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• A Southern blot is a method used in molecular biology for detection of a specific DNA sequence in DNA samples.

• Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization.

Southern Blot (DNA)

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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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Southern Blot (DNA)

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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 filedby 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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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 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• 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 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 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 (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.

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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.

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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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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 inGel 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• 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.

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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).

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.

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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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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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northern blot

• The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample.

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To what extent is the gene expressed?N

ORT

HER

N B

LOT

RNA

DNA ... Southern blot

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Summary

Southern• DNA on membrane.• Digest DNA.• Convert dsDNA to ssDNA.• Probe with DNA or RNA.

Northern• RNA on membrane.• No need to digest DNA.• Denature “folded” RNA

with formaldehyde.• Probe with DNA or

RNA.

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western blot

• The western blot (sometimes called the protein immunoblot) is a widely used analytical technique used to detect specific proteins in a sample of tissue homogenate or extract.

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Laboratory procedure that allows you to:

Western Analysis

1. Verify the expression of a protein

2. Determine the relative amount of a protein present in different samples

3. Analyze protein-protein interactions

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Two Main Types of Westerns

1. Denaturing (Most Commonly Used)

2. Non-Denaturing

- SDS-PAGE

- Native PAGE

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SDS-PAGE Western Blot Method

-

--

-

-

Cells in Culture

Cell Removal

Human Cells Containing Protein

Cell Lysis by Detergents and

Sonication

Heat Denaturation of

Proteins

+

-Load Proteins on Gel

SDS or LDS

Apply Electric Current

- - -

- -

Proteins Separate by Size

Detergents Bind Proteins

--

-

--- - -

--

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- - -

- - Transfer or Blot Protein from Gel to

Nitrocellulose and/or PVDF Membrane

- - -

- -

Block Membrane with Non-

Specific Proteins

- - -

- -

- - -

- -

Incubate Membrane with 1o Antibody

1o Antibody Binds Antigen (i.e. Protein of Interest)

Non-Specific Proteins Bind to Unbound Regions of

Membrane

1o Antibody is a Rabbit Anti-Human b-Actin

Antibody

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- - -

- -

- - -

- -

Add HRP-Conjugated 2o Antibody

- -

HRP

Luminol Light

Detected by Film

Add Chemiluminescent

Substrate

2o Antibody is a Goat Anti-Rabbit-HRP-

Conjugated Antibody

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b-Actin

Magic Mark XP Western Protein

Standard

2030

405060

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kDa

Invitrogen.com

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Western Blot (Protein)

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RNA

DNA

PROTEINS

WESTERN BLOTTING

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