MOL 114 LAB Experiment

Genomic DNA Isolation and Quantitation

Academic Year 2022-2023
Semester 1
Purpose
The main aim of this laboratory session is to learn and practise DNA extraction and purification
from buccal cells (inner cheek) using a commercial kit.

Objectives
At the end of this practical, you will be able to:
- Explain the importance of studying DNA in clinical research
- Determine biological sources of DNA
- List different methods for DNA extraction
- Extract DNA from human buccal cells using silica column
- Identify the role of each reagent used in DNA extraction
- Understand the importance of determining extracted DNA concentration and purity
- Determine the concentration and purity of the extracted DNA

References

- https://www.qiagen.com/jp/resources/download.aspx?id=62a200d6-faf4-469b-b50f-
2b59cf738962&lang=en
 Introduction

DNA represents the hereditary materials in every cell that carries the genetic information from
generation to generation and is essential for the development and functioning of a living
organism. DNA is mainly located in the nucleus and minorly located in the mitochondria.
The study of DNA is essential for a better understanding of how genes function and how they are
controlled this knowledge is used by help researchers develop new strategies to treat and prevent
human disease. DNA can be isolated from numerous sources, including whole blood,
hair, sperm, bones, nails, tissues, blood stains, saliva, buccal (cheek) swabs, epithelial cells,
urine, bacteria, and tissues from animals or plants. The purity and quantity of DNA are important
for further studies and storage of samples. They are usually determined by Nanodrop (a
spectrophotometer) at an absorbance of A 260/A280. DNA molecules best absorb light at A 260 nm
and proteins best absorb light at A280 nm. The acceptable ratio of DNA purity should fall into the
value range of 1.8 to 2.0.
DNA quantity is essential for normalizing the sample volume before beginning further
experiments for accurate comparisons with other samples. Below are some of the most used
DNA extraction procedures:
1. Organic (phenol-chloroform) extraction
2. Non-organic (proteinase K and salting out)
3. Silica-based extraction

Silica-based extraction
Silica-based nucleic acid purification methods employ a simple bind-wash-elute process.
Nucleic acids bind to the silica membrane in the presence of chaotropic salts. The high ionic
strength of chaotropic salts cause DNA to bind to silica membranes while other contaminants
pass through the column. These Chaotropic salts have two main roles. Firstly, they destabilize
week bonds such as hydrogen bonds, van der Waals forces and hydrophobic interactions, this
leads to the destabilization of proteins, including nucleases. Secondly, they set the stage for the
binding of DNA to a silica substance by disrupting the association of nucleic acids with water.
Polysaccharides and proteins do not bind well to the column, and residual traces are removed
during alcohol-based wash steps, along with the salts. After binding and washing, nucleic acids
are selectively eluted under low-salt conditions, using water or TE buffer. Silica-based
methods are fast, easy to perform, and economical. The high ionic strength and presence of
chaotropic salt cause DNA to bind to the silica membrane while other contaminants pass through
the column.

All silica-based DNA extraction procedures have common elements, which are:
1. Lysis: Lysis break down cells to access DNA in the nucleus. This is done using proteinase k
enzyme and a lysis buffer containing chaotropic salts
2. Separation: Separate DNA from cell material and any PCR inhibitors, where DNA binds to the
membrane and the remaining lysate is discarded
3. Purification: There are typically 2 washes with a centrifuge step after each:
 Wash 1 contains a low amount of chaotropic salt to remove any remaining proteins and
coloured contaminants
 Wash 2 contains a high concentration of ethanol to remove the remaining salts

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4. Elution: Elution buffer is added to the nucleic
acids to become hydrated and will release from
the membrane

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DNA purification from buccal swabs using silica-gel membrane spin columns
Buccal cells are easily collected by a buccal cotton swab or by a mouthwash procedure. The
isolation of DNA by buccal swabs provides many advantages, such as cost-effectiveness, small
sample volume required, long-term archiving, painlessness, and self-collection. Buccal swabs
provide sufficient DNA for performing Polymerase chain reaction (PCR) as they need only a few
nanograms of DNA.

Reagents

1. PBS: Phosphate buffer saline

This is water-based salt solution which is used to preserve the cell and avoid cell rupture

2. Proteinase K
Proteinase K is an enzyme that belongs to the peptidase family. It cleaves the peptide
bonds between amino acids for breaking down proteins

3. Lyse C (Lysis buffer)

The lysis buffer is a solution that breaks cellular and nuclear membranes

4. Absolute Ethanol (100% Ethanol)

Ethanol allows for the precipitation of DNA in aqueous solution, it also enhances the
binding of nucleic acids to silica

5. Pre-washing buffer
Pre-washing buffer is used for removing protein residues and degraded RNA residues

6. Washing buffer
Buffer is used for removing salt residues

7. Elute buffer (elution buffer)

This buffer helps to lose the affinity of DNA to the silica gel membrane thus allowing the
elution of DNA for further storage or use

Tubes

Eppendorf tube

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DNA extraction procedure
1. To collect a sample, scrape the swab firmly against the inside of each cheek 6 times. Air-
dry the swab for at least 2 h after collection. Ensure that the person providing the sample
has not consumed any food or drink in the 30 min prior to sample collection
2. To prepare the sample place buccal swab in a 2 ml Eppendorf tube. Add 400 μL PBS to
the sample
N.B: Cotton swabs are separated from the stick by hand or with scissors.
3. Add 20 μL proteinase K and 400 μL of lysis solution C buffer to the sample. Mix
immediately by vortexing for 15sec
N.B: To ensure efficient lysis, it is essential that the sample and lysis solution C buffer
are mixed immediately and thoroughly
4. Incubate at 56°C for 10 minutes and briefly centrifuge to remove drops from inside the
lid for 1 minute at 8000rpm speed
5. Add 400 μL of absolute ethanol to the sample, mix again by vortexing. Briefly
centrifuge to remove drops from inside the lid for 1 minute at 8000rpm speed
6. Pipette 700 μL of the mixture from step 5 to the mini spin column (in a 2 mL collection
tube) without wetting the rim. Close the cap, and centrifuge at 8000 rpm for 1 min. Place
the mini spin column in a clean 2 mL collection tube, discard the tube containing the
filtrate
7. Repeat step 6 by pipetting the remaining mixture from step 5 into the mini spin column
8. Carefully open the mini spin column and add 500 μL pre-wash solution concentrate
without wetting the rim. Close the cap and centrifuge at 8000 rpm for 1 min. Place the
mini spin column in a clean 2 mL collection tube, discard the collection tube containing
the filtrate
9. Carefully open the mini spin column and add 500 μL wash solution concentrate without
wetting the rim. Close the cap and centrifuge at full speed 13,300rpm for 3 min
10. Recommended: Place the mini spin column in a new 2 mL collection tube and discard the
old collection tube with the filtrate. Centrifuge at full speed for 1 min. This step helps to
eliminate the chance of possible washing buffer carryover
11. Place the mini spin column in a clean 1.5 mL Eppendorf tube, discard the collection tube
containing the filtrate. Carefully open the mini spin column and add 150 μL of elution
buffer. Incubate at room temperature (15–25°C) for 5 minutes, and then centrifuge at
8000 rpm for 1 min

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DNA Quantitation
Quantitation of DNA is a very important step in many procedures, as it is necessary to know the
amount of DNA that is present when trying to amplify DNA using PCR or cut DNA using
restriction enzymes. There are several methods for quantifying DNA, the most common methods
are:
1. Gel electrophoresis: the comparison of an aliquot of the
extracted sample with standard DNA of known
concentration
2. Spectrophotometric determination using Nanodrop

DNA Quantitation Procedure

1. Turn on the Nano-Drop software and select the Nucleic Acid tab and tap dsDNA
2. Clean the upper and lower pedestal of the Nano-Drop by pipetting 2μL of clean deionized
water onto the lower pedestal arm
3. Close the lever arm, ensuring that the upper pedestal comes in contact with the deionized
water. Lift the lever arm and wipe off upper and lower pedestal with a clean, dry Kim-
Wipes
4. Pipette 2μL of blanking solution onto the lower pedestal. Lower the lever arm and tap
"Blank". Once the blank measurement is complete, clean both upper and lower pedestal
with a clean, dry Kim-Wipes
5. Pipette 2μL of nucleic acid sample onto the lower pedestal and lower the lever arm.
6. Tap "Measure" in the application software. The software will automatically calculate the
nucleic acid concentration and purity ratios (260/280 ratio)
7. When you are finished measuring samples, tap "End experiment"
8. Lift the arm and clean both pedestals with a new wipe

N.B:
 For long-term storage of DNA, eluting in Buffer AE and placing at -30 to -15°C is
recommended
 One buccal swab typically yields 0.5–3.5 μg of DNA in 150 μl of buffer (3–23 ng/μl),
with A260/A280 ratios of 1.8–2