:
manipulate small amounts of
Spectrophotometer reading in absorbance units ×
fluid (10-9 to 10-18L), using appropriate conversion factor
channels measuring from tens
If the DNA / RNA preparation require dilution before
to hundreds of micrometers
spectrophotometry, to determine concentration:

Absorbance reading x conversion factor x dilution

factor

Example 1:

A DNA preparation diluted 1/100 yields an absorbance

reading of 0.200 at 260 nm. What is the concentration
of DNA in µg/mL?

To obtain the concentration in µg/mL

I. Electrophoresis
0.200 absorbance units x 100 = 1,000 µg/mL
• DNA and RNA can be analyzed for quality (detection &
size analysis by resolving an aliquot of the isolated The yield of the sample is calculated using the volume
of the preparation. If the DNA was eluted /
sample on an agarose gel
resuspended in a volume of 0.5 mL in the case
• Uses an electric current to propel charged illustrated previously, the yield would be:
biomolecules through a porous gel matrix at a rate that
1,000 µg/mL × 0.5 mL = 500 µg
is the function of the charge, size, and shape of the
Example 2:
molecules
• Fluorescent dyes used: ethidium bromide, SybrGreen An RNA preparation diluted 1/10 yields an absorbance
reading of 0.500 at 260 nm. What is the concentration
I, silver stain
of DNA in µg/mL?
• Appearance of DNA on agarose gels depends on the
To obtain the concentration in µg/mL:
type of DNA isolated
0.500 absorbance units × 40 µg/mL
II. Spectrophotometry Per absorbance units × 20 = 200 µg/mL
• Nucleic acids absorb light at 260 nm through adenine
The yield of the sample is calculated using the volume
residues of the preparation. If the DNA was eluted /
• Beer-Lambert Law: A= ∈bc
MOLECULAR BIOLOGY – MIDTERM TOPIC 1
Mercado, Ada Audrey Nichole J. BMLS 10-2D

resuspended in a volume of 0.2 mL in the case (iii) 3 0.398 0.174

illustrated previously, the yield would be:
For each sample based on the A260 / A280 ratio, is each
200 µg/mL × 0.2 mL = 40 µg
preparation suitable for further use? If not, what is the
• May also be used to estimate the purity of nucleic contaminating DNA?
acid
Solution: Get the A260 / A280 ratio for each sample
• Detection of contaminants: reading the
concentration over a range of wavelength i. 0.419 / 0.230 = 1.82 – suitable
• Indication of contamination: absorbance over ii. 0.258 / 0.225 = 1.15 – not suitable ; protein
background at any wavelength other than the iii. 0.398 / 0.174 = 2.29 – may be suitable if RNA does
A260 maxima of the nucleic acid. not interfere with the subsequent assay
o Absorbance of DNA at 260 nm = 1.6 to 2.00
times more than the absorbance at 280 nm • UV Spectrophotometry
o RNA = 2.0 to 2.3 o Standard nucleic acid quantitation
o If the 260 nm / 280 nm ratio is <1.6, the o Nucleic acid sample is placed into quartz cuvette,
nucleic acid preparation may be which is then placed inside the UV
contaminated with unacceptable amounts of spectrophotometer
protein & not of sufficient purity for use o UV light passed through the sample at a specified
• Most likely contaminant: protein (absorbs light at path length, & the absorbance of the sample at
260 nm through the aromatic tryptophan & specific wavelengths is measured
tyrosine residues) o Does not require additional reagents / incubation
time
Common Contaminants and Their Wavelengths of Peak
• NanoDrop Spectrophotometry
Absorbance
o Similar in principle with the previous, but has many
Wavelength (nm) Contaminant
additional capabilities:
230 Organic compounds
▪ Functions by combining filter optic technology
270 Phenol
& natural surface tension properties
280 Protein
▪ Accompanied by special software to enable
>330 Particulate matter
analysis of signal from small quantities of
sample

• Absorbance of DNA at 260 nm = 1.6 to 2.00 times ▪ Displays the entire absorbance spectrum of the

more than the absorbance at 280 nm. sample in graphical form – allows detection of

• RNA = 2.0 to 2.3 contaminants

• If the 260 nm / 280 nm ratio is <1.6, the the nucleic ▪ Capable of determining a wide range of sample

acid preparation may be contaminated with concentrations w/o requiring serial dilutions

unacceptable amounts of protein & not of sufficient III. Fluorometry (Fluorescent Spectroscopy)

purity for use • Measures fluorescence related to DNA

concentration in association with DNA-specific
Example: fluorescent dyes

Three DNA preparations have the following A260 and A280 • Early methods: 3,5-diaminobenzoic acid 2HCl

readings: (DABA), combined with alpha-methylene aldehydes

(deoxyribose) to yield a fluorescent product
Sample OD260 OD280
• Modern methods: DNA-specific dye Hoechst 33258

(i) 1 0.419 0.230 combined with adenine-thymine base pairs in the

minor grove of the DNA double helix
(ii) 2 0.258 0.225
MOLECULAR BIOLOGY – MIDTERM TOPIC 1
Mercado, Ada Audrey Nichole J. BMLS 10-2D

o Fluorometric determination of DNA

concentration down to 200 ng DNA/mL
• Other DNA-specific dyes
o PicoGreen: detection down to 25 pg/mL
concentrations
o OilGreen: detection down to 100 pg/mL of
ssDNA
• RNA: SybrGreen II RNA gel stain is used

Absorption and fluorometry readings may not always agree

• These methods recognize different targets

o Single nucleotides do not bind to fluorescent dyes,
but they can absorb ultraviolet light
o Absorption measurements do not distinguish
between DNA & RNA
o Deciding which instrument to use is at the discretion
of the laboratory:
▪ Most use spectrophotometry because the
samples can be read directly without staining
or mixing with dye
▪ Methods requiring accurate measurements of
low amounts of DNA/RNA (in the range of 10
to 100 ng/mL), fluorometry may be preferred
IV. Microfluidics
• Sample is applied to a multiwell chip & then moves
through microchannels across a detector
• Instrument software generates image in
electropherogram (peak) or gel (band) configurations
• RNA integrity number: quantification estimate for RNA,
determined as a standard measure of RNA integrity
• Uses a minimal volume of sample (as low as 1 µL) &
can test multiple samples simultaneously
• Useful for analysis of studies on small TNAs
(microRNAs) in eukaryotes & gene expression in
bacteria.