Human DNA Profiling
DNA Fingerprinting
What is DNA Fingerprinting
• The chemical structure of everyone's DNA is the
same.
• The only difference between people (or any
animal) is the order or sequence of the base pairs.
• There are so many millions of base pairs in each
person's DNA that every person has a different
sequence.
• Using these sequences, every person could be
identified solely by the sequence of their base
pairs.
What is DNA Fingerprinting?
• However, because there are so many millions of
base pairs, the task would be very time-consuming.
• Instead, scientists are able to use a shorter method,
because of repeating patterns in DNA called
VNTRs which are known to vary among
individuals a great deal
• These patterns do not, however, give an individual
"fingerprint," but they are able to determine
whether two DNA samples are from the same
person, related people, or non-related people.
How is it done?
1. Performing a Southern Blot
2. Making a Radioactive Probe
3. Creating a Hybridization Reaction
Southern Blot
• The Southern Blot is one way to analyze the genetic
patterns which appear in a person's DNA. It involves…
1. Isolating the DNA from rest of the cell:
• This can be done either chemically, by using a detergent
to wash the extra material from the DNA, or
mechanically, by applying a large amount of pressure in
order to "squeeze out" the DNA.
2. Denaturing the DNA:
• To render the entire DNA as a single strand.
• This can be done either by heating or chemically treating
the DNA in the gel.
Southern Blot
3. Cutting the DNA into several pieces of different sizes.
• This is done using one or more restriction enzymes.
4. Sorting the DNA pieces by size – Size Fractionation
• The process by which the size separation is done is called
gel electrophoresis.
• The DNA is poured into a gel, such as agarose, and an
electrical charge is applied to the gel, with the positive &
negative charges
• Because DNA has a slightly negative charge, the pieces of
DNA will be attracted towards positive charge
• The smaller pieces, however, will be able to move more
quickly and thus further towards the positive charge
Southern Blot
5. Blotting the DNA:
• The gel with the size-fractionated DNA is applied to a
sheet of nitrocellulose paper, and then baked to
permanently attach the DNA to the sheet.
• The Southern Blot is now ready to be analyzed.
Radioactive Probe
• In order to analyze a Southern Blot, a radioactive genetic
probe is used in a hybridization reaction with the DNA in
question.
• If an X‐ray is taken of the Southern Blot after a radioactive
probe has been allowed to bond with the denatured DNA
on the paper, only the areas where the radioactive probe
binds will show up on the film.
• This allows researchers to identify, in a particular person's
DNA, the occurrence and frequency of the particular
genetic pattern contained in the probe.
Southern Blot & Probing
Hybridization
• Hybridization is the coming
together, or binding, of two genetic
sequences.
• The binding occurs because of the
hydrogen bonds [pink] between
base pairs.
• Between a A base and a T base,
there are two hydrogen bonds;
between a C base and a G base,
there are three hydrogen bonds.
Hybridization
• Denaturing is a process by which
the hydrogen bonds of the
original double-stranded DNA
are broken, leaving a single
strand of DNA whose bases are
available for hydrogen bonding.
• Once the DNA has been
denatured, a single-stranded
radioactive probe [light blue] can
be used to see if the denatured
DNA contains a sequence similar
to that on the probe.
VNTRs
• Every strand of DNA has pieces that contain
genetic information which informs an organism's
development (exons) and pieces that, apparently,
supply no relevant genetic information at all
(introns).
• Although the introns may seem useless, it has been
found that they contain repeated sequences of base
pairs.
• These sequences, called Variable Number Tandem
Repeats (VNTRs), can contain anywhere from
twenty to one hundred base pairs.
VNTRs
• VNTRs are hereditary and hence unique
• Hence an individual will have VNTRs inherited from
the mother or father, or a combination of both, but
never a VNTR either of the parents do not have.
• The more VNTR probes used to analyze a person's
VNTR pattern, the more distinctive and individualized
that pattern, or DNA fingerprint, will be.
• US CODIS - Combined DNA Information System –
Uses 13 STR Loci
VNTRs
• Every human being has some VNTRs.
• To determine if a person has a particular VNTR, a
Southern Blot is performed, and then the Southern
Blot is probed, through a hybridization reaction,
with a radioactive version of the VNTR in
question.
• The pattern which results from this process is what
is often referred to as a DNA fingerprint.
CODIS
Practical Applications
• Paternity and Maternity
• Criminal Investigations and Forensics
• Personal Identification
DNA Fingerprints