武大分子生物学课件application1.pptVIP

I1 Genomic libraries I1-1 Representative gene libraries I1-2 Size of library I1-3 Genomic DNA I1-4 Vectors I1-4 Vectors According to genome’s size,we can select a proper vector to construct a library . Vectors Plasmid phageλ cosmid YAC insert (kb) 5 23 45 1000 The most commonly chosen genomic cloning vectors are λ relacement vectors which must be digested with restriction enzymes to produce the two λ end fragment or λ arms between which the genomic DNA will be digested I1 Genomic libraries cos cos Long (left) arm short (right) arm Exogenous DNA (~20-23 kb) λ phage vector in cloning cos cos Long (left) arm short (right) arm Exogenous DNA (~20-23 kb) λ replacement vector cloning 2. Packing with a mixture of the phage coat proteins and phage DNA-processing enzymes 3. Infection and formation of plaques Library constructed Ligation 0.preparation of arm and genomic inserts I 2 cDNA libraries I2-1 mRNA isolation, purification I2-2 Check theRNA integrity I2-3 Fractionate and enrich mRNA I2-4 Synthesis of cDNA I2-5 Treatment of cDNA ends I2-6 Ligation to vector Gene libraries and screening cDNA libraries No cDNA library was made from prokaryotic mRNA. Prokaryotic mRNA is very unstable Genomic libraries of prokaryotes are easier to make and contain all the genome sequences. I 2 cDNA libraries cDNA libraries are very useful for eukaryotic gene analysis Condensed protein encoded gene libraries, have much less junk sequences. cDNAs have no introns ? genes can be expressed in E. coli directly Are very useful to identify new genes Tissue or cell type specific (differential expression of genes) cDNA libraries I 2 cDNA libraries I2-1 mRNA isolation Most eukaryotic mRNAs are polyadenylated at their 3’ ends oligo (dT) can be bound to the poly(A) tail and used to recover the mRNA. AAAAAAAAAAn 5’ cap I 2 cDNA libraries I 2 cDNA libraries 1.Traditionally