分子生物学10-gene libraries.pptVIP

Chapter 9 DNA libraries and screening Contents Genomic DNA library Construction of genomic libraries Properties of cDNA cDNA library cDNA library construction Screening strategies sequence-dependent screening expression libraries screening Gene library (基因文库) Genomic libraries: made from genomic DNA cDNA libraries: made from cDNA （DNA complementary to mRNA, copy of mRNA） Genomic Library A genomic library contains clones of all the genes from a species genome Restriction fragments of a genome can be packaged into phage using about 16 – 20 kb per fragment This fragment size will include the entirety of most eukaryotic genes Once a library is established, it can be used to search for any gene of interest Packaging, transfection or transformation Restriction Enzyme Digestions Packaging, transfection or transformation λ phage used as a vector Representative gene libraries --- Contain all the original sequences from an organism Advantages using high capacity vector To increase average insert size so that the number of the clones will be decreased, which will make the screening for target gene easier. ? replacement vectors, cosmids: appropriate choice for many experiments. Bacterial artificial chromosomes (BACs) P1 derived artificial chromosomes (PACs) Yeast artificial chromosomes (YACs) For genome mapping, sequencing and the assembly of clone contigs. Limitations of Genomic Library Since all chromosomal content is present: -- Coding information unprocessed -- Interrupting sequences are present -- Protein genes harder to understand … Composition of genomic DNA Organization and content of the human genome Split gene Noncoding DNA ：introns，5‘ untranscripted area， 3’ untranscripted area，etc. cDNA library Properties of cDNA Protein coding regions of most eukaryotic genomes make up only a small percentage of the total DNA (1.5% in humans) Most cells only express a small subset of an organism’s genes By usi