BIOCHEMISTRY FOR MEDICAL LABORATORY SCIENCE [LAB]

MLSBCHML | FINALS | LESSON 11

DNA ISOLATION AND UV MEASUREMENT

DEOXYRIBONUCLEIC ACID (DNA)

Conc. H2SO4 Marbles
• GENOME Conc. HNO3 Quartz cuvettes
o The complete set of genetic information Ba(OH)2 Marbles
and encoded in the base sequence of the pH paper UV- Vis microplate
dsDNA. spectrophotometer
o In bacteria and other haploid organisms Diphenylamine
▪ there is one copy of genome per reagent
cell. • Standard solutions:
o In diploid organisms such as humans o Phosphate, deoxyribose, adenine, guanine,
▪ There are two copies of genome cytosine, and uracil
in each cell o Standard saline citrate (SSC): 0.15 NaCI
• The study on GENOMIC DNA: and 0.015M sodium citrate
o Can provide valuable information on the o Tris-EDTA buffer (TE buffer), pH 8.0: 0.01M
promoter and other regulatory Tris-HCI and 0.001M EDTA
sequences, introns and intergenic
MICROBIAL DNA ISOLATION
sequences.
o Supply information regarding the • Reagents and materials
structure of individual genes and physical o Bacterial cells, wet- packed, 3-5 g. Use
relationships among neighboring genes. Bacillus subtilis or Escherichia coli broth
o Provides valuable databases and cultures (up to the late log phase of growth;
information on the organization of the if lyophilized cells are available, use 1g).
genome and the evolution of species o Saline – EDTA: 0.15 M NaCl and 0.1 M
through DNA manipulation, cloning and ethylenediaminetetraacetic acid (EDTA),
sequencing studies. pH 8
• DNA o Lysozyme solution, 10 mg/mL in water
o A high molecular weight biopolymer of o Sodium dodecyl sulfate (SDS), 25% in
deoxyribonucleotides that stores genetic water
information. o 5M NaCIO4
o It is found in highly cellular organs such o 95% ethanol
as the spleen, liver, thymus, and other o Chloroform: isoamyl alcohol (24:1)
lymphoid tissues. STEPS ACTION
o In plants, young tissues samples with 1 Suspend 3-5 g wet packed cells or 1 g
actively dividing meristematic cells lyophilized cells in 25 mL saline-EDTA
condense and often duplicate DNA, solution in a 125-mL Erlenmeyer flask.
producing a higher yield of DNA with less 2 Add 1.0 mL of lysozyme solution (10 mg) and
polysaccharides. incubate at 37 °C for 30-45 minutes.
o Bacterial cells usually have one larger 3 Add 2.0 mL of 25% SDS solution; heat in a 60
circular DNA, and one smaller, circular °C water bath for 10 minutes.
4 Stir gently to avoid excessive foaming.
extrachromosomal DNA called plasmid.
5 Cool the mixture to room temperature or
VIRTUAL EXPERIMENT under running water.
6 Add 9.0 mL of 5 M NaCIO, to achieve -1 M
REAGENTS AND MATERIAL final salt concentration.
7 Mix gently and add equal volume of
1M HCL 10% (NH4)2 MoO4
chloroform: isoamyl alcohol.
solution
8 Shake gently for 10 minutes in a tightly
1M KOH Br2 water
stoppered flask.
10% KOH Glacial acetic acid
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BIOCHEMISTRY FOR MEDICAL LABORATORY SCIENCE [LAB]
MLSBCHML | FINALS | LESSON 11
DNA ISOLATION AND UV MEASUREMENT
9 Centrifuge for 5 minutes at 10,000 x g to STEPS ACTION
separate into three layers. 1 Place 50 mL of homogenizing solution in a
10 Transfer the upper aqueous layer to a test 125-mL Erlenmeyer flask; heat in a water
tube; precipitate DNA by layering 2 volumes bath to 60 °C
of 95% ethanol. 2 Mince an onion and weigh out 25 g.
11 Mix gently using a glass stirring rod; spool
3 Add minced onion to the pre-heated
DNA onto the rod, pressing against the test
homogenizing solution; stir and let sit in
tube.
water bath for 5 minutes.
12 Dissolve spooled DNA in 10-15 mL of TE
4 Add 1.5 g papain (or 4 mL meat tenderizer
buffet or SSC solution.
solution) and keep in the 60 °C water bath
for an additional 10 minutes.
PLANT DNA ISOLATION
5 Place the flask in an ice bath for 5 minutes;
• Reagents and materials swirl gently to cool evenly.
o Freshly harvested young papaya leaves (or 6 Quickly pour the contents into a blender,
other young plant samples) homogenize for 45 seconds.
o Cetyltrimethylammonium bromide (CTAB) 7 Filter the homogenate through 4 layers of
o 1 M Tris-HCI, pH 8.0 cheesecloth into a 250-mL beaker, cool on
o 0.5 M EDTA ice.
o Chloroform: isoamyl alcohol (24:1) 8 Add 15-20 mL of ice-cold ethanol using a
o 95% ethanol pipette, allowing it to drip down the tube's
o 70% ethanol side.
STEPS ACTION 9 Let the tube sit undisturbed for 3-5 minutes
1 Prepare CTAB buffer. Mix 2 g CTAB, 10 mL 1 (or overnight in the refrigerator) to
M Tris-HCl (pH 8), 4 mL 0.5 M EDTA, 26 mL 5 precipitate DNA.
M NaCl, and 58 mL distilled water. Pre-heat 10 Spool the DNA using a bent Pasteur pipette;
at 60 °C. transfer to a clean test tube.
2 Harvest 10-15 g young papaya leaves; wash 11 Resuspend the DNA in TE buffer or SSE
with tap water, rinse with distilled water, solution
and blot dry. Cut off large veins.
3 Pack leaves in dry ice until frozen or ANIMAL DNA ISOLATION
thoroughly chilled.
4 Grind frozen leaves in a chilled mortar and • Reagents and materials
pestle; transfer to a chilled 50-ml test tube. o Chicken liver (or other sources)
5 Add 15 mL pre-heated CTAB buffer to the o Lysing buffer: 0.5 M Tris-HCl, pH 8.0; 0.5 M
sample and mix gently. EDTA; 5 M NaCl; and 5% SDS
Place the mixture in a 60 °C water bath for o 5 M NaCIO4
6
30 minutes, gently swirl occasionally. o Chloroform: isoamyl alcohol (24:1)
o Ice- cold 95% ethanol
7 Centrifuge at 16,000 x g for 15 minutes at
o 70% ethanol
room temperature; transfer supernatant to a
clean test tube. STEPS ACTION
1 Pre-heat the lysing buffer in a 60 °C water
bath.
DNA ISOLATION FROM AN ONION Pack fresh chicken liver in dry ice until
2
• Reagents and materials thoroughly chilled.
o 2 yellow onions 3 Homogenize 25 g of chilled liver in a blender
o Homogenizing solution: 5% SDS (sodium with enough ice-cold distilled water for a
dodecyl sulfate or sodium lauryl sulfate), uniform suspension.
0.15 M NaCl, 0.15 M Na3C6H5O7 (sodium 4 Transfer the homogenate to a pre-chilled
citrate), and 0.001 M EDTA. Erlenmeyer flask.
o Commercial papain or meat tenderizer (6% 5 Immediately add pre-heated lysing buffer
in water) and mix gently.
o Ice- cold 95% ethanol 6 Place the mixture in a 60 °C water bath for

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BIOCHEMISTRY FOR MEDICAL LABORATORY SCIENCE [LAB]
MLSBCHML | FINALS | LESSON 11
DNA ISOLATION AND UV MEASUREMENT
30 minutes; gently swirl occasionally. 5 Calculate the concentration of nucleic acid
7 Add 10 mL of 5 M NaCIO and return to the 60 in the solution.
°C water bath for another 10 minutes.
8 Centrifuge at 13,000 rpm for 10 minutes.
POST-LAB EXPERIMENT
9 Collect the supernatant; add an equal
volume of chloroform: isoamyl alcohol. DNA ISOLATION/EXTRACTION
10 Mix gently and centrifuge at 13,000 rpm for 5
minutes; observe three layers. • DNA was first isolated in 1869 by chemist Friederich
11 Using a wide-bore pipette, transfer the Miescher from eukaryotic nuclei.
upper aqueous phase to a clean test tube. • DNA is also negatively charged as opposed to the
12 Add 2 volumes of ice-cold 95% ethanol; mix positive charge of histones and is stabilized by
gently with a glass rod. MAGNESIUM.
13 Spool the DNA precipitate and wash it twice • DNA isolation requires the removal of various cellular
with enough 70% ethanol. materials to free up the DNA material inside the cell.
14 Resuspend the precipitate in SSC TE buffer • The process of DNA isolation removes potential
inhibitors to the polymerase chain reaction (PCR)
ULTRAVIOLET MEASUREMENT OF ISOLATED DNA amplification and produces a stable solution of high-
quality DNA that can be stored for prolonged
• For high-volume determination (using a quartz
durations without degrading.
cuvette or a UV- Vis/Microplate
Spectrophotometer):
STEPS ACTION DNA ISOLATION AND UV MEASUREMENT
1 Dissolve 0.5 mL of DNA aliquot in 4.5 mL of • Test Objective:
SSC solution or TE buffer (1:20 dilution). o To isolate DNA from bacteria, plants, animal
2 Transfer the solution to a quartz cuvette. and onion.
3 Measure absorbance at 230, 260, and 280 • Test principle:
nm; use the buffer as the blank o The extraction/isolation of DNA from the
4 Calculate DNA purity using A260/A280 and sources through these steps: Cell lysis,
A260/A230 ratios. enzyme treatment, Phenol: Chloroform
5 Calculate the concentration of nucleic acid extraction, alcohol precipitation. After
in the solution. isolating the DNA, it is measured using UV
6 Place the mixture in a 60 °C water bath for spectroscopy/photometry.
30 minutes, gently swirl occasionally. • Steps in extraction of DNA:
7 Centrifuge at 16,000 x g for 15 minutes at 1. Lysis of Cells
room temperature; transfer supernatant to a o DNA must be RELEASED from cells,
clean test tube. nuclei or organisms as the first step of
the DNA isolation process.
o Lysis can be a single step or involve
ULTRAVIOLET MEASUREMENT OF ISOLATED multiple steps.
DNA o When using a blood sample, for
example, red blood cells need to be
• For low-volume determination (using a Drop™ Plate
lysed first, yielding white blood cells
or a Microplate Spectrophotometer):
which contain the DNA.
STEPS ACTION
o The WBCs are then lysed to free the
1 Dilute DNA samples in a 1:2 ratio with TE DNA from the cells
buffer. o Techniques that can be employed to
2 Transfer 2-4 mL of the diluted samples to lyse the cells include:
the Drop™ Plate low-volume measurement o Chemical lysis -include
area. chaotropic agents, detergents,
3 Determine absorbance at 230, 260, and 280 salts and strong bases.
nm; use the buffer as the blank. o Enzymes such as proteinase K
4 Calculate purity of DNA samples using and lysozyme.
A260/A280 and A260/A230 ratios. o External physical forces can also
be used to lyse cells such as
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BIOCHEMISTRY FOR MEDICAL LABORATORY SCIENCE [LAB]
MLSBCHML | FINALS | LESSON 11
DNA ISOLATION AND UV MEASUREMENT
through milling, sonication, DNA EXTRACTION FROM DIFFERENT SOURCES
boiling, and freeze thaw cycling.
2. Separation of DNA from Other Cell
Components
o The isolation of DNA is prone to protein
contamination due to the presence of
histones and other accessory proteins.
o Because the presence of proteins is
detrimental to nucleic acid preparation
as well as to downstream analysis, it is
imperative to either denature or remove
proteins during the extraction of DNA.
o Unwanted proteins can also be 1. Prokaryotic cells (bacteria)
removed by protease digestion. A o The simplest cells, such as bacteria, are
common protease used is the serine prokaryotes.
protease proteinase K originally o These cells are composed of a lipid bilayer
extracted from the fungus outer membrane and a cytoplasm containing
Engyodontium album. a circular chromosome, proteins, inorganic
o It breaks down proteins into smaller salts, metal ions, carbohydrates and other
molecules by cleaving peptide bonds cellular components.
and is thus useful in reducing the o Lysis of prokaryotic cells releases
protein background as well as in the chromosomal material where DNA can be
lysis of cells extracted
o Protein Removal o Differences in the structure of the cell wall of
▪ Detergents such as sodium gram-positive and gram- negative bacteria
dodecyl sulfate (SDS) and plays a role in the optimal selection of
Triton X-100 can be used to extraction/isolation methods to be used
remove proteins from DNA o The isolation of genomic DNA from bacteria is
through solubilization. traditionally achieved using organic
▪ Chemicals such as chaotropic extraction of the soluble DNA while the
acids (e.g. guanidine insoluble cell debris is removed.
hydrochloride and guanidinium o The DNA is then purified from soluble
thiocyanate) can be used to proteins and RNA by ethanol precipitation
denature proteins. They can o Commercial kit-based extraction methods
also be employed to lyse are currently available for the convenient and
bacteria and yeasts. rapid extraction of genomic DNA from
3. Isolation of the DNA bacteria.
o After lysis, the DNA needs to be isolated 2. Eukaryotic cells (plant, onion, animal)
from other sample or cellular materials. o Eukaryotic cells such as those found in
o There are basically two separation humans, animals and plants are also
methods that can be employed: composed of a lipid bilayer outer membrane
▪ Liquid – liquid extraction and a cytoplasm containing various proteins,
▪ Solid – phase extraction carbohydrates, lipids and other inorganic
materials.
o Differences in cell structure often presents
difficulties in extracting the DNA.
o The cell walls of plants for example
complicate the DNA extraction process by
presenting a barrier which makes it more
difficult to lyse the cells.
o Many plant species have a high content of
polysaccharides and polyphenols which are
not removed by phenol extraction.
o It is much easier to lyse and extract genomic

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BIOCHEMISTRY FOR MEDICAL LABORATORY SCIENCE [LAB]
MLSBCHML | FINALS | LESSON 11
DNA ISOLATION AND UV MEASUREMENT
DNA from human and animal cells because Dilution Factor accounts for any dilution
of the absence of cell walls and made during the preparation of the sample.50
chloroplasts. is the conversion factor for dsDNA (for RNA, it
o Most animal cells do not have a cell wall like is 40, and for ssDNA, it is 33).
microbial cells, and consequently, are • DNA Concentration Sample:
easier to lyse and can be lysed using only o Calculate the concentration of DNA in the
detergents. sample by the O.D. at 260 nm.
DNA ISOLATION AND UV MEASUREMENT ▪ Diluting the sample 1 ul in 100 ul (the
dilution factor is 100).
• Sample: Bacterial suspension (Bacillus subtilis or ▪ OD260 1.6
Escherichia coli broth), young papaya leaves, onion, ▪ 1 O.D. at 260 nm for double-stranded
and fresh chicken liver DNA = 50 ng/uL of
• Reagents and their use: ▪ dsDNA.
o EDTA – use as resuspending buffer to ▪ OD260 * 50 ng/uL * dilution factor.
prevent immediate lysis of cells. Also used ▪ The concentration is: 1.6 * 50 * 100 =
as chelating agent to destabilize cell 8000 ng/ul or 8 ug/ul
membrane and inhibit DNase activity. • The purity of DNA is assessed by calculating the ratio
o Lysozyme – disrupt the cell wall of the of absorbance at 260 nm to the absorbance at 280 nm
bacteria. • Formula: A260/A280 ratio
o SDS – used as lysis buffer to fully disrupt • A260/A280
and denature protein in cell membrane. • ratio is used to determine protein contamination, as
o NaCLO4 (Sodium perchlorate) – promotes proteins absorb at 280 nm and RNA contamination at
phase separation of RNA from protein and 260 nm.
DNA. • A pure DNA sample typically has an A260/A280 ratio
o Chloroform: isoamyl alcohol – to separate of 1.8 to 2.0.
DNA from proteins. • If the ratio is <1.8, this suggests protein
o 95% Ethanol – for alcohol precipitation contamination.
o CTAB (Cetytrimethylammonium bromide) • If the ratio is >2.0, it may indicate RNA contamination
– is a cationic detergent that facilitates cell or chemical contamination (phenols, carbs, or salt).
lysis and prevents secondary metabolites
from interfering with DNA extraction.
o Tris-HCl – a common biological buffer used
throughout the DNA extraction process. It is
used to maintain the right pH for DNA
isolation
o TE buffer – Serve as a storage or dilution
buffer solution
o NaCl – remove proteins bound to DNA
o Papain or meat tenderizer – serves as a
protease enzyme that helps break down
proteins, particularly histones and other
proteins that are bound to the DNA.
UV SPECTROPHOTOMETRY
• To compute for DNA concentration:
o DNA concentration is usually measured by
determining the absorbance at 260 nm
(A260), as DNA absorbs UV light strongly at
this wavelength.
o The formula for calculating DNA
concentration is: DNA Concentration
(ng/µL)=A260×Dilution Factor×50
• Where:
o A260 is the absorbance reading at 260 nm.
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