Chapter 20 techniques of molecular biology 2 Introduction • The methods depend upon, and were developed from, an understanding of the properties of biological macromolecules themselves 3 4 Topic 1 nuc[.]
Introduction • The methods depend upon, and were developed from, an understanding of the properties of biological macromolecules themselves Topic nucleic acids • • • • • • • Electrophoresis Restriction Hybridization DNA Cloning and gene expression PCR Genome sequence & analysis Comparative genome analysis 1.Electrophoresis through a Gel separate DNA and RNA molecules according to size • Gel matrix an inert, jolly-like porous material that sieve the DNA molecules according to its volumn DNA characteristics negatively charged, when subject to an electrical field, it migrates through the gel toward the positive pole Two types of normal gel matrices • Polyacrylamide has high resolving capability but can separate DNAs only over a narrow size range • Agarose has less resolving power than polyacrylamide but can separate from one another DNA molecules of up to tens, and even hundreds, of kilobases Fig 20-1: DNA separation by gel electrophoresis http://a32.lehman.cuny.edu/molbio_course/agarose.htm Some fundamental steps of electrophoresis Whereas very long DNAs are unable to penetrate the pores in agarose • DNA molecules above a certain size (30 to 50 kb) usually use pulsedfield electrophoresis to separate electrophoresis DNA and R NA 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 10 2.Restriction endonucleases cleave DNA molecules at particular sites • endonucleases – To make large DNA molecules break into manageable fragments • Restriction endonucleases: the nucleases that cleave DNA at particular sites by the recognition of specific sequences • The target site recognized by endonucleases is usually palindromic 11