CHAPTER20: Techniques of Molecular Biology Topic1: Nucleic acids 1. Electrophoresis 2. Restriction 3. Hybridization 4. DNA Cloning and gene expression 5. PCR 6. Genome sequence analysis
6 Topic1: Nucleic acids CHAPTER20: Techniques of Molecular Biology 1.Electrophoresis 2.Restriction 3.Hybridization 4.DNA Cloning and gene expression 5.PCR 6.Genome sequence & analysis
Electrophoresis 1. 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 througl h. Agarose and polyacrylamide are two different gel matrices
7 1. 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 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. The mobility of the same molecular weight dna molecule with different shapes is supercoiled> Iinear> nicked or relaxe
8 ➢ 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. The mobility of the same molecular weight DNA molecule with different shapes is: supercoiled> linear> nicked or relaxed Electrophoresis
Fig 20-1: DNA separation by gel electrophoresis electrophoresis chamber buffer solution DNA fragments agarose gel electrode electrode large moderate small After electr electrode electrode small DNA fragments move further through the gel than large fragments Copyright C 2004 Pearson Education, Inc, publishing as Benjamin Cummings
9 Fig 20-1: DNA separation by gel electrophoresis large moderate small After electr
Electrophoresis To separate DNA of different SIze ranges a 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 10
10 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 Electrophoresis