1. Restriction digestion and Southern blot
Dr Nikola Tanić
Institut za biološka istaživanja
“Siniša Stanković”, Beograd

2. Techniques of molecular biology Nucleic acids
Electrophoresis
Restriction
Hybridization
DNA Cloning
PCR
Genome sequence & analysis
Gene expression

3. Electrophoresis
Gel electrophoresis separates DNA and RNA molecules according to size, shape and topological properties
Gel matrix is an inserted, jello-like porous material that support and allows macromolecules to move through. Agarose and polyacrylamide are two different gel matrices

4. Electrophoresis

5. Electrophoresis

6. Electrophoresis
DNA and RNA molecules are negatively charged, thus move in the gel matrix toward the positive pole (+)
Linear DNA molecules are separated according to size
The mobility of circular DNA molecules is affected by their topological structures.

7. Electrophoresis
The mobility of the same molecular weight DNA molecule with different shapes is: supercoiled> linear> nicked or relaxed

8. To separate DNA of different size ranges:
Electrophoresis
To separate DNA of different size ranges:
Narrow size range of DNA: use polyacrylamide
Wide size range of DNA: use agarose gel
Very large DNA(>30-50kb): use pulsed-field gel electrophoresis

9. Pulsed-field gel electrophoresis

11. Restriction digestion
Restriction endonucleases cleave DNA molecules at particular sites
Different enzymes recognize their specific target sites with different frequency
EcoRI recognize hexameric sequence: 46 = 4096bp
Sau3A1 Recognize terameric sequence: 44 = 256bp
Thus Sau3A1 cuts the same DNA molecule more frequently

12. Restriction digestion
blunt ends
sticky ends

13. RFLP - Restriction Fragment Length Polymorphism
AFLP - Amplified Fragment Length Polymorphism
STRP - Short Tandem Repeat Polymorphism

14. RFLP genotyping

15. DNA hybridization
Hybridization: the process of base-pairing between complementary ssDNA or RNA from two different sources
Probe: a labeled, defined sequence used to search mixtures of nucleic acids for molecules containing a complementary sequence

16. Labelling of DNA/RNA probes
radioactive labeling: display and/or magnify the signals by radioactivity
Non-radioactive labeling: display and/or magnify the signals by antigen labeling – antibody binding – enzyme binding - substrate application (signal release)

17. End labeling put the labels at the ends
5’-end labeling: polynucleotide kinase (PNK)
3’-end labeling: terminal transferase

18. Uniform labeling put the labels internally Nick translation:
DNase I to introduce random nicks DNA polI to remove dNMPs from 3’ to 5’ and add new dNMP including labeled nucleotide at the 3’ ends.
Hexanucleotide primered labeling:
Denature DNA  add random hexanucleotide primers and DNA pol  synthesis of new strand incorporating labeled nucleotide

19. Southern blot
Southern analysis

20. Southern blot

21. RFLP