Experiment 5.1: DNA - The Genetic Record
Introduction
In recent years, forensic investigations have relied increasingly upon DNA evidence both
to convict and exonerate suspects. DNA, the genetic ticker-tape” of life, provide often
compelling information about crimes and suspects. In order to understand how DNA is
employed in forensic investigations, an understanding of some of the basics about DNA itself is
first necessary.
In 1985, former Marine Kirk Bloodsworth was convicted and sentenced to death
for the rape and murder of a nine year old Dawn Hamilton. He received a second
trial after a successful appeal based on the grounds of evidence having been
withheld at his first trial. However, he was sentenced again, this time for two
consecutive life terms.
After several years of fighting for a DNA test, samples from the scene of the
crime were sent to a lab for testing. The final reports concluded that the DNA
found at the scene of the crime did not match Bloodsworth's DNA, and he was
released and pardoned by the Governor of Maryland shortly thereafter.
Bloodsworth's ordeal lasted 9 years, including two years on death row. The DNA
did match that of Kimberly Shay Ruffner, and on May 20, 2004, he pleaded guilty to
the crime for which Bloodsworth was convicted.
Objectives
The purpose of this laboratory experiment is to extract and isolate several samples of
DNA from commonly available substances. Additionally, a simple qualitative test will be
performed to confirm the presence of DNA in the sample.
Background
The discovery of DNA is attributed to Johann
Miescher (1844 – 1895), a chemist from Switzerland, while
working on pus cells. Prior to his work, it was believed that
cells were made up largely of protein, long chains of linked
amino acid molecules, but Miescher found that certain
extracts from cells “cannot belong among any of the protein
substances known hitherto”. He successfully showed that
these fractions were not protein because they were not
digested by protease enzymes (enzymes that break up
proteins). These particular extracts were also shown to arise
from cell nuclei and were, therefore, named nuclein. Later,
work by Albrecht Kossel on nuclein showed that the material
contained four nucleic acid bases, and was subsequently
renamed a nucleic acid by Richard Altmann. The structure
we recognize today as the famous double helical arrangement
was explained in 1953 by James Watson and Francis Crick.
DNA Extraction and Isolation
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Copyright James T. Spencer 2004
HO
OH
CH2
OH
CH2
O
O
C H
H C
C H
H C
H C
C
H C
C
H
H
OH
Ribose
phosphoric acid residue (PO42-), a fivecarbon sugar unit (possible sugars shown in
Figure 1), and a nitrogen-containing base
(Figure 2) – together these are called a
nucleotide. The phosphate group and the
deoxyribose sugar, when linked together in
an alternating fashion, form a straight chain
backbone for the DNA polymer. Off of
this backbone are then fastened the
nitrogen bases. A complete nuclei acid
unit is shown in Fig. 3. Thus, all the units
of the backbone are identical (alternating
NH2
O
-1
P
O
N
N
O
-1
N
CH2
N
O
C
H
H
H
C
C
OH
H
C
H
Figure 3. Nuclei Acid Unit
a nuclear DNA polymer, millions and
millions of these basic units are strung
together.
These long strands of DNA
polymer are found in nature in
complementary pairs, meaning that two
are required to form the observed DNA
structure. But these strands don’t just
come together in any random fashion,
but are instead very specific in their
interactions. Specifically, two nitrogen
bases can come together to form a close
electrostatic interaction, called a
hydrogen bond, based upon their
chemical structure. Thus, cytosine and
DNA Extraction and Isolation
H
OH
OH
Figure 1. Deoxyribose
O
DNA is made up, at its most
fundamental level, of repeating units of
nucleic acids arrayed in a polymeric
fashion. Thus, one nucleic acid unit is
linked to another, much the way
railroad cars are linked together, to
form a very long biopolymer. These
fundamental nucleic acid units are each
composed of three basic parts: a
HO
NH2
N
O
N
N
NH2
N
N
N
N
N
N
NH2
N
O
O
O
H3C
N
N
N
O
N
O
Figure 2. Nitrogen bases used in DNA and RNA
(clockwise from upper left): adenine, guanine,
cytosine, thymine (DNA only) and uracil (RNA
only).
phosphate and sugar) while the pendant nitrogen
bases can be different. In fact, DNA uses only
four different nitrogen base units: adenine,
guanine, cytosine and thymine (RNA, a relative
of DNA, replaces thymine with uracil). These
bases are shown in Fig. 2.
The entire
fundamental structure of DNA, therefore,
consists of the phosphate-sugar backbone with
nitrogen bases hanging off this backbone from
the sugar subunit, Figure 4. In a typical strand of
O
O
O
O
O
O
H
O
C
C
P
H
O
C
C
P
O
C
C
P
O
C
H2
H
O
C
H2
O
H
O
C
H2
H
H
C
H
O
H
H
H
C
C
N
N
H
O
H
H
C
H
C
N
N
N
NH2
H
C
N
N
N
N
NH2
N
N
NH2
N
Figure 4. Structure of DNA polymer.
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Figure 5. DNA transcription process.
DNA double helix is first opened up in the
region containing the genetic information that is
to be converted into chemical compounds such
as proteins. A complimentary strand is then
generated (matching A’s with T’s and G’s with
C’s – see below). The new strand (messenger
RNA or mRNA) is transferred to a ribosome in
the cytoplasm. Each group of three nitrogen
bases in the messenger RNA strand designates a
specific amino acid compound that is placed in
order by a transfer RNA unit (tRNA). The order
of the bases in DNA is ultimately transferred
into a very specific sequence of amino acids that
are linked together to form a new polymer. The
polymer formed by linking the amino acids
together is called a protein. Protein composition,
structure and function (including that of
enzymes, structural proteins, and many others)
DNA Extraction and Isolation
guanine are built to allow a close interaction
– close enough to form a hydrogen bond
holding the two bases together. Likewise
adenine and thymine can also form a
hydrogen bonded unit.
Having a
complementary sequence on the two DNA
strands where a cytosine is opposite every
guanine and an adenine is opposite every
thymine yields a double strand effectively
“pinned together” through the nitrogen
bases. This is shown schematically in Figure
5. When these complementary strands of
DNA come together, they form the wellknow double helical structure (like a twisted
flight of stairs).
The sequence of the nitrogen bases is
the basis of controlling all cellular processes
and ultimately determining things like what
color eyes we have or how tall we might
become. Our genes, the genetic information
encoded in DNA, are really composed of just
four letters (adenine, guanine, cytosine and
thymine) that write all the chemical
information required to regulate cellular
function. The way that this translates from
letters into chemical reactions requires a
process called transcription. In a very brief
summary, it works something like this. The
Figure 6. Hydrogen bonded bases pairs
between two strands of DNA.
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is, therefore, completely dictated by the DNA base orders.
As mentioned previously, DNA is found in the nuclei of cells and also in the cell’s
mitochondria (called mitochondrial DNA, mtDNA). In the nucleus, it forms the familiar
chromosomes that carry all the genetic information. The very long chain DNA molecule in the
chromosomes are coiled up tightly to for the chromosomal structures observed as shown in
Figure 7.
Our DNA, as it turns
out, contains far more “data”
than is contained within the
gene portions of the DNA.
The DNA information that
makes up a gene, the part that
regulates protein sequences, is
separated from other genes by
regions of DNA that contain
essentially nonsense codes
(hypervariable region) – codes
that do not translate into
proteins. These portions may
either have had a function in
the past but are now not
Figure 7. DNA coiling to form chromosomal structures.
necessary for cellular function
or may have arisen from other means such as mutation or viral insertion.
Since protein structure and function derives directly from the ordering of the nitrogen
bases in DNA, very small changes in the base ordering in genetic regions of DNA may cause
catastrophic changes in cellular function. One base unit incorrectly placed can mean the
difference between disease or no disease or even life versus death. For example, the substitution
of a single nitrogen base for the correct base in the gene regulating red blood cells may result in a
person having sickle cell anemia. Within a species such as humans, there is very little variation
of the DNA code within the genetic portions. Mutations, changes in the order of the nitrogen
bases in the genetic region of the DNA chain, are not tolerated and usually do not result in viable
offspring. Changes (mutations) in the “nonsense” hypervariable DNA regions between the
genes, however, usually make no difference in the survival of an organism since these DNA
codes play no role in regulating cells or in developing the traits we observe in an organism.
Since there is no survival advantage of one code versus another in the hypervariable region, over
time these nonsensical regions have become extremely diverse such that essentially no two
people have the same DNA codes in these inter-gene regions. The basis, therefore, of forensic
DNA is to look at the DNA codes in these inter-gene regions (since looking at the gene regions
would not be able to discriminate one persons DNA from another – we’re essentially all the same
in the gene regions).
In forensic applications of DNA, one of the first tasks is to isolate the DNA from samples
gathered at a crime scene. In this laboratory experiment, we will isolate DNA samples from two
different sources, one plant and one animal. We will then confirm the presence of DNA in the
samples through a chemical qualitative analytical test.
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DNA: The Genetic Record
Experimental Methods
I. DNA Extraction:
In this experiment, you will extract DNA from a biological sample. Record your
measurements and observations on the data sheets provided.
Procedure:
I.A. Preparation of DNA buffer solution.
To make the DNA buffer solution, you will need to measure out 2.5 mL shampoo
(without conditioner) or 1.5 mL liquid dishwashing detergent. To this, add 3.75 g NaCl
and 25 mL of water. Mix this solution very gently, but thoroughly, so as to avoid
forming soapy bubbles as much as possible. This solution is the DNA buffer solution.
I.B. Extraction Strawberry DNA.
Place one strawberry in a zip lock baggie and smash the strawberry vigorously for
about two minutes. To this, add 10 mL of the DNA buffer solution (made in I.A. above)
to the bag and reseal. Smash the contents to completely mix then for about one minute.
Filter the mixture from the baggie through cheesecloth into a beaker (plastic cup) and
then pour the filtrate (the solution in the beaker) into test tube so that it is 1/8 full. Very
slowly pour cold alcohol (either 95% ethanol or 95% isopropyl alcohol that has been
cooled for at least 15 min. in an ice bath) into the tube so as to form two layers until the
tube is about one-half full. At the interface of the layers, you will see the DNA
precipitate out of solution and float. Spool the DNA onto a glass rod or pipet tip and
collect.
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DNA: The Genetic Record
Data Sheet
Name
Instructor
Laboratory Section
Lab Period
Step
Observation
IA
IB - strawberries
IIA - strawberries
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DNA: The Genetic Record
Post-lab Assignment
Name
Instructor
Laboratory Section
Lab Period
(1) Suppose a student added the ethyl alcohol straight to the solution from the baggie
WITHOUT filtering it through the cheesecloth first. What do you think the student
would observe? Would the student be successful in extracting the DNA? Explain briefly
why you think this.
(2) Would you expect similar results if you were to use other cells, such as liver, onion, or
yeast? Briefly explain your reasoning.
(3) Consider a single celled organism, such as a bacterium, whose DNA is not enclosed in a
membrane-bound nucleus:
a. Would you predict that it would be easier or harder to extract the DNA from the
bacterium compared to the extraction of DNA from the strawberry or cheek cell?
b. Would the single cell organism have as much DNA as the multi-celled organism
examined in this experiment? Briefly explain.
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DNA: The Genetic Record
Pre-lab Assignment
Name
Instructor
Laboratory Section
Lab Period
(1) Using a MSDS sheet, can be found online, describe the specific hazards associated with
the following reagents:
a. Ethyl alcohol
b.
Diphenylamine Reagent
(2) Why do we perform the following steps:
a. Addition of the buffer solution to the baggie
b. Filtering the solution through cheesecloth
c. Adding the ethyl alcohol
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