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.

Southern Blot is a copy of DNA profile

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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 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

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

Collect DNA
Collect DNA sample

Blood, hair, tissue, semen DNA found at a crime scene usu. dirty Must be clean before analyzed

Clean DNA

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

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 Apply large amounts of pressure to squeeze out DNA

Mechanically

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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

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

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

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 Comparing DNA fingerprints

Hybridization Reaction

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The Hybridization Reaction

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

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/rdna/rdnadir.html http://www.biology.washington.edu/fingerprint/dnaintro.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.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

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