Basic Principles and Applications of Electrophoresis Stephen K.W. Tsui Department of Biochemistry

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Theory of Electrophoresis The movement of a charged molecule subjected to an electric field is represented by the following equation: V = Eq f V: the velocity of the molecule E: the electric field in volts/cm q: the net charge on the molecule f: frictional coefficient, which depend on the mass and shape of the molecule

Applications of Gel Electrophoresis Southern blot is produced when DNA on a nitrocellulose blot is hybridized with a DNA probe. Northern blots are generated when RNA is hybridized with a complementary DNA probe produced by the reverse transcription of messenger RNA. A slightly different but related technique, known as a Western blot, involves separating proteins by gel electrophoresis and probing with labeled antibodies for specific proteins.

Blotting Techniques Southern Blot Northern Blot Western Macromolecules on the blot DNA RNA Protein Probe Labeled DNA Labeled antibodies Source of labels Radioactively or fluorescent labeled deoxynucleotide Radioactively or fluorescent labeled amino acids

Tissue distribution of Messenger RNA Revealed by Northern Blot 1: heart 2: brain 3: placenta 4: lung 5: liver 6: skeletal muscle 7: kidney 8: pancreas 9 : spleen 10: thymus 11: prostate 12: testis 13: ovary 14: small intestine 15: colon 16: leukocyte 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Coomassie Blue Dye Stained protein gel A Protein can be Specifically Recognized by an Antibody in a Western Blot M 1 2 3 4 M 1 2 3 4 Coomassie Blue Dye Stained protein gel Western blot

Gel Electrophoresis of DNA Agarose slab gel submerged in buffer Wells for sample loading Cathode (-) Direction for DNA migration Anode (+)   Agarose is a polysaccharide derived from seaweed, which forms a solid gel when dissolved in aqueous solution. When an electric field is applied to an agarose gel in the presence of a buffer solution which will conduct electricity, DNA fragments move through the gel towards the positive electrode (DNA is highly negatively charged) at a rate which is dependent on its size and shape.

Gel Electrophoresis of DNA For linear DNA molecules, they have uniform shape and charge to mass ratio. The electrophoretic mobility of the DNA molecule is influenced primarily by the molecular size: The larger molecules are retarded by the molecular sieving effect of the gel, and the small molecules have greater mobility.

Gel Electrophoresis of DNA The DNA can be stained by the inclusion of ethidium bromide in the gel, or by soaking the gel in a solution of ethidium bromide after electrophoresis. The DNA shows up as an orange band on illumination by UV light. Alternatively, methylene blue can be used to stain DNA. Gels composed of polyacrylamide can separate DNA molecules that differ in length by only one nucleotide and are used to determine the base sequence of DNA. Agarose gels are used to separate DNA fragments that have larger size differences.

Digestion of DNA by Restriction Enzymes GAATTC CTTAAG EcoRI G AATTC CTTAA G Before the electrophoresis, DNA is digested by restriction enzymes into small fragments of DNA.

Procedures of DNA Fingerprinting http://www.bios.co.uk/illustrations/1859962025/05_12t.gif

Procedures of DNA Fingerprinting In order to detect specific sequences, DNA is usually transferred to a solid support, such as a sheet of nitrocellulose or nylon paper. The paper is treated with an alkaline solution to denature DNA, that is, separate the two strands of each double helix. The single-stranded DNA can be hybridized with a probe, and the regions on the nitrocellulose blot containing DNA that base-pairs with the probe can be identified.

DNA Polymorphisms Polymorphisms are variations in DNA sequences. There may be millions of different polymorphisms in the human DNA. Polymorphisms in the human DNA serve as the basis for the diagnosis of diseases and the identity of individuals.

Detection of Polymorphism Restriction Fragment Length Polymorphisms Occasionally, a point mutation occurs in a recognition site for a restriction enzyme. The enzyme, therefore, can cut at other recognition sites but not at the site of the mutation. Consequently, the restriction fragment produced by the enzyme is larger for a person with the mutation than for a normal person. Mutations can also create restriction sites that are not present in the normal gene. In this case, restriction fragments will be smaller for the person with the mutation than for the normal individual. These variations in the length of restriction fragments are known as restriction fragment length polymorphisms (RFLPs).

Application of DNA Fingerprinting Mutation Detection http://www.bios.co.uk/illustrations/1859962025//05_14f.gif

Highly Variable Regions Human DNA contains many sequences that are repeated in tandem a variable number of times at certain loci in the genome. These regions are called hypervariable regions because they contain a variable number of tandem repeats (VNTR).

Detection of Highly Variable Regions Digestion with restriction enzymes that recognize sites which flank the VNTR region produces fragments containing these loci, which differ in size from one individual to another, depending on the number of repeats that are present. Probes used to identify these restriction fragments bind to or near the sequence that is repeated.

Application of DNA Fingerprinting Forensic Analysis This restriction fragment technique has been called "DNA fingerprinting" and is gaining widespread use in forensic analysis. Family relationships can be determined by this method, and it can be used to convict suspects in criminal cases. Individuals who are closely related genetically will have restriction fragment pattern that are more similar than those who are more distantly related.

Other Applications of DNA Fingerprinting Parentage test Endangered species or Chinese herbs identification

Animation 1: Southern Blotting http://www.dnalc.org/resources/BiologyAnimationLibrary.htm

Animation 2: DNA Detective http://www.dnalc.org/resources/BiologyAnimationLibrary.htm

Online Courses: DNA from the Beginning http://www.dnaftb.org/dnaftb/

Download Illustrations: Human Molecular Genetics http://www.bios.co.uk/illustrations.asp

Good Website: Gel Electrophoresis http://dlab.reed.edu/projects/vgm/vgm/VGMProjectFolder/VGM/RED/RED.ISG/gel.html

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Agarose Gel Electrophoresis Workshop Agarose Gel Electrophoresis Department of Biochemistry (2001-2002)

Properties of DNA- Double helix Building block(dA, dC, dG and dT) negatively charged at neutral pH AT and GC complementary pairing Restriction enzymes - enzymes isolated from bacteria that cut DNA at specific sites(restriction sites) EcoRI - 5'- G A A T T C -3‘ 3'- C T T A A G -5'

Baterial plasmid DNA Plasmids are molecules of DNA that are found in bacteria separate from the bacterial chromosome. They: are small (a few thousand base pairs) usually carry only one or a few genes are circular have a single origin of replication

Plasmid DNA for digestion – pBluescript II SK+ + BglI 2166 BglI 472 EcoRI 701

Agarose A linear polymer extracted from seaweed Migration of DNA in agarose dependent on four factors - molecular size of the DNA - agarose concentration - conformation of the DNA - applied current

Cathode(-) wells DNA fragments of different sizes 1.5% agarose gel stained with methylene blue Anode(+)

Preparation of plasmid DNA http://dlab.reed.edu/projects/vgm/vgm/VGMProjectFolder/VGM/RED/RED.ISG/gel.html http://dlab.reed.edu/projects/vgm/vgm/VGMProjectFolder/VGM/RED/RED.ISG/gel.html Restriction enzyme digestion Agarose gel casting DNA sample loading electrophoresis Methylene blue staining

Agarose gel electrophoresis unit Electrophoresis tank Plugs and wire Gel casting unit and comb

Agarose gel casting unit Step 4 Comb Tape Gel casting unit Seal both ends of the gel casting unit with tape

Preparation of 1.5% agarose gel Step 5 http://dlab.reed.edu/projects/vgm/vgm/VGMProjectFolder/VGM/RED/RED.ISG/gel.html

Electrophoresis(5V/cm) Step 10 Sample loading, wash syringe with 1X TBE buffer between successive loading http://dlab.reed.edu/projects/vgm/vgm/VGMProjectFolder/VGM/RED/RED.ISG/gel.html Electrophoresis(5V/cm)

Wells Tracking dye Xylene cyanol FF Bromophenol blue

Methylene blue staining to visualize the DNA fragments, stain agarose gel overnight with 1X methylene blue staining solution safe alternative for DNA staining easy available non-carcinogenic DNA fragments

DNA fragments of different sizes DNA fragments of known sizes A B C D M Base pairs distance migrated(mm) 1808 23 1634 25 656 38 316 47

Calibration curve for DNA size determination http://www.pangloss.com/seidel/Protocols/webmap.html

Size determination of the candidate DNA fragments http://www.pangloss.com/seidel/Protocols/webmap.html