2. What is it?
The Human Genome Project
was an effort to determine
the complete sequence of
DNA in the human genome.
Its goal was to discover and
map all of the approximately
35,000 human genes and
make them available for
further biological study.
3. Techniques used:
Restriction Fragment
Length Polymorphisms
Automated DNA
Sequencing
Polymerase Chain
Reaction
4. Restriction Fragment Length
Polymorphisms (RFLPs)
Each restriction enzyme is specific to a certain base sequence, a “restriction site”, and will cut
up DNA at all such sites to produce a number of “restriction fragments”.
No one will have the exact same base sequence unless they are identical twins.
Because of the DNA variability, restriction fragments from a given region of an individual’s
genome can be separated using get electrophoresis to reveal a unique pattern (a finger print).
Inheritance of RFLPs can be followed through families. By using the RFLPs scientists can create
linkage maps.
5. Step 1: Isolate the DNA
To extract DNA from its
location, several
laboratory procedures
are needed to break the
cell wall and nuclear
membrane.
Appropriately separate
the DNA from other cell
components.
When doing so, make
sure that the process
doesn’t damage the
DNA at all.
6. Step 2: Restriction Digestion
and Gel Electrophoresis
The extracted DNA is
digested with specific
restriction enzymes.
Each restriction enzyme
will recognize and cut up
DNA in a predictable
way, resulting in a
reproducible set of DNA
fragments, or restriction
fragments, or different
lengths.
7. Step 2: Restriction Digestion and Gel
Electrophoresis (continued)
The millions of
restriction fragments
produced are
commonly separated
by electrophoresis on
agarose gels.
8. Step 3: Transfer DNA by
Southern Blotting
The gel is denatured in a basic
solution and placed in a tray.
A porous nylon or nitrocellulose
membrane is laid over the gel,
and the whole thing is
weighted down.
All the DNA restriction
fragments in the gel are
transferred as single strands by
capillary action to the
membrane.
All fragments retain the same
pattern on the membrane as
on the gel.
9. Step 4: DNA Hybridization
Themembrane
with the target
DNA is incubated
with the DNA
probe.
10. DNA Probe
The DNA probe usually
comes from a DNA library,
which is a collection of
vectors that contain a
representation of an original
DNA molecule cut into
pieces.
Vectors may be transformed
into bacteria and may
multiply the piece of DNA
they contain many times.
The DNA probe is also
converted into a single-
stranded molecule,
conveniently labeled, using
any standard method.
11. Step 4: DNA Hybridization
(continued)
If strands on the membrane
are complementary to those
of the probe, hybridization will
occur and labeled duplexed
formed.
If strands are highly stringent,
hybridization with distantly
related or non-homologous
DNA does not happen.
The DNA probe picks up
sequences that are
complementary and ideally
homologous to it among the
thousands or millions of
undetected fragments that
migrate through the gel.
