YAC vectors Plasmid with Yac vector elements Expose yeast cells to recombinant YACs; a small percentage of YACs Centromere enter yeast cells Origin of Foreign DNA Restriction replication sites Selectable marker 1 Selectable Telomeres marker 2 Partial digestion Cut with several with EcoRI restriction enzymes to release two 8② ector arms Recombinant YACs replicate in yeast cells. 2 Combine vector arms and fragments; ligate with phosphatase 2 2 2 Plate onto medium containing two antibiotics Cells containing YACs with two selectable markers survive
1-— mb yac insert 20-kb lambda insert 1-kb 2-kb Plasmid insert Plasmid insert Figure 8.10 Subcloning: From YAC clones to lambda clones to plasmids. The large genomes of higher eukaryotes( such as those
Identifying transformants foreign DNA insert Ill. Distinguishing cells carrying insert- Recombinant containing recombinant molecules from Disrupted plasmid cells carrying vectors without inserts lacz gene AmpR Intact vector. Vector EcoRI no insert with insert sites origin of Disrupted licatio lacz ge lacz gene lacz gene split by Transformation: foreign dNA enters the host cell lacz gen foreign DNA insert intact no product lacz gene transcript lacz X-Gal Blue Selecting cells that have received a plasmid pigment
(a) Separating plasmid from bacterial chromosome Purifying cloned DNA Bacterial cells containing plasmids Lyse cells, extract DNA (b)Separating insert from plasmid vector Treat with ethidium bromide →閔閤·閤閤A Cut with EcoRI Separate fragments by size Add to solution of Cscl and centrifuge Cscl forms density gradient DNA settles according to its density. Cloned fragment Vector →8 Host chromosomal DNA
Construction of recombinant dNa Libraries Genomic library: A collection of clones representing the entire chromosomal DNA sequence. These clones include the entire gene, including introns(eukaryotes) and regulatory regions CDNA library(complimentary DNa A collection of clones representing DNA copies of all the different mrna molecules in the cell. these clones do not include the introns(eukaryotes)nor the regulatory regions of the gene because they are derived from mrNA
Construction of Recombinant DNA Libraries • Genomic library: • A collection of clones representing the entire chromosomal DNA sequence. These clones include the entire gene, including introns (eukaryotes) and regulatory regions. • cDNA library (complimentary DNA) • A collection of clones representing DNA copies of all the different mRNA molecules in the cell. These clones do not include the introns (eukaryotes) nor the regulatory regions of the gene because they are derived from mRNA