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Basic Principles and Applications of Electrophoresis

Basic Principles and Applications of Electrophoresis. Stephen K.W. Tsui Department of Biochemistry. Order Form for Electrophoresis Kits and Reagents. Please click this sentence to download the form. Eq. V. =. f. Theory of Electrophoresis.

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Basic Principles and Applications of Electrophoresis

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

  2. Order Form for Electrophoresis Kits and Reagents Please click this sentence to download the form

  3. Eq V = f Theory of Electrophoresis The movement of a charged molecule subjected to an electric field is represented by the following equation: 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

  4. Applications of Gel Electrophoresis • Southern blot is produced when DNA on a nitrocellulose blot ishybridized with a DNA probe. • Northern blots are generated when RNA is hybridized with a complementary DNA probeproduced 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.

  5. Blotting Techniques

  6. 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

  7. 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

  8. Direction for DNA migration Wells for sample loading Anode (+) Agarose slab gel submerged in buffer Cathode (-) Gel Electrophoresis of DNA 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.

  9. Gel Electrophoresis of DNA For linear DNA molecules, they have uniform shape and charge to mass ratio. The electrophoreticmobility 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.

  10. 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.

  11. Procedures of DNA Fingerprinting

  12. 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.

  13. 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.

  14. 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).

  15. Application of DNA Fingerprinting Mutation Detection

  16. 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).

  17. 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.

  18. 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.

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

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

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

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

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

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

  25. The End

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

  27. 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' Properties of DNA- Double helixBuilding block(dA, dC, dG and dT)negatively charged at neutral pHAT and GC complementary pairing

  28. 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

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

  30. 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

  31. Cathode(-) wells DNA fragments of different sizes 1.5% agarosegel stained with methylene blue Anode(+)

  32. 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

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

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

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

  36. 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)

  37. Wells Tracking dye Xylene cyanol FF Bromophenol blue

  38. DNA fragments 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

  39. 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

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

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

  42. Workshop DNA Fingerprinting & Agarose Gel Electrophoresis Department of BiochemistryCUHK(TDC2003)

  43. Animal cell nucleus • Human genome ~ 3 billion base pairs. • 5 % of the genome are protein coding sequence (30,000 genes). • 95% non-coding DNA. • 20-30% are repetitive.

  44. Rich source of genetic variation Repetitive DNA Interspersed elements(~15-20% of the genome) Tandemly repeated sequences (~10% of genome) Satellite DNA Minisatellites / VNTRs SINES eg. Alu (3-6%) LINES eg L1(1-2%) VNTRs : Variable number tandem repeats (J.C.S. Fowler et al.)

  45. Identical twins You and me 0.1 % vary person to person • Each of us have unique DNA fingerprint / personal barcode.

  46. A, T T, A Two kinds of DNA polymorphic regions (regions of different) Sequence polymorphism Length polymorphism Simple variations in the physical length of the DNA(eg. VNTRs) …ACGTAGCAGCAGCG… …TGCATCGTCGTCGC… Simple substitution of one or two bases in the gene

  47. M P M P M P AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT DNA fingerprint/Length polymorphism in a single locus Person 1Homozygous Person 2Heterozygous Person 3Heterozygous VNTR repeating unit AT M Maternal chromosome copy P Paternal chromosome copy

  48. M P AT AT AT AT AT AT AT AT AT AT AT AT Variable number tandem repeats (VNTRs) • VNTRs are not distributed evenly across human population. • Each of allele occurs at a certain frequency in a population. • Each locus usually has approximately 30 different alleles. • Frequency of allele A at one locus = 0.1 (10%) Frequency of allele B at second locus = 0.05 (5%) Frequency of the two alleles of the loci occur together = 0.1 X 0.05(DNA profile frequency) = 0.005 or 1 in 200

  49. X Y Z Father 10, 14 18, 24 8, 9 Mother 12, 20 22, 22 8, 15 Son 10, 20 22, 24 8, 8 Daughter 12, 14 22, 22 26, 9 Locus X Locus Y Chromosome A Chromosome B Locus Z DNA profile frequency for 3 loci: ~1 in 3 million

  50. Application of DNA fingerprinting • Paternity and maternity test. • Criminal identification and forensics. • Personal identification. Source of human DNA for fingerprinting • Whole blood • Buccal epithilial cells • Hair follicles • Semen

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