Download DNA Profiling: How many CATS

DNA Profiling: How many CATS?
Objective
The purpose of today’s exercise is to present basic concepts related to DNA profiling or
fingerprinting. These concepts include DNA base pairing, restriction enzyme digestion,
gel electrophoresis, and probe hybridization. In addition, you will discuss the analytical
and ethical questions associated with DNA fingerprinting.
Background Information
The differences in physical attributes or characteristics observed between people are due,
in part, to differences in their genes. Given such diversity in human populations, one
would expect great diversity in the genes that regulate the observed physical differences.
In spite of this, individuals in a population share approximately 99.9% of their DNA with
one another. This means that each individual differs on average in 1 out of 1000 base
pairs with any other individual. In addition, much of our DNA is considered “junk” DNA
because it is not transcribed into RNA; thus, “junk” DNA does not influence protein
expression and has no known function. These non-functional DNA segments are variable
(i.e. not conserved) and contain repeated sequences called tandem repeats that are
arranged one behind another (e.g. CATCAT). If we were to consider an individual that
has two different alleles at gene A (i.e. heterozygote for gene A), the length of their two
alleles may be different because of the variation in the number of tandem repeats
(VNTR) in those sequences (see Figure 1). For example, allele 1 has two tandem repeats
(CATCAT) while allele 2 has six repeats (CATCATCATCATCATCAT) that separate
two restriction sites. A restriction site is a specific sequence of DNA that is recognized
and cut by a restriction enzyme within the recognition sequence. When a restriction
enzyme is used to cut the DNA of the restriction site, the number of copies of repeated
sequences alters the length of the DNA fragment at those alleles. Because we are
measuring differences in fragment length at the different alleles, it is necessary to have
restriction sites that are not within the variable region but are on either side of the variable
region. Within the human genome, each restriction enzyme has many restriction sites. If
we were to use the restriction enzyme Hind III to cut our DNA sample, we would get a
huge number of different fragments. Not only would our digested sample have a large
number of fragments but it would also have millions of copies of each fragment. This is
because the tissue sample that the DNA was extracted from has millions of cells.
Once the sample has been digested, it is run on an agarose gel using a technique called gel
electrophoresis. A positive electric current is applied along the top of to the gel
(perpendicular to the length of gel) where the DNA that has been placed in a well or
depression. Because DNA has a negative charge, it is repelled by the positive current and
begins to move toward the negative electric current that runs parallel along the base of the
gel. The gel creates a matrix and the movement of DNA through the gel is dependent on
its size with large fragments taking more time to travel the length of the gel than small
fragments. In order to know the fragment sizes, it is necessary to run a standard of known
fragment sizes along side the unknown samples.
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What should your DNA sample look like after electrophoresis and staining is complete?
The DNA sample should appear as a smear. Remember that each restriction enzyme cuts
at every restriction site that is encountered so there should be millions of different sized
fragments that have separated along the gel.
So how can we see the fragments we are interested in? First, the DNA samples have to
be transferred to a nylon membrane. The nylon membrane forms links to the DNA which
holds it in place and prevents it from being washed away during the labeling process.
Otherwise, the DNA would slowly leach out of the gel and into the surrounding medium
or the gel would dry out and the DNA would be destroyed. Once the DNA has been
transferred to the nylon membrane, it can be probed with a radioactively labeled singlestranded DNA sequence. Radioactive probes recognize and bind to complementary
sequences (e.g. a probe with the sequence CAT will bind to GTA) under proper
conditions. Excess probes that do not bind to the DNA are washed away in the labeling
process leaving only DNA-probe complexes.
Target:
Probe:
5' CATCAT 3'
3' GTAGTA 5'
Following the labeling process, the fragments are visualized using audioradiography. The
radioactive probe emits energy that is transferred as light energy onto film. This is
similar to what would happen if you exposed a roll of unprocessed film to light; that is,
the film fogs or blackens. Since each probe is attached to a specific sequence of DNA,
that area of the film shows a black spot following development. Furthermore, the nylon
membrane can be washed (i.e. removal of radioactive probe) and re-probed with a new
radioactively labeled single-stranded DNA sequence for a different VNTR. When
enough VNTR regions of different alleles are sampled (6-12 in any given person), a
composite of an individual’s molecular signature or fingerprint is created. A DNA
fingerprint most commonly allows the identification of an individual in paternity and
criminal cases. Other applications include the identification of the illegal killing and
collecting of endangered animals and plants, respectively.
Materials
Scissors
Magnetic Strips (~36 cm per group)
Tap
Pencil
Copies of nucleotide sequences
Copies of standard (marker)
Copies of probe
Poster board
Procedure
You will simulate the molecular profiling techniques with the following exercise. In a
group of two, you will find the father of a pregnant woman recently raped. You will be
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given a ‘DNA sample’ from the child, the pregnant woman, her husband, and the rapist.
Each sample will be of a different color to prevent confusion of the samples.
Note: Only single-stranded DNA will be used for this simulation to simplify the
procedure.
1. Cut out the strips of sequences of the ‘DNA samples’ from the child, mother, husband
and rapist.
For each DNA sample:
2. Find the CAT repeat sequences and tape a 1 cm piece of magnetic strip in the middle
of each CAT sequence.
3. With a pencil, mark the recognition sites for the restriction enzyme Hind III
(AAGCTT).
4. At each restriction site, cut the strip between the two As. Place all the cut pieces into
an a labeled envelop which will represent each well of your gel.
Running the gel:
5. On a large sheet of poster board (at least 62 X 74 cm), assemble the fragments from
the standard and from steps 1-4. This will appear as five lanes on your gel.
6. For the standard, arrange the fragments so that the largest pieces are closest to the
wells (top of gel or poster board) with progressively smaller fragments further away
from the wells. This should look like a ladder that spans the entire length of the
poster board with the exception of 5 cm of space at the bottom. Tape the standards in
place. Label the top of this lane as Lane 1 or Standard.
7. In the next lane, arrange the mother’s sample. If mom has a 12-base fragment, it
should be in the same position as the 12-base fragment of the standard. Similarly
each fragment should be placed relative to the standard. Label this lane as Mother.
8. Repeat step 7 for the husband, suspect, and child.
Probing the gel:
9. Cut out strips of the probe with the sequence 3' GTAGTA 5' and attach a piece of
magnetic strip to the back.
10. Attach the probe to its complementary strand (5' CATCAT 3') on the gel. Only two
fragments per lane should be labeled. Each label represents one allele of a
homologous pair of a chromosome.
11. Analyze the pattern according to Mendelian principles.
Can you tell who the fathered the child? ____________________
Discussion Questions
1. Why does the child not have a DNA fingerprint that is exactly the same as her/his
mom and dad?
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2. Why do you think it is necessary to look at samples that are cut with more than one
restriction enzyme?
3. Given what you have learned, should DNA fingerprinting be used to convict or
exonerate a suspect of a crime? Why or why not?
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