Southern Blot - PowerPoint PPT Presentation

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Southern Blot

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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. – PowerPoint PPT presentation

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Title: Southern Blot


1
Southern Blot
  • By Jacqueline Jai

2
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

3
Interesting Facts about DNA Analysis
4
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
5
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.

6
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
7
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.

8
DNA Profile
9
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
10
Collect the DNA
11
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
12
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

13
Isolate the DNA
14
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

15
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

16
Cut the DNA
17
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

18
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
19
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.

20
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

21
Sort the DNA
22
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
23
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.

24
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.
25
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

26
Transfer the DNA
27
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.

28
Method of Transferring
  • DNA must be transferred onto a SOLID support.
  • A commonly used solid support is nitrocellulose
    paper (filter paper).

29
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
30
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

31
The Hybridization Reaction
32
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???

33
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.

34
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
35
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.

36
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.

37
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.

38
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.

39
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

40
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

41
Compare DNA Profile
42
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!

43
Thank You
  • Thank you for listening to my presentation!
  • I hope you now have clear understanding as to
    how to make a DNA profile!

44
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
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