Southern Blot By: Jacqueline Jai. JJ-2 Southern BlotJanuary 28, 2003 Southern Blot Southern Blot-a piece nitrocellulose paper containing spots of DNA.

Southern Southern BlotBlot

By: Jacqueline Jai

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

Interesting Facts about DNA Analysis


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


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.


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


Paternity TestingEvidence can be used to compare the DNA of

the suspected parent(s) and that of the child and determine the real parent.

DNA Profile


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

Collect the DNA


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


Agenda Revisited

Collect the DNA

Isolate the DNACut DNA


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

Isolate the DNA


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


Agenda Revisited

Collect the DNA

Isolate the DNA

Cut DNA Variable Number Tandem Repeats (VNTRs)


Transfer DNA to a solid support Methods of transferring




Cut the DNA


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



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


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.


Agenda Revisited

Collect the DNA

Isolate the DNA


Sort DNA through Gel ElectrophoresisTransfer DNA to a solid support

Methods of Transferring




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


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.


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.


Agenda Revisited

Collect the DNA

Isolate the DNA







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.


Method of Transferring

DNA must be transferred onto a SOLID support.

A commonly used solid support is nitrocellulose paper (filter paper).


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


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


Continuation of the Hybridization ReactionComparing DNA fingerprints

The Hybridization Reaction


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???


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.


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


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.


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.


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.


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.


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


Agenda Revisited

Collect the DNA

Isolate the DNA







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 Place membrane on the special sheet of film for a short

period of time And you have a picture!


Thank You

Thank you for listening to my presentation!

I hope you now have clear understanding as to how to make a DNA profile!


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

Southern Blot By: Jacqueline Jai. JJ-2 Southern BlotJanuary 28, 2003 Southern Blot Southern Blot-a piece nitrocellulose paper containing spots of DNA.

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