Southern Blot By: Jacqueline Jai. JJ-2 Southern BlotJanuary 28, 2003 Southern Blot Southern Blot-a...
-
Upload
chrystal-chambers -
Category
Documents
-
view
218 -
download
0
Transcript of Southern Blot By: Jacqueline Jai. JJ-2 Southern BlotJanuary 28, 2003 Southern Blot Southern Blot-a...
Southern Blot January 28, 2003 JJ-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
Southern Blot January 28, 2003 JJ-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 TestingFirst criminal identification card filedby the NY State Bertillon Bureau
Southern Blot January 28, 2003 JJ-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.
Southern Blot January 28, 2003 JJ-6
IdentificationUsed 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-7
Paternity TestingEvidence can be used to compare the DNA of
the suspected parent(s) and that of the child and determine the real parent.
Southern Blot January 28, 2003 JJ-9
The Basics to Creating a DNA Profile (Agenda)
Collect the DNAIsolate the DNACut DNA
Variable Number Tandem Repeats (VNTRs)
Sort DNA through Gel ElectrophoresisTransfer DNA to a solid support
Methods of TranferringThe Hybridization Reaction
Denatured and Nicked DNA Radioactive Probe
Continuation of the Hybridization ReactionComparing DNA fingerprints
A DNA Profile
Southern Blot January 28, 2003 JJ-11
Collect DNACollect 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-12
Agenda Revisited
Collect the DNA
Isolate the DNACut 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-14
Isolate the DNAIsolate 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
Southern Blot January 28, 2003 JJ-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
Southern Blot January 28, 2003 JJ-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
Southern Blot January 28, 2003 JJ-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
Southern Blot January 28, 2003 JJ-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.
Southern Blot January 28, 2003 JJ-20
Agenda Revisited
Collect the DNA
Isolate the DNA
Cut DNA Variable Number Tandem Repeats (VNTRs)
Sort DNA through Gel ElectrophoresisTransfer 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-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
Southern Blot January 28, 2003 JJ-23
Sorting DNA Through Gel Electrophoresis (Cont’d)
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.
Southern Blot January 28, 2003 JJ-24
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-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
Southern Blot January 28, 2003 JJ-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.
Southern Blot January 28, 2003 JJ-28
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-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 inGel electrophoresis
Southern Blot January 28, 2003 JJ-30
Agenda RevisitedCollect the DNAIsolate the DNACut DNA
Variable Number Tandem Repeats (VNTRs)Sort DNA through Gel ElectrophoresisTransfer DNA to a solid support
Methods of transferring
The Hybridization Reaction Denatured and Nicked DNA Radioactive Probe
Continuation of the Hybridization ReactionComparing DNA fingerprints
Southern Blot January 28, 2003 JJ-32
The Hybridization ReactionHybridization 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-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.
Southern Blot January 28, 2003 JJ-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
Southern Blot January 28, 2003 JJ-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.
Southern Blot January 28, 2003 JJ-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.
Southern Blot January 28, 2003 JJ-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.
Southern Blot January 28, 2003 JJ-38
Hybridization Rxn cont.Add DNA polymerase into tube (which now contains nicked DNA ready to be radiolabeled).
Every G base will bond with a C* base.
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.
Southern Blot January 28, 2003 JJ-39
Hybridization Rxn cont.
Locate a specific VNTR sequence on a single stranded DNA fragmentMake a DNA probe out of DNA sequence Labeling probe with radioactive compoundLetting probe bind to like DNA sequences on membraneUse radioactive tag to find where probe has attached
Southern Blot January 28, 2003 JJ-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
Southern Blot January 28, 2003 JJ-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!
Southern Blot January 28, 2003 JJ-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!
Southern Blot January 28, 2003 JJ-44
Bibliographyhttp://merriam-webster.com/cgi-bin/dictionaryhttp://www.howstuffworks.com/dna-evidence.htmhttp://www.botany.uwc.ac.za/mirrors/MIT-bio/bio/rdna/rdnadir.htmlhttp://www.biology.washington.edu/fingerprint/dnaintro.htmlwww.accessexcellence.org/AB/GG/restriction.htmlwww-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