Cloning large DNA fragments (Eukaryotic Genome project) Analysis of eukaryotic genes and the genome organization of eukaryotes requires vectors with a larger capacity for cloned DNA than plasmids or phage n Human genome(3 x 10 bp): large genome and large gene demand vectors with a large size capacity. Genomic library VS CDNA library
Analysis of eukaryotic genes and the genome organization of eukaryotes requires vectors with a larger capacity for cloned DNA than plasmids or phage . Human genome (3 x 109 bp): large genome and large gene demand vectors with a large size capacity. Cloning large DNA fragments (Eukaryotic Genome project) Genomic library VS cDNA library
Cosmid vectors 1. Utilizing the properties of the phage n cos sites in a plasmid vector 2. A combination of the plasmid vector and the cos site which allows the target DNA to be inserted into the n head 3. The insert can be 37-52 kb
Cosmid vectors 1. Utilizing the properties of the phage cos sites in a plasmid vector. 2. A combination of the plasmid vector and the COS site which allows the target DNA to be inserted into the head. 3. The insert can be 37-52 kb
Formation of a cosmid clone COs polylinker Digestion Cleave at polylinker a Cosmid (b) Ligation Target DNA Cleaved co smid 个 COS COS C) Packaging and infect
Digestion Ligation C) Packaging and infect Formation of a cosmid clone
YAC vectors (yeast artificial chromosome) Accommodates genomic DNA fragments of more than 1 Mb and can be used to clone the entire human genome, but not good in mapping and analysis
YAC vectors Accommodates genomic DNA fragments of more than 1 Mb, and can be used to clone the entire human genome, but not good in mapping and analysis. (yeast artificial chromosome)
Essential components of yAc vectors Centromers(CEN), telomeres (teland autonomous replicating sequence(ARS)for proliferation in the host cell ampr for selective amplification and markers such as TRP1 and URA3 for identifying cells containing the yac vector in yeast cells Recognition sites of restriction enzymes (e. g, EcoRI and BamHr
Essential components of YAC vectors : • Centromers (CEN), telomeres (TEL) and autonomous replicating sequence (ARS) for proliferation in the host cell. • ampr for selective amplification and markers such as TRP1 and URA3 for identifying cells containing the YAC vector in yeast cells. • Recognition sites of restriction enzymes (e.g., EcoRI and BamHI)