Southern Blot (DNA) 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 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 (DNA)

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

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.

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

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.

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

Collect DNA Collect DNA sample Clean DNA Blood, hair, tissue, semen DNA found at a crime scene usu. dirty Must be clean before analyzed A piece of DNA

Agenda Revisited Isolate the DNA Cut DNA 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

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

Agenda Revisited Cut DNA Variable Number Tandem Repeats (VNTRs) 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

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 (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

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 Sort DNA through Gel Electrophoresis 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

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 Transfer DNA to a solid support 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

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 in Gel electrophoresis

Agenda Revisited The Hybridization Reaction 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

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.

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). 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.

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

Agenda Revisited Comparing DNA fingerprints 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

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!

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.

To what extent is the gene expressed? NORTHERN BLOT RNA DNA ... Southern blot

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.

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.

Western Analysis Laboratory procedure that allows you to: 1. Verify the expression of a protein 2. Determine the relative amount of a protein present in different samples 3. Analyze protein-protein interactions

Two Main Types of Westerns 1. Denaturing (Most Commonly Used) - SDS-PAGE 2. Non-Denaturing - Native PAGE

- + SDS-PAGE Western Blot Method - - - - - - - - Cell Lysis by Detergents and Sonication Cells in Culture Cell Removal Human Cells Containing Protein - SDS or LDS - - Heat Denaturation of Proteins - Detergents Bind Proteins - - - Load Proteins on Gel + - - Proteins Separate by Size Apply Electric Current

- - 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 is a Rabbit Anti-Human b-Actin Antibody 1o Antibody Binds Antigen (i.e. Protein of Interest) Non-Specific Proteins Bind to Unbound Regions of Membrane

- - - Add HRP-Conjugated 2o Antibody HRP Luminol Light Detected by Film 2o Antibody is a Goat Anti-Rabbit-HRP-Conjugated Antibody Add Chemiluminescent Substrate

Magic Mark XP Western Protein Standard kDa 60 50 45 b-Actin 40 30 20 Invitrogen.com

Western Blot (Protein)

RNA PROTEINS WESTERN BLOTTING DNA