public has always been captivated by the drama that occurs in
the courtroom. There is even a whole channel, CourtTV, devoted
to showing real court cases as they wend their way through the
legal system. TV shows and movies depict passionate attorneys
sparring verbally as they fight to convict or acquit the
accused. However, the most tense moments of a criminal trial
are likely those that go unseen: the jury deliberations.
After both sides present their evidence and argue their
cases, a panel of jurors must weigh what they have heard and
decide whether or not the accused person is guilty as charged.
This can be difficult. The evidence presented is not always
clear-cut, and sometimes jurors must decide based on what a
witness says they saw or heard. Physical evidence can be
limited to strands of hair or pieces of fabric that the
prosecution must somehow link conclusively to the defendant.
What if there were a way of tying a person to the scene of
a crime beyond a shadow of a doubt? Or, more importantly, what
if you could rule out suspects and prevent the wrong person
from being locked up in jail? This dream is beginning to be
realized through the use of DNA
evidence. In this issue of HowStuffWorks,
we'll look at how DNA "fingerprinting" works and what DNA
evidence can be used for.
Proving that a suspect's DNA
matches a sample left at the scene of a crime requires two
- Creating a DNA profile using basic molecular
- Crunching numbers and applying the principles of
population genetics to prove a match mathematically
Your Own Personal
We all like to think that we are unique,
not like anyone else in the world. Unless you are an identical
twin, at the nuclear level, you are! Humans have 23 pairs of
chromosomes containing the DNA blueprint that encodes all the
materials needed to make up your body as well as the
instructions for how to run it. One member of each chromosomal
pair comes from your mother, and the other is contributed by
Every cell in your body contains a copy of this DNA (see How Cells
Work for details). While the majority of DNA doesn't
differ from human to human, some 3 million base pairs
of DNA (about 0.10 percent of your entire genome) vary from
person to person. The key to DNA evidence lies in comparing
the DNA left at the scene of a crime with a suspect's DNA in
these chromosomal regions that do differ.
There are two kinds of polymorphic regions (areas
where there is a lot of diversity) in the genome:
- Sequence polymorphisms
- Length polymorphisms
Sequence polymorphisms are
usually simple substitutions of one or two bases in the
genes themselves. Genes are the pieces of the
chromosome that actually serve as templates for the production
of proteins. Amazingly, despite our complexity, genes make up
only 5 percent of the human genome. Individual
variations within genes aren't very useful for DNA
fingerprinting in criminal cases.
95 percent of your genetic makeup doesn't code for any
protein. Because of this, these non-coding sequences
used to be called "junk DNA," but it turns out that these
regions do actually have important functions such as:
- Regulation of gene expression during development.
- Aiding or impeding cellular machinery from
reading nearby genes and making protein.
- Serving as the bricks and mortar of chromosomal
Non-coding DNA is full of length
polymorphisms. Length polymorphisms are simply variations
in the physical length of the DNA molecule.
DNA evidence uses a special kind of length polymorphism
found in non-coding regions. These special variations come
from stretches of short, identical repeat sequences of DNA. A
particular sequence can be repeated anywhere from one to 30
times in a row, and so these regions are called variable
number tandem repeats (VNTRs).
The size of a DNA fragment will be longer or shorter,
depending on how many copies of a VNTR there are. In the case
of DNA evidence, the great thing is that the number of tandem
repeats at specific places (called loci) on your
chromosomes varies between individuals. For any given VNTR
loci in your DNA, you will have a certain number of repeats.
You inherit one copy of each chromosome from your mother
and father. This means that you have two copies of each VNTR
locus, just like you have two copies of real genes. If you
have the same number of sequence repeats at a particular VNTR
site, you are called homozygous at that site; if you
have a different number of repeats, you are said to be
Creating a DNA Profile: The Basics
procedure used to isolate an individual's DNA fingerprint is
called Restriction Fragment Length Polymorphism (RFLP)
analysis. This is a complicated way of saying that
investigators determine the number of VNTR repeats at a number
of distinctive loci to come up with an individual's DNA
Here is the key to DNA evidence: If you are looking
at a particular person's DNA, and a particular VNTR area in
that person's DNA, there is going to be a certain number of
repeats in that area. What you do to make a DNA fingerprint is
to count the number of repeats for a specific person for a
specific VNTR area. For each person, there are two numbers of
repeats in each VNTR region (one from mom and one from dad),
so you are getting both counts. If you do this for a number of
different VNTR regions, you can build a profile for a person
that is statistically unique. The resulting DNA fingerprint
can then be compared with the one left by the "perp" at a
crime scene to see if there might be a match.
Here's how it works in general:
- Isolate the DNA.
- Cut the DNA up into shorter fragments containing known
- Sort the DNA fragments by size.
- Compare the DNA fragments in different samples.
The way we sort by size is gel electrophoresis, and
then we look at the results using a Southern Blot.
Creating a DNA Profile: Step by Step
let's look at the exact steps used...
- DNA is isolated from a sample such as blood, saliva,
semen, tissue, or hair. DNA has to be cleaned up,
because, unlike in a pristine laboratory, samples at a crime
scene are often contaminated by dirt and other debris.
Sometimes, DNA must be isolated from samples dried to
patches of cloth or carpet, and getting the sample safely
out of these fabrics adds additional steps to the isolation
and purification processes.
- The huge genome is cut up with restriction
enzymes to produce short, manageable DNA fragments.
These bacterial enzymes recognize specific four to six base
sequences and reliably cleave DNA at a specific base pair
within this span. Cleaving human DNA with one of these
enzymes breaks the chromosomes down into millions of
differently sized DNA fragments ranging from 100 to more
than 10,000 base pairs long. You have to carefully select an
enzyme that doesn't cut within any of the VNTR loci that are
being studied; for RFLP analysis, the enzyme(s) chosen will
ideally cut close to the end on the outside of a VNTR
- These DNA fragments are then sorted by size using gel
electrophoresis. In this process, DNA is loaded into a
slab of Jell-O-like agarose and placed in an electric
field. The DNA is separated by size because:
- DNA, being negatively charged, is pulled through the
gel toward the positively charged electrode.
- Larger fragments move more slowly than smaller ones
through the porous agarose.
Once you have separated the DNA, you can determine the
relative size of each fragment based on how far it has moved
through the agarose.
- DNA fragments that have been separated on an agarose gel
will begin to disintegrate after a day or two. To
permanently save the DNA fragments in this segregated state,
you need to transfer and permanently affix DNA to a nylon
membrane. First, the DNA is denatured from its native
double helix into a single-stranded state (this frees up
nucleotides to base-pair with DNA probes for step 5 of the
process). The positively charged nylon membrane is then
placed on top of the agarose gel and used to sop up the
negatively charged DNA fragment, like you might blot ink off
with Silly Putty.
- Unlike Silly Putty, however, you can't actually see any
of the DNA on your membrane. In order to figure out which
fragments contain a particular VNTR locus, you have to flag
them with some kind of tag that you can visualize. How do
you do this? You simply make use of the basic
structure and chemistry of DNA. DNA normally occurs as a
double-stranded molecule, as two strings of nucleotides
twisted around each other. The structure is held together by
weak bonding between nucleotides on opposing strands. Only
certain pairs of nucleotides can interact (adenine with
thymine and guanine with cytosine), so these nucleotides are
said to be complementary.
To locate a specific VNTR sequence on a single stranded
DNA fragment, you can find it by simply:
- Making a DNA probe out of a DNA sequence
complementary to that of a VNTR locus
- Labeling the probe with a radioactive
- Letting the probe bind to like DNA sequences on the
- Using the radioactive tag to find where the
probe has attached
- Once the radioactive probe is stuck to its target
on the membrane, you can take a picture of it using special
X-ray film. You don't need a camera or
other machinery to accomplish this feat --all you have to do
is place the membrane against a special sheet of film for a
short period of time! How does this work? In a regular
camera, the film has a special coating that undergoes
chemical changes when it absorbs the energy of a
photon of light (to learn more, see How
Photographic Film Works). When you take a picture with a
camera, the light you let in by opening the shutter forms an
image on the film. X-ray
film, on the other hand, picks up radiation emitted from the
natural decay of
the isotope used in your probe. What you see on the film
is a darkened band that indicates the places on the
membrane where the probe has bound to DNA containing the
The results of RFLP analysis of one VNTR
locus in a sexual assault
In the image above, DNA from suspects 1 and 2 are compared
to DNA extracted from semen evidence. You can see in this
sample that suspect 1 and the sperm DNA found at the scene
match. Suspect 2 has a profile totally different from the
semen sample; his DNA fragments have run much farther down the
gel, meaning that they are shorter. You can also tell that he
is a homozygote because there is only one darker band
indicating the presence of two copies of the same fragment.
The other samples tested come from heterozygotes, because they
have two bands of distinct sizes in each lane. DNA isolated
from the victim as well as a human DNA (K562) that serves as a
standard size reference are included as controls.
The results from just one
VNTR locus by itself don't pinpoint a suspect anymore than
having one digit of someone's Social
Security number (SSN) would let you figure out who they
were. For example, a certain percentage of people are likely
to have the number 2 as the first digit in their SSN.
Similarly, for any given VNTR locus, a fragment length
corresponding to a certain number of sequence repeats occurs
in a certain number of individuals. What gives DNA
fingerprinting its power is the combined analysis of a
number of VNTR loci located on different chromosomes.
The final DNA profile is compiled from the results of
four or five probes that are applied to a membrane
sequentially. Each probe targets a different VNTR
locus. Using four probes (as in the figure below) actually
gives you eight pieces of information about an individual,
since each of us has two separate copies of each VNTR region.
To add to the complexity, it turns out that each VNTR
locus usually has approximately 30 different length
variants (alleles). Each of these alleles occurs at a
certain frequency in a population. To get the
probability that a given 8 band profile will occur, you
multiply the eight different allele frequencies together.
While the number of repeats at a single VNTR
locus can't distinguish an individual from the rest of
the population, the combined results from a number of
loci produce a pattern unique to that
Using four loci, the probabilitythat you'd
find a given allele combination in the general population is
somewhere around 1 in 5,000,000. In the United States,
the FBI incorporates 13 sites on average into its profiles.
With 26 different bands studied, you'd be incredibly hard
pressed to find two unrelated individual with the same DNA
profile; the odds of a match in this case are well more than
one in a hundred billion. The bottom line is that, unless you
have a twin, you're statistically two thousand times more
likely to win the Publisher's Clearinghouse sweepstakes (1 in
50,000,000) than to have a DNA profile that matches anyone
Advances in DNA Evidence
In 1985, DNA
entered the courtroom for the first time as evidence in a
trial, but it wasn't until 1988 that DNA evidence actually
sent someone to jail. This is a complex area of forensic
science that relies heavily on statistical predictions; in
early cases where jurors were hit with reams of evidence
heavily laden with mathematical formulas, it was easy for
defense attorneys to create doubt in jurors' minds. Since
then, a number of advances have allowed criminal investigators
to perfect the techniques involved and face down legal
challenges to DNA fingerprinting. Improvements include:
- Amount of DNA needed - RFLP analysis requires
large amounts of relatively high-quality DNA. Getting
sufficient DNA for analysis has become much easier since it
became possible to reliably amplify small samples using the
polymerase chain reaction (PCR). With PCR, tiny
amounts of a specific DNA sequence can be copied
exponentially within hours.
- Source of DNA - Science has devised ingenious
ways of extracting DNA from sources that used to be too
difficult or too contaminated to use.
- Expanded DNA databases - Several countries,
including the U.S. and Britain, have built elaborate
databases with hundreds of thousands of unique individual
DNA profiles. This adds a lot of weight to arguments
formerly based on mathematical theory alone, but it does
raise questions of civil liberty as authorities ponder
whether everyone in a community should be forced to submit a
sample for the sake of completeness.
- Training - Crime labs have come up with formal
protocols for handling and processing evidence, reducing the
likelihood of contamination of samples. On the courtroom
side, prosecutors have become more savvy at presenting
genetic evidence, and many states have come up with specific
rules governing its admissibility in court cases.
- Science education - In recent years, a number of
debates have erupted around the world over issues like using
DNA evidence, cloning animals or selling genetically
modified crops. It has dawned on many that to be active,
informed participants in such ethical debates, the general
public must understand the basic tenets of genetics,
statistics, and the like. Students in some schools today
aren't just learning about dominant and recessive genes in a
lecture; they are performing PCR and RFLP analysis on
samples to look for that recessive gene!
Using DNA Evidence
Given the high profile
DNA evidence had during the O.J. Simpson trial, most people
know DNA profiles are used by criminal investigators to:
DNA evidence is also
useful beyond the criminal courtroom in:
- Prove guilt - Matching DNA profiles can link a
suspect to a crime or crime scene. The British police have
an online database of more than 360,000 profiles that they
compare to crime scene samples; more than 500 positive
matches come up a week.
- Exonerate an innocent person - At least 10
innocent people have been freed from death row in the United
States after DNA evidence from their cases was studied. So
far, DNA evidence has been almost as useful in excluding
suspects as in fingering and convicting them; about 30
percent of DNA profile comparisons done by the FBI result in
excluding someone as a suspect.
- Paternity testing and other cases where
authorities need to prove whether or not individuals are
related - One of the more infamous paternity cases of late
revolved around a 1998 paper in the journal "Nature" that
studied whether or not Thomas Jefferson, the third president
of the United States, actually fathered children with one of
his slaves (in case you're wondering, according the
researchers, the answer is a resounding yes).
Photo courtesy Genelex,
DNA evidence can
pinpoint whether or not someone is a
- Identification of John or Jane Does - Police
investigators often face the unpleasant task of trying to
identify a body or skeletal remains. DNA is a fairly
resilient molecule, and samples can be easily extracted from
hair or bone tissue; once a DNA profile has been created, it
can be compared to samples from families of missing persons
to see if a match can be made. The military even uses
DNA profiles in place of the old-school dog tag. Each new
recruit must provide blood and saliva samples, and the
stored samples can subsequently be used as a positive ID for
soldiers killed in the line of duty. Even without a DNA
match to conclusively identify a body, a profile is useful
because it can provide important clues about the victim,
such as his or her sex and race.
- Studying the evolution of human populations -
Scientists are trying to use samples extracted from
skeletons and from living people around the world to show
how early human populations might have migrated across the
globe and diversified into so many different races.
- Studying inherited disorders - Scientist also
study the DNA fingerprints of families with members who have
inherited diseases like Alzheimer's Disease to try
and ferret out chromosomal differences between those without
the disease and who are have it, in the hopes that these
changes might be linked to getting the disease.
For more information, check out the links on the next page.
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