1. The Human
Genome
Project
Holli Cook

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.