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

2. Order Form for Electrophoresis Kits and Reagents
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3. 電泳

4. 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

5. 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.

6. 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

7. 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

8. Coomassie Blue Dye Stained protein gel
A Protein can be Specifically Recognized by an Antibody in a Western Blot M M
Coomassie Blue Dye Stained protein gel
Western blot

9. 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.

12. 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.

13. 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.

14. 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.

15. Procedures of DNA Fingerprinting

16. 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.

17. 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.

18. 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).

19. Application of DNA Fingerprinting Mutation Detection

20. 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).

21. 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.

22. 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.

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

24. Animation 1: Southern Blotting

25. Animation 2: DNA Detective

26. Online Courses: DNA from the Beginning

27. Download Illustrations: Human Molecular Genetics

28. Good Website: Gel Electrophoresis

29. The End

30. Agarose Gel Electrophoresis
Workshop
Agarose Gel Electrophoresis
Department of Biochemistry
( )

31. 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'

32. 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

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

34. 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

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

36. Preparation of plasmid DNA
Restriction enzyme digestion
Agarose gel casting
DNA sample loading
electrophoresis
Methylene blue staining

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

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

39. Preparation of 1.5% agarose gel
Step 5

40. Electrophoresis(5V/cm)
Step 10
Sample loading, wash syringe with 1X TBE buffer between successive loading
Electrophoresis(5V/cm)

41. Wells
Tracking dye
Xylene cyanol FF
Bromophenol blue

42. 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

43. DNA fragments of different sizes DNA fragments of known sizes
A B C D M
Base pairs distance migrated(mm)

44. Calibration curve for DNA size determination

45. Size determination of the candidate DNA fragments