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

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

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

Electrophoresis

Electrophoresis

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.

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

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

Pulsed-field gel electrophoresis

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

Restriction digestion blunt ends sticky ends

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

RFLP genotyping

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

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)

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

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

Southern blot Southern analysis

Southern blot

RFLP