Download Southern Blots

Hybridization And
Southern Blots
Timothy G. Standish, Ph. D.
©2000 Timothy G. Standish
Hybridization
The bases in DNA will only pair in very specific ways: G
with C and A with T
In short DNA sequences, imprecise base pairing will not
be tolerated
Long sequences can tolerate some mispairing only if
hydrogen bonding of the majority of bases in a sequence
exceeds the energy required to overcome mispaired bases
The source of any single strand of DNA is irrelevant,
merely the sequence is important, thus complimentary
DNA from different sources can form a double helix
This phenomenon of base pairing of single stranded DNA
strands to form a double helix is called hybridization as it
may be used to make hybrid DNA composed of strands
from different sources
©2000 Timothy G. Standish
Hybridization
DNA from source “X”
CTGATGGTCATGAGCTGTCCGATCGATCAT
TACTCGACAGGCTAG
Hybridization
TACTCGACAGGCTAG
DNA from source “Y”
©2000 Timothy G. Standish
Hybridization
Because DNA sequences will seek out and hybridize with
other sequences with which they base pair in a specific
way much information can be gained about unknown DNA
using single stranded DNA of known sequence
Short sequences of single stranded DNA can be used as
“probes” to detect the presence of their complimentary
sequence in any number of applications including:
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–
–
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Southern blots
Northern blots (in which RNA is probed)
In situ hybridization
Dot blots . . .
In addition, the renaturation, or hybridization, of DNA in
solution can tell much about the nature of organism’s
genomes
©2000 Timothy G. Standish
Library Screening
The most common method of library screening
involves hybridization of probes to target DNA
Hybridization refers to the specific way DNA
sequences base pair with their exact compliment
Probes - Single stranded nucleic acids used to
hybridize with a target DNA. Generally probes
are radioactive or marked in some other way so
that they can easily be identified after binding to
target DNA
To design probes for hybridization screening,
something must be known in advance about the
target sequence
©2000 Timothy G. Standish
Hybridization Screening
Takes advantage of the fact that
complimentary strands of DNA can
recognize one another
By sticking DNA from many colonies or
plaques in a library to a membrane
Making the DNA single stranded
Then hybridizing a probe to the DNA on the
membrane thus marking target DNA on the
membrane, colonies or plaques containing
the target DNA can be identified
©2000 Timothy G. Standish
Hybridization
Screening
Transfer cells to
Membrane
membrane
Lyse cells - DNA
and protein stick
to membrane
Locate
colony with
target clone
Block membrane Prevents probe
from sticking to
membrane
Develop
X-ray
film
Add probe
Cover with
X-ray film
Wash off
excess probe
©2000 Timothy G. Standish
Southern Blots
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2
3
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5
Called Southern blots after their inventor
Involve four steps:
Digestion of DNA using restriction enzymes
Separation of the DNA fragments by size using
gel electrophoresis
Transfer of fragments to a nitrocellulose or nylon
membrane
Hybridization of a probe to the fragment or
fragments of interest
Probe detection (autorad development)
©2000 Timothy G. Standish
Making A Southern Blot 1 + 2
Digestion and Electrophoresis
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Marker
2
Control
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2
3
Experimental
©2000 Timothy G. Standish
Making A Southern Blot 3
DNA Transfer To Membrane
DNA
Paper Towels
Gel
Membrane
Gel
Membrane
Buffer
©2000 Timothy G. Standish
Making A Southern Blot 4
Probe Hybridization
Addition of
blocking reagent
Probe addition
After washing
Membrane with
bound DNA
Parts of the
membrane not
already covered
with DNA now
bind blocking
reagent
Probe covers the
membrane, but
only binds to
complimentary
DNA
Probe only remains
annealed to
complimentary
DNA
©2000 Timothy G. Standish
Making A Southern Blot 5
Autorad Development
Membrane with
probe bound to
complimentary DNA
X-ray film is placed
over the membrane
and left until radiation
from the probe has
exposed the film
Fragments
complimentary to the
probe appear as bands
on the autorad
©2000 Timothy G. Standish
So What is the Big Deal?
Southern blots tell both the size and something about the
sequence of a fragment mixed in with many other
fragments, thus they can be used for many purposes
RFLPs - Restriction Fragment Length Polymorphisms When mutations change the size of specific sequences in
the genome, they may be identified by a change in the size
of a RE digested fragment identified using a Southern blot
RFLPs - Can be correlated to specific genetic defects thus
allowing diagnosis of genetic disease
RFLPS - Are used as genetic markers in mapping genes
Some parts of DNA called Variable Number Tandem
Repeats (VNTRs) are highly polymorphic (variable) in
length and can be used for genetic fingerprinting
©2000 Timothy G. Standish
Using DNA Fingerprinting
A murder victim is found to
have human tissue under his
fingernails having, during the
course of struggle, managed to
scratch his murderer
Two suspects have been
identified as having a motive
and suspicious scratches on
their faces:
Suspect 1 - The victims wife
Suspect 2 - The wife’s
boyfriend
Victim
Under Suspect Suspect
Nails
1
2
A VNTR Probe was used to
make this DNA fingerprint
©2000 Timothy G. Standish
Does This Prove The Boyfriend Did It?
Not exactly
DNA fingerprinting is very good for elimination of
suspects. Thus the wife’s tissue was not under her
husbands fingernails
That the tissue under the husbands fingernails was
the murderer’s is an assumption
There are other plausible scenarios including - The
husband and boyfriend were fighting, the wife came
home and shot her husband to protect her boyfriend
The boyfriend’s DNA matches tissue under the
victims nails, while this eliminates millions of other
people it does not prove it is actually his tissue
©2000 Timothy G. Standish
DNA Fingerprinting Can Show
Relationships
During the 1970s and 80s
a “dirty war” was fought
in Chile directed by
General Pinochet
Many people disappeared
and their children were
adopted by innocent
people
These children have been
reunited with their
grandparents using DNA
fingerprinting
Paternal
Grandfather
Paternal
Grandmother
Maternal
Maternal
Grandfather Grandmother
Father
Mother
Child
©2000 Timothy G. Standish
©2000 Timothy G. Standish