Chapter 4 - Molecular cloning methods. This chapter review the fundamentals of gene structure and function, we are ready to start a more detailed study of molecular biology. The main focus of chapter will be the experiments that molecular biologists have performed to elucidate the structure and function of genes. | Molecular Biology Fourth Edition Chapter 4 Molecular Cloning Methods Lecture PowerPoint to accompany Robert F. Weaver Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Gene Cloning Gene cloning links eukaryotic genes to small bacterial or phage DNAs and inserting these recombinant molecules into bacterial hosts One can then produce large quantities of these genes in pure form 4- The Role of Restriction Endonucleases Restriction endonucleases, first discovered in the late 1960s, are named for preventing invasion by foreign DNA by cutting it into pieces These enzymes cut at sites within the foreign DNA instead of chewing from the ends By cutting DNA at specific sites they function as finely honed molecular knives 4- Naming Restriction Endonucleases Restriction endonucleases are named using the 1st three letters of their name from the Latin name of their source microorganism Hind III First letter is from the genus H from Haemophilus | Molecular Biology Fourth Edition Chapter 4 Molecular Cloning Methods Lecture PowerPoint to accompany Robert F. Weaver Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Gene Cloning Gene cloning links eukaryotic genes to small bacterial or phage DNAs and inserting these recombinant molecules into bacterial hosts One can then produce large quantities of these genes in pure form 4- The Role of Restriction Endonucleases Restriction endonucleases, first discovered in the late 1960s, are named for preventing invasion by foreign DNA by cutting it into pieces These enzymes cut at sites within the foreign DNA instead of chewing from the ends By cutting DNA at specific sites they function as finely honed molecular knives 4- Naming Restriction Endonucleases Restriction endonucleases are named using the 1st three letters of their name from the Latin name of their source microorganism Hind III First letter is from the genus H from Haemophilus Next two letters are the 1st two letters of the species name in from influenzae Sometimes the strain designation is included “d” from strain Rd If microorganism produces only 1 restriction enzyme, end the name with Roman numeral I Hind I If more than one restriction enzyme is produced, the others are numbered sequentially II, III, IV, etc. 4- Restriction Endonuclease Specificity Restriction endonucleases recognize a specific DNA sequence, cutting ONLY at that sequence These enzymes can recognize 4-bp, 6-bp, 8-bp sequences The frequency of cuts lessens when the recognition sequence is longer 4- Restriction Enzyme Terminology A 6-bp cutter will yield DNA fragments averaging 4000-bp or 4 kilobases (4kb) in length Heteroschizomers recognize the same DNA sequence but use a different cutting site – they are also called isochizomers These enzymes cut DNA strands reproducibly in the same place, which is extremely useful in gene analysis 4- Use of Restriction Endonucleases Many .
