基因克隆原理.pptVIP

基因克隆原理 Definition of cloning CLONE a population of identical cells or DNA molecules descended from a a single progenitor. Also viruses or organisms that are genetically identical and descended from a single progenitor DNA CLONING Recombinant DNA technique in which specific cDNAs or fragments of genomic DNA are inserted into a cloning vector, which then is incorporated into cultured host cells and maintained during growth of the host cells; also called gene cloning Cloning vectors Plasmids lambda (l) vectors cosmids BACs and YACs Plasmid conformation affects gel mobility Plasmid vectors Advantages: Small, easy to handle Straightforward selection strategies Useful for cloning small DNA fragments ( 10kbp) Disadvantages: Less useful for cloning large DNA fragments ( 10kbp) lambda (?) vectors Cosmids: combine the properties of plasmid vectors with the useful properties of the cos site BACs and YACs: Bacterial Artificial Chromosomes; YACs Yeast Artificial Chromosomes l vectors Advantages: Useful for cloning large DNA fragments (10 - 23 kbp) Inherent size selection for large inserts Disadvantages: Less easy to handle Cosmid vectors Advantages: Useful for cloning very large DNA fragments (32 - 47 kbp) Inherent size selection for large inserts Handle like plasmids Disadvantages: Not easy to handle very large plasmids (~ 50 kbp) BACs and YACs Advantages: Useful for cloning extremely large DNA fragments (100 - 2,000 kbp) This is very important for genome sequencing projects Disadvantages: Not easy to handle extremely large DNA molecules Restriction-recognition sites are short DNA sequences recognized and cleaved by various restriction endonucleases Types of Restriction Endonuclease Restriction and modification done by separate enzymes. -Unlike Types I and III Type II do not require cofactors. Most importantly, Type II enzymes recognise a defined symetrical sequence and cut within it. Advantages -4 different types; Type I, II, IIs and III. -O