Southern Blot

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Southern Blot

• By Jacqueline Jai

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Southern Blot

• Southern Blot-a piece nitrocellulose paper
  containing spots of DNA ready for identification
  by a suitable molecular probe.
• Southern Blot is a copy of DNA profile

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Interesting Facts about DNA Analysis
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DNA Evidence

• DNA evidence-has many uses within the legal
  system and criminal cases.
• Proving someone guilty or innocent for a crime
  they have or have not committed.
• Identification
• Paternity Testing

First criminal identification card filed by the
NY State Bertillon Bureau
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Criminal Cases

• 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.

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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
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Paternity Testing

• Evidence can be used to compare the DNA of the
  suspected parent(s) and that of the child and
  determine the real parent.

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DNA Profile
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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
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Collect the DNA
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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
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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

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Isolate the DNA
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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
• Apply large amounts of pressure to squeeze out
  DNA

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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

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Cut the DNA
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Cut DNA

• Cut large genome into shorter DNA fragments with
  restriction enzymes.
• Enzymes will recognize four to six specific base
  sequences and cleave the DNA at these specific
  boundaries

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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
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VNTRs cont.

• Every human has unique VNTR sequence (because
  VNTRs are inherited genetically).
• They may be used in the production of a DNA
  Fingerprint
• The VNTRs must go through Southern Blotting,
  probing, and a hybridization reaction in order to
  result in a DNA fingerprint.

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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

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Sort the DNA
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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
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Sorting DNA Through Gel Electrophoresis (Contd)

• The DNA molecules will then be separated by size
• In the gel agarose
• 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.

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Sorting DNA Through Gel Electrophoresis (Contd)

• 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.
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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

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Transfer the DNA
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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.

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Method of Transferring

• DNA must be transferred onto a SOLID support.
• A commonly used solid support is nitrocellulose
  paper (filter paper).

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Electrophoresis Capillary Blotting

• The transferring process usually goes via
  electrophoresis or capillary blotting
• 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.

Equipment used in Gel electrophoresis
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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

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The Hybridization Reaction
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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???

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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.

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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
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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.

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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.
• 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.

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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.

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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.

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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

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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

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Compare DNA Profile
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Visualize Banding by Exposure X-ray Film

• Take a picture of probe stuck to its target on
  the membrane using specialized X-ray film
• Place membrane on the special sheet of film for a
  short period of time
• And you have a picture!

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Thank You

• Thank you for listening to my presentation!
• I hope you now have clear understanding as to
  how to make a DNA profile!

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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/rd
  na/rdnadir.html
• http//www.biology.washington.edu/fingerprint/dnai
  ntro.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.gi
  f
• esg-www.mit.edu8001/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.jp
  g
• www.sun.ac.za/kie/unistel/medical_labs/
  paternity3.htm
• www.majintl.com/dogtag.htm