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. 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

4. 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.

5. Southern Blot Northern Blot Western Blot Macromolecules on the blot DNARNAProtein ProbeLabeled DNA Labeled antibodies Source of labelsRadioactively or fluorescent labeled deoxynucleotide Radioactively or fluorescent labeled amino acids Blotting Techniques

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

8. 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.

9. 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.

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. 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. Procedures of DNA Fingerprinting

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. 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. Detection of Highly Variable Regions

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/BiologyAnimatio nLibrary.htm

21. Animation 2: DNA Detective http://www.dnalc.org/resources/BiologyAnimatio nLibrary.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. http://dlab.reed.edu/projects/vgm/vgm/VGMPr ojectFolder/VGM/RED/RED.ISG/gel.html Good Website: Gel Electrophoresis

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 helix Building block(dA, dC, dG and dT) negatively charged at neutral pH AT 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 2166 BglI 472 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. wells Anode(+) Cathode(-) 1.5% agarose gel stained with methylene blue DNA fragments of different sizes

32. Preparation of plasmid DNA Restriction enzyme digestion » Agarose gel casting » DNA sample loading » electrophoresis Methylene blue staining http://dlab.reed.edu/projects/vgm/vgm/V GMProjectFolder/VGM/RED/RED.ISG/ gel.html http://dlab.reed.edu/projects/v gm/vgm/VGMProjectFolder/ VGM/RED/RED.ISG/gel.htm l

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

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

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

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

37. Wells Bromophenol blue Xylene cyanol FF Tracking dye

38. 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

39. Base pairs distance migrated(mm) A B C D M 31647 65638 180823 163425 DNA fragments of different sizes DNA fragments of known sizes

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 Biochemistry CUHK (TDC2003)

43. 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. Animal cell

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

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

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

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

48. 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 M P AT

49. Locus X Locus Y Locus Z XYZ Father10, 1418, 248, 9 Mother12, 2022, 228, 15 Son10, 2022, 248, 8 Daughter12, 1422, 2226, 9 Chromosome A Chromosome B 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

51. Double stranded target DNA Two DNA targets available for PCR PCR amplification Cycle (35 – 40 cycles) DNA denatured 95 o C Step 1 : Denaturation Primers bind to target DNA ~55 o C Step 2 : Primer Annealing Double stranded DNA duplicated 72 o C Step 3 : DNA Extension Amount of amplified DNA = 2 n x C where n = number of PCR cycles; and C = the initial number of copies of DNA template present in the tube. So, you will get 1,048,576 copies of DNA after 20 cycles of PCR reaction even you start with only one copy of DNA template initially Polymerase chain reaction

52. Maternal tandem repeat fragment Paternal tandem repeat fragment Ethidium bromide stained agarose gel

53. Electrical wire 1ml syringe filtted with tip 10 X TBE concentrate Electrophoresis unit Gel casting unit Home made gel electrophoresis kit

54. 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

55. Seal both ends of the gel casting unit with tape Agarose gel casting unit Gel casting unit Comb Tape

56. Preparation of 1.5% agarose gel

57. Wells Electrophoresis(5V/cm) Sample loading DNA is negatively charged

58. Power supply Nine 9V batteries connected in series Three 24V adaptor connected in series

59. Wells Bromophenol blue Xylene cyanol FF Tracking dye 1cm Cathode (-) Anode (+)

60. Methylene blue staining » to visualize the DNA fragments, stain agarose gel overnight with 1X methylene blue staining solution and destain in distilled water for 3 – 4 hours » non-toxic » easy available » non-carcinogenic

61. Lane MK: DNA marker Lane M: Mother Lane F: Father Lane D1: Daughter 1 Lane D2: Daughter 2 Lane S1: Son 1 Lane S2: Son 2 2100 bp 1700 bp 1100 bp 850 bp 600 bp 250 bp MK M F D1 D2 S1 S2 Results (Biologic child / adopted child / stepchild)

62. Calibration curve for DNA size determination

63. Size determination of the candidate DNA fragments online size determination

64. THE END