MLAB 2337 Molecular Diagnostics Techniques

Laboratory 5: DNA Isolation

Objectives
1. List and describe 9 parameters which should be evaluated when trying out a new kit or
strategy for preparing nucleic acids for a test application.
2. List and describe 3 major factors that interfere with nucleic acid purification.
3. State the two fundamental steps involved in DNA purification.
4. List three considerations when selecting a DNA isolation method.
5. State why simplicity and speed are top priorities in selecting a DNA isolation method.
6. State the two considerations in selecting a method for DNA purification.
7. State the method used to render genomic DNA insoluble.
8. List two advantages of spin column methods for purifying DNA.
9. State a significant, potential problem in the use of silica columns for purifying DNA.
10. State the sample source of DNA in the PV92 procedure.
11. What is the purpose of the InstaGene solution?
12. State the purposes of the 56°C and 100°C incubations.

Introduction:

An efficient method of nucleic acid extraction that produces purified, high-quality nucleic acid
product is crucial to the success of PCR, sequencing, and many other molecular diagnostics
techniques involving enzymatic transformations. Many methods of nucleic acid extraction have
been developed, and the specific method of choice depends on the type of tissue source, as well
as the intended subsequent use of the purified nucleic acid. Although many vendors offer kits
for nucleic acid extraction, it is important to understand the principle of the extraction techniques
in order to evaluate the best one to use for any particular application. Each extraction strategy
comes with its own advantages and drawbacks, possible problems associated with them that you
need to watch out for.

Some specific parameters that should be evaluated when trying out a new kit or strategy for
preparing nucleic acids for a test application include:

1. Simplicity. The greater the number of steps, the greater the time required and the more
possibilities of a failure. Multiple steps also tend to reduce yields.
2. Efficiency. The greater the yield of nucleic acid from the tissue, the less sample and
reagents required. Different tissue types will extract with different efficiencies,
depending on the exact extraction strategy selected. In general, the more efforts to purify
DNA, the low the expected yields.
3. Purity of nucleic acids extracted. Some molecular assays require highly pure sample,
while others are more robust at lower degrees of purification. Some extraction
techniques can address the specific impurities commonly contaminating cellular extracts
better than do others.
4. Specificity. The type of nucleic acid being extracted (DNA vs RNA) will guide your
selection of extraction method. In extracting pathogenic nucleic acids from clients’
tissues, the extraction method should be chosen that can selectively purify from the more
abundant gDNA of the client.

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 5. Sensitivity. Some types of tissue extract easily, while others require much harsher
treatments in order to get nucleic acids out of cells. The harsher treatments are more
laborious and pose a greater risk of carryover of reagents or nucleic acid degradation that
can interfere with subsequent analysis.
6. Integrity of the nucleic acids. Harsh treatments, especially those that introduce shear
forces, can greatly decrease the average size of genomic DNA prepared.
7. Adaptation to high-throughput techniques. As the demand for molecular analyses
increases, the ability to automate extraction techniques becomes more important.
8. Reliability. Since extraction methods deal with a range of tissue sources and tissue
storage conditions, an extraction technique that is the most fool-proof is always preferred.
The method that has the least fail-rate will save lab time and reduce the number of
specimens that require retesting.
9. Expense. A great deal of both time and expense can be saved by determining exactly
what cellular contaminants actually need to be removed. For example, some PCR
applications might only require cellular lysis and no subsequent purification steps,
depending on the tissue being tested and how quickly the extract can be processed.

The major factors that interfere with nucleic acid purification are:

1. Nucleases. The isolation of nucleic acid is a race against internal degradation from
nucleases. Lysis buffers are the key to avoiding this problem, by promoting a rapid and
thorough lysis of cells, and by including inhibitors of nucleases. Most lysis buffers either
contain protein-denaturing reagents such as detergents and enzyme-inhibiting
components such as EDTA. These components must be carefully selected in order to
avoid inhibition of enzymes used later in testing. Keeping samples cold prior to nuclease
inactivation is also key to winning the race against nucleases, as the enzyme activity is
slowed at the lower temperatures. To avoid reintroduction of nucleases later on during
sample processing, reagent purity must be guarded, and materials must be kept nuclease-
free. The use of disposable materials or the use of heat and chemical treatments of non-
disposable materials can effectively prevent nuclease problems. Signs of nuclease
contamination is apparent in smears or lack of signal in electrophoresis gels, or failure to
amplify by PCR.
2. Shearing. Shearing the chromosomes will break up the genomic DNA into small pieces,
making it harder to isolate. The quality of genomic DNA can be improved by avoiding
vortexing, repeated pipetting through narrow tips, and freeze-thawing of sample.
Mechanical techniques for disrupting cell wall materials found in bacteria, plants, and
fungi contribute to shearing of genomic DNA (gDNA). Sonication, grinding in liquid
nitrogen, and shredding devices such as rigid spheres or beads are used to lyse difficult
samples, but lead to low quality gDNA. Enzymatic cell wall disruption, such as the use
of lysozyme to break down bacterial cell walls, can improve the size of gDNA isolated.
3. Chemical contaminants. Cells can be sources of contaminants that interfere with
nucleic acid isolations and down-stream applications. Polysaccharides and cell wall
materials can react irreversibly to create an unusable final product, so CTAB
(cetyltrimethyl ammonium bromide) is often used to precipitate gDNA and separate it
from these interfering contaminants. Low levels of certain purification reagents can lead
to failures in subsequent enzymatic treatments, and must be removed. Phenol-based
purifications leads not only to phenol interferences with many enzymes, but oxidation
products of phenol can also damage nucleic acids. Freshly-prepared phenol solutions
must be used, and traces of phenol must be removed by chloroform extraction, followed

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 by precipitation of nucleic acids in ethanol. Many impurities, such as guanidinium
isothiocyanate, can be removed by ethanol precipitation of nucleic acids, followed by
ethanol washes of the nucleic acid precipitate. Ethanol itself, however, can interfere with
subsequent enzymatic treatments, and must be carefully removed. Drying in the open air
is commonly used to remove traces of ethanol, but overdried nucleic acids can be difficult
to redissolve. Phosphate buffers inhibit many polymerases, including Taq polymerase,
and should be avoided. High levels of EDTA should be avoided, as it can inhibit many
enzymes that act on nucleic acids. Heparin should be avoided, because it inhibits Taq
polymerase and has been shown to lead to nicked DNA molecules.

Overview

The fundamental steps of DNA purification are 1) sample lysis and 2) purification of the DNA
from contaminants. There are a myriad of protocols available for isolating DNA from organisms
in the molecular lab. The more “classical” methods have remained essentially unchanged for
decades, and the more modern methods involve kits that are commercially available. Basically,
the best method for any particular application depends on these fundamental considerations:
a. Where the DNA is isolated from, i.e. sample required, will determine the cell lysis
techniques used.
b. The purity requirements of the intended use of the DNA being isolated will determine
how many purification steps will be involved.
c. The type of DNA being isolated: genomic DNA has different physical properties from
those of plasmid DNA.

First Phase – Lysis - Releasing DNA from the cell

The successful isolation of DNA requires methods that prevent nuclease degradation of the
DNA. Some buffer constituents used to promote lysis and denaturation of nucleases include
 Detergents are used to solubilize cell membranes: Popular choices are SDS (sodium
dodecyl sulfate, aka SLS, sodium lauryl sulfate), Triton X-100, and CTAB (cetyltrimethyl
ammonium bromide)
 Proteinase K is sometimes added to cleave glycoproteins and to help the detergents to
inactivate DNAses.
 Denaturants such as urea, guanidinium salts, and other chaotropes are sometimes applied
to inactivate enzymes.
 Heat is often applied to enhance the lysis of cells and the denaturation of proteins.
 For microbial sources of DNA, enzymes must be added to break down cell walls in order
to make the cells susceptible to lysis.
 RNAses are often added to a lysis buffer to remove contaminating RNAs, which can
interfere with the intended use of the DNA being isolated.

In selecting a lysis method for a particular application, top priority should be given to choosing a
method that has simplicity and speed (fewest numbers of steps and solutions required).
Remember that every constituent added to cells during the lysis procedure could become a
culprit by sabotaging the activity of an enzyme later on. You will want to remove anything
added to your DNA at the beginning of DNA isolation sometime later, so try to keep the number
of constituents in your lysis buffer to those that are absolutely needed. The number of steps in a
cell lysis protocol should also be kept to a minimum, since any delays during this part of the
DNA isolation procedure runs the risk of DNA degradation by nucleases in the cells. DNA will
not be safely stabilized until it has been purified from all protein contaminants. In general,

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animal tissues are easily lysed, due to the fact that they have no cell wall, and a gentle detergent
treatment is usually sufficient to break open cells. Yeast and microbial cells, on the other hand,
have rigid cell walls that must be weakened enzymatically before the cell will release its DNA.
In the case of bacteria, lysozyme enzyme is added, while in the case of yeast a more complex
mixture of enzymes must be used to degrade cell wall polymers. Plant cell walls are generally
abraded mechanically by grinding frozen plant tissue, often with glass beads or sand and a
mortar and pestle.

Second Phase – Purification of DNA

The second phase of DNA isolation protocols is the purification of the DNA released from the
cell from other components of the cell and the lysis buffer. The method you select for your
application depends on the 1) size and 2) source of the DNA to be isolated. When plasmid DNA
is being isolated from bacteria such as Escherichia coli (E. coli), an alkaline solution of SDS is
sufficient to release plasmid DNA, leaving behind the genomic DNA still associated with the
cellular debris. The genomic DNA is then conveniently removed from the plasmid DNA by a
quick centrifugation step. Genomic DNA can frequently be rendered insoluble and quickly
“spooled” from the lysed cells by addition of alcohol to the mixture. The spooled DNA can be
transferred to a fresh buffer to redissolve the genomic DNA.

For some applications, this low level of DNA purity will suffice. Often, though, there are
proteins or polysaccharides (especially in plant sources of DNA) that coprecipitate with the DNA
and interfere with subsequent enzymatic treatments. Classically, the further purification of DNA
involves the removal of proteins by aqueous phenol solutions, followed by numerous alcoholic
precipitation steps to remove traces of phenol from the isolated DNA. Alcohol precipitations of
DNA also serve to concentrate the DNA into a smaller volume, and to purify the DNA from any
water-soluble contaminants. The phenol extraction is an inefficient method of purification and
suffers from a poor yield of DNA. Also, phenol reagents are unstable, and fresh solutions must
be used or the quality of the reagent must be monitored, generally by observed changes in pH.
This, along with safety concerns in the use of phenolic solutions, is a serious drawback in this
method of DNA purification.

An alternative procedure is the use of so-called “spin columns”, which are small chromatography
columns that purify the DNA from other solutes. While this procedure is more expensive than
phenol extractions and alcohol precipitations, the purification and yields of product by spin
columns are improved. Also, the reagents used are more stable, so provide a more reliable, or
“robust”, method. The final DNA prepared with spin columns is free of protein and salt
contaminants and can be used directly in restriction digests, Southern blotting, and PCR
applications. All components of this system are stable at room temperature for one year.
Another advantage of purchased kits for plasmid preparation is that the quality of reagents can be
tested and assured the venders. For these reasons, many biotechnology labs routinely use kits for
their plasmid preparations.

Binding and elution from silica beads has become the method of choice for isolation of genomic
DNA from animal tissues. A high concentration of chaotropes serves to bind nucleic acids to
silica surfaces. The adsorption step to bind DNA to the silica particles is followed by wash steps,
usually with salt/ethanol solutions which will not interfere with the strong binding of nucleic
acids but will wash away remaining impurities and excess chaotrope. Elution of DNA from
silica columns requires the use of nuclease-free water or low ionic strength buffers such as TE.
This is an advantage since it means that the isolated DNA can be used directly in further

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manipulations without further cleanup. The spin-column method does not require the use of
time-consuming and toxic phenol/chloroform extractions or ethanol precipitations. The final
genomic DNA prep is free of protein and salt contaminants and can be used directly in restriction
digests, Southern blotting, and PCR applications.
A potential problem with the use of silica columns for the binding of DNA is the possibility of
overloading the column with DNA, resulting in a wash-through of non-adsorbed DNA and
reducing the overall yield of DNA. There is also some loss of material that does not elute from
the silica resin. The smaller the DNA size is, the tighter is its interaction with silica surfaces.
Although size is not a problem with isolations of genomic DNA, loading the silica resin with too
little DNA can also lead to a low overall yield of DNA eluted from a silica column.

Protocols for the use of spin columns are unique to each vendor, and so the vendor’s protocol
should be followed when they are used.

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PV92 Procedure

Procedure 1: DNA Extraction and Template Preparation

To obtain DNA for use in the polymerase chain reaction you will extract the DNA from your
own living cells. There are several steps involved:
1. Rinse mouth with saline to collect cheek cells.
2. Mix cheek cells with InstaGene to remove interfering substances.
3. Heat at 56 °C to soften cell membrane.
4. Boil at 100 °C to rupture cells, releasing DNA from nucleus.

The cheek cells will be collected and processed to isolate the DNA. This is accomplished by
swishing saline in your mouth while gently biting the inside of your cheek. It is crucial to collect
an adequate volume of cheek cells to ensure an adequate volume of DNA.

Once an adequate amount of cheek cells are collected a set amount is transferred into the
InstaGene matrix. This particulate matrix is made up of negatively charged microscopic beads
that "chelate", or grab metal ions out of solution. It acts to trap metal ions, such as Mg 2+, which
are required as catalysts or cofactors in enzymatic reactions.

The cheek cells are then lysed by heating to release all of their cellular constituents, including
enzymes that were once contained in the cheek cell lysosomes. Lysosomes are sacs within the
cells cytoplasm that contain powerful enzymes, such as DNAases, which are used by cells to
digest the DNA of invading viruses. When you rupture the cells, these DNAases can digest the
released DNA of interest. However, when the cells are lysed in the presence of the chelating
beads, the cofactors are adsorbed and are not available to the enzymes. This virtually blocks all
enzyme degradation of the extracted DNA and results in a population of intact genomic DNA
molecules that will be used as the template in your PCR reaction.

Once the cheek cells are added to the InstaGene matrix the mixture is incubated at 56 °C
for 10 minutes. This "pre-incubation" step helps to soften the plasma membranes and release
clumps of cells from each other. The increased temperature also acts to inactivate enzymes such
as DNAases, which will degrade the DNA template. After this 10 minute incubation period, the
cells are then placed into a boiling (100 °C) water bath for 6 minutes. The boiling ruptures the
cells and releases the DNA from the cell nucleus. Your extracted genomic DNA will then be used
as the target template for PCR amplification.

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Name _________________________________ Date __________________________

MLAB 2337 Molecular Diagnostics Techniques

Laboratory 5: DNA Isolation
Study Questions
Points: /24
Instructions:
 Copy/paste the following study questions into word processing document.
 Answer the questions
 Save the file as “Lab5DNA_IsolationYOURNAME”
 Submit to “Assignments” in BlackBoard

1. List and describe 4 of the 9 parameters which should be evaluated when trying out a new kit
or strategy for preparing nucleic acids for a test application. (4 points)
a.
b.
c.
d.
2. List and describe 3 major factors that interfere with nucleic acid purification. (3 points)
a.
b.
c.
3. State the two fundamental steps involved in DNA purification.(2 points)
a.
b.
4. List three considerations when selecting a DNA isolation method. (3 points)
a.
b.
c.
5. State why simplicity and speed are top priorities in selecting a DNA isolation method. (2
points)
a.
b.

6. State the two considerations in selecting a method for DNA purification. (2 points)
a.
b.

7. State the method used to render genomic DNA insoluble. (1 point)

8. List two advantages of spin column methods for purifying DNA. (2 points)
a.
b.

Lab 5: DNA Isolation Page 7

9. State a significant, potential problem in the use of silica columns for purifying DNA. (1
point)

10. State the sample source of DNA in the PV92 procedure. (1 point)

11. What is the purpose of the InstaGene solution? (1 point)

12. State the purposes of the 56°C and 100°C incubations. (2 points)
a.
b.

Lab 5: DNA Isolation Page 8