Southern Blot
By: Jacqueline Jai
Southern Blot
Southern Blot-a piece nitrocellulose
paper containing spots of DNA ready for
identification by a suitable molecular
probe.

Southern Blot
Southern Blot is a copy of DNA profile
January 28, 2003
JJ-2
Interesting Facts
about DNA Analysis
DNA Evidence
DNA evidence-has
many uses within the
legal system and
criminal cases.
First criminal identification card filed
by the NY State Bertillon Bureau
Southern Blot

Proving someone guilty
or innocent for a crime
they have or have not
committed.

Identification

Paternity Testing
January 28, 2003
JJ-4
Criminal Cases
Southern Blot

DNA evidence has exonerated people accused
of committing crimes.

Only about 30% of all DNA tests run by the FBI
have exonerated an accused person; DNA
evidence is still not as useful as fingerprinting.
January 28, 2003
JJ-5
Identification
Used to determine the sex, race, or even name of
unnamed victims of crimes.
Used in military to identify those who have died in
battle, similar to the purpose of dog tags.
Typical dog tags
Southern Blot
January 28, 2003
JJ-6
Paternity Testing

Southern Blot
Evidence can be used to compare the DNA of
the suspected parent(s) and that of the child
and determine the real parent.
January 28, 2003
JJ-7
DNA Profile
The Basics to Creating a
DNA Profile (Agenda)
Collect the DNA
Isolate the DNA
Cut DNA
 Variable Number Tandem Repeats
(VNTRs)
Sort DNA through Gel Electrophoresis
Transfer DNA to a solid support
 Methods of Tranferring
The Hybridization Reaction
 Denatured and Nicked DNA
 Radioactive Probe
 Continuation of the Hybridization
Reaction
Comparing DNA fingerprints
A DNA Profile
Southern Blot
January 28, 2003
JJ-9
Collect the DNA
Collect DNA
Collect DNA sample

Blood, hair, tissue, semen
Clean DNA


DNA found at a crime scene usu. dirty
Must be clean before analyzed
A piece of DNA
Southern Blot
January 28, 2003
JJ-11
Agenda Revisited
Collect the DNA
Isolate the DNA
Cut DNA
 Variable Number Tandem Repeats (VNTRs)
Sort DNA through Gel Electrophoresis
Transfer DNA to a solid support

Methods of Transferring
The Hybridization Reaction
 Denatured and Nicked DNA
 Radioactive Probe
 Continuation of the Hybridization Reaction
Comparing DNA fingerprints
Southern Blot
January 28, 2003
JJ-12
Isolate the DNA
Isolate the DNA
Isolate DNA from the rest of
cellular material in nucleus. Done
chemically or mechanically.
Chemically

Use detergent to wash extra material
from DNA
Mechanically

Southern Blot
Apply large amounts of pressure to
“squeeze” out DNA
January 28, 2003
JJ-14
Agenda Revisited
Collect the DNA
Isolate the DNA
Cut DNA
 Variable Number Tandem Repeats (VNTRs)
Sort DNA through Gel Electrophoresis
Transfer DNA to a solid support

Methods of transferring
The Hybridization Reaction
 Denatured and Nicked DNA
 Radioactive Probe
 Continuation of the Hybridization Reaction
Comparing DNA fingerprints
Southern Blot
January 28, 2003
JJ-15
Cut the DNA
Cut DNA
Cut large genome into
shorter DNA fragments
with restriction
enzymes.

Southern Blot
Enzymes will recognize
four to six specific base
sequences and cleave
the DNA at these specific
boundaries
January 28, 2003
JJ-17
Variable Number Tandem Repeats
(VNTRs)
Variable Number Tandem Repeats-repeated sequences of base
pairs found at the introns (the “useless” part of of the DNA
strand).
VNTRs contain from 20-100 base pairs.
An example of VNTRs
Southern Blot
January 28, 2003
JJ-18
VNTRs cont.
Every human has unique VNTR sequence
(because VNTRs are inherited genetically).
They may be used in the production of a DNA
Fingerprint

Southern Blot
The VNTRs must go through: Southern Blotting, probing,
and a hybridization reaction in order to result in a DNA
fingerprint.
January 28, 2003
JJ-19
Agenda Revisited
Collect the DNA
Isolate the DNA
Cut DNA
 Variable Number Tandem Repeats (VNTRs)
Sort DNA through Gel Electrophoresis
Transfer DNA to a solid support

Methods of Transferring
The Hybridization Reaction
 Denatured and Nicked DNA
 Radioactive Probe
 Continuation of the Hybridization Reaction
Comparing DNA fingerprints
Southern Blot
January 28, 2003
JJ-20
Sort the DNA
Sort DNA Through Gel
Electrophoresis
Gel Electrophoresis separates DNA molecules by size NOT by
molecular weight.

Prior to process, must first:



Prepare slab of gel material cast
Set gel up for electrophoresis by having electrodes apply an electric
field.
DNA is slightly negative (REMEMBER!!!)
Slab of agarose
Southern Blot
January 28, 2003
JJ-22
Sorting DNA Through Gel
Electrophoresis (Cont’d)
The DNA molecules will then
be separated by size
In the gel agarose:


Southern Blot
Negative (-) electrode is on left
side, positive (+) electrode on
right side
Since DNA molecules have a (-)
charge (you already memorized
that), they will want to move from
left to right.
January 28, 2003
JJ-23
Sorting DNA Through Gel
Electrophoresis (Cont’d)


Gel has pores restraining
larger molecules from
moving all the way to the
right side
Hence, smaller DNA
molecules will flow through
quickly, this separates the
molecules by SIZE
DNA molecules moving
through agarose.
Southern Blot
January 28, 2003
JJ-24
Agenda Revisited
Collect the DNA
Isolate the DNA
Cut DNA
 Variable Number Tandem Repeats (VNTRs)
Sort DNA through Gel Electrophoresis
Transfer DNA to a solid support
 Methods of Transferring
The Hybridization Reaction
 Denatured and Nicked DNA
 Radioactive Probe
 Continuation of the Hybridization Reaction
Comparing DNA fingerprints
Southern Blot
January 28, 2003
JJ-25
Transfer the DNA
Transferring DNA Onto a
Solid Support
DNA is sorted into single strands either by heating or
chemical treatment in gel.
After DNA molecules are separated by size, the
protein must be transferred onto some solid support
in preparation for hybridization. This process is called
blotting.
Southern Blot
January 28, 2003
JJ-27
Method of Transferring
DNA must be transferred onto a SOLID support.
A commonly used solid support is nitrocellulose
paper (filter paper).
Southern Blot
January 28, 2003
JJ-28
Electrophoresis –
Capillary Blotting
The transferring process
usually goes via
electrophoresis or capillary
blotting


Southern Blot
Electrophoresis is the transfer
separation of molecules by size
Capillary blotting is the process
in which the molecules are
transferred in a flow of buffer
from wet filter paper to dry filter
paper.
January 28, 2003
Equipment used in
Gel electrophoresis
JJ-29
Agenda Revisited
Collect the DNA
Isolate the DNA
Cut DNA
 Variable Number Tandem Repeats (VNTRs)
Sort DNA through Gel Electrophoresis
Transfer DNA to a solid support
 Methods of transferring
The Hybridization Reaction


Denatured and Nicked DNA
Radioactive Probe
 Continuation of the
Comparing DNA fingerprints
Southern Blot
Hybridization Reaction
January 28, 2003
JJ-30
The Hybridization
Reaction
The Hybridization Reaction
Hybridization reaction-the binding of
two genetic sequences, specifically
the denatured and Nicked DNA and
the radioactive probe.
Binding occurs between A and T
and C and G through Hydrogen
bonds. There are two hydrogen
bonds between A and T and three
H-Bonds between C and G.
•HOW am I supposed to
•Explain this thing???
Southern Blot
January 28, 2003
JJ-32
Denatured and Nicked DNA
However first DNA must be denatured. To denature
DNA, the existing H-Bonds must first be broken
through chemical processes or heating. This leaves a
single strand of DNA whose bases are available for
hydrogen bonding
Nicked DNA-DNA that has been cut in certain areas
for further use.
Southern Blot
January 28, 2003
JJ-33
Radioactive Probe
Creation
How a radioactive probe is created
The nicked DNA strand is essentially repaired by the DNA polymerase, and
at the same time, making it radioactive by including the C* bases.
The nicked DNA is then heated and split apart resulting in single stranded
radioactive and non-radioactive pieces. The radioactive DNA piece is called
the probe.
Probe
Southern Blot
January 28, 2003
JJ-34
The Hybridization Rxn
continued
The single stranded radioactive probe can be used to
see if the denatured DNA contains a sequence
similar to that on the probe.
Southern Blot
January 28, 2003
JJ-35
Hybridization Rxn cont.
If a positive match does comes up and the DNA
probe contains a sequence similar to that of the
denatured DNA, the two will form H-Bonds and bind.


Southern Blot
Although if the fit between the two sequences is poor, there
will be fewer H-Bonds.
The ability for low-homology probes to still bind to DNA
sequences may be altered through varying amounts of
saline solution or varying temperatures.
January 28, 2003
JJ-36
Hybridization Rxn cont.
Obtain some DNA polymerase, place radiolabeled
DNA into a tube
Make horizontal breaks along a strand of DNA to be
radiolabeled. While doing this, add individual
nucleotides to the nicked DNA.
Southern Blot
January 28, 2003
JJ-37
Hybridization Rxn cont.
Add DNA polymerase into tube (which now contains
nicked DNA ready to be radiolabeled).
Once DNA polymerase is added, it will immediately be
attracted to the nicks in the DNA and attempt to “repair” the
DNA. In doing so, it will destroy all existing bonds in front of it
and will place the new nucleotides (added earlier) behind it.
Every G base will bond with a C* base.
Southern Blot
January 28, 2003
JJ-38
Hybridization Rxn cont.
Locate a specific VNTR sequence on a single
stranded DNA fragment
Make a DNA probe out of DNA sequence
Labeling probe with radioactive compound
Letting probe bind to like DNA sequences on
membrane
Use radioactive tag to find where probe has attached
Southern Blot
January 28, 2003
JJ-39
Agenda Revisited
Collect the DNA
Isolate the DNA
Cut DNA
 Variable Number Tandem Repeats (VNTRs)
Sort DNA through Gel Electrophoresis
Transfer DNA to a solid support

Methods of Transferring
The Hybridization Reaction
 Denatured and Nicked DNA
 Radioactive Probe
 Continuation of the Hybridization Reaction
Comparing DNA fingerprints
Southern Blot
January 28, 2003
JJ-40
Compare DNA Profile
Visualize Banding by
Exposure X-ray Film
Take a picture of probe stuck to its target on the
membrane using specialized X-ray film


Southern Blot
Place membrane on the special sheet of film for a short
period of time
And you have a picture!
January 28, 2003
JJ-42
Thank You
Thank you for listening to my
presentation!
I hope you now have clear
understanding as to how to make a
DNA profile!
Southern Blot
January 28, 2003
JJ-43
Bibliography
http://merriam-webster.com/cgi-bin/dictionary
http://www.howstuffworks.com/dna-evidence.htm
http://www.botany.uwc.ac.za/mirrors/MIT-bio/bio/rdna/rdnadir.html
http://www.biology.washington.edu/fingerprint/dnaintro.html
www.accessexcellence.org/AB/GG/restriction.html
www-hhmi.princeton.edu/grp2/size of dna molecule.htm
www.frontiernet.net/~plasmid/pictures/ansvr.jpg
www.biology.washington.edu/ fingerprint/radio01.gif
esg-www.mit.edu:8001/esgbio/ rdna/probe.gif
www.hsa.gov.sg/hsa/Images/ cfs/dna_profiling2.jpg
www.labcorp.com/paternity/ body_index.html
criminaljustice.state.ny.us/ ops/history/bert_1.jpg
www.sun.ac.za/kie/unistel/medical_labs/ paternity3.htm
www.majintl.com/dogtag.htm
Southern Blot
January 28, 2003
JJ-44