cDNA library

cDNA Library MB206 Angelia 09 1 Angelia 09 2 Making a DNA library Angelia 09 3 Types of Libraries (5’UTR) (3’UTR) Angelia 09 4 Genomic DNA libraries -Contains the whole genome of an organism. -A restriction-enzyme is used to cut the genome (the DNA) at numerous locations. -Contains the whole genome of an organism. -A restriction-enzyme is used to cut the genome (the DNA) at numerous locations. Angelia 09 5 Genomic Libraries Genomic DNA Digest DNA fragments Clone in vector polyA polyA polyA mRNA Genomic DNA Reverse transcribe (and more) cDNA cDNA library Genomic DNA library Angelia 09 6 cDNA Libraries polyA polyA polyA Genomic DNA Genomic DNA mRNA Reverse transcribe (and more) Digest DNA fragments cDNA Clone in vector cDNA library Genomic DNA library Angelia 09 7 cDNA Libraries  Purify mRNA  mRNA-> single stranded cDNA using reverse transcriptase.  Single stranded cDNA -> double stranded cDNA (DNA polymerase and other “cloning tricks”).  Linkers added to cDNA & clone into vectors as seen in genomic DNA libraries Angelia 09 8 “complementary” DNA [mRNA it is used to create it] Lodish, et al. 1999 Figure 7-14, 7-15 Angelia 09 9 Genomic Libraries “The Details” Genomic Libraries “The Details” Angelia 09 10 [...]... clone a particular gene rather to make a complete cDNA library gene rather to make a complete cDNA library Fractionate on the gel: performed on the basis of size, mRNAs of the interested sizes are recovered from agarose gels Enrichment: carried out by hybridization Example: clone the hormone induced mRNAs (substrated cDNA library) Angelia 09 29 Synthesis of cDNA : First stand synthesis: materials as reverse... inserts  YAC-Vectors (yeast artificial chromosome) Up to 500kbp inserts  Angelia 09 20 Lambda Library Lodish, et al Fig 7-12 Plasmid Library Lodish, et al Fig 7-1 Angelia 09 21 Genomic Libraries Genomic DNA library Clone in vector Genomic DNA Digest DNA fragments Angelia 09 22 Angelia 09 23 cDNA libraries 1 No cDNA library was made from prokaryotic mRNA • Prokaryotic mRNA is very unstable • Genomic libraries...Genomic Libraries cDNA library Genomic DNA mRNA polyA Reverse transcribe cDNA (and more) polyA polyA Genomic DNA library Clone in vector Genomic DNA Digest DNA fragments Angelia 09 11 Digest genomic DNA with restriction enzymes  Which restriction enzyme should we select?... full-length cDNA is to ‘tail’ the 3’-end of the first strand and then use a complementary primer to make the second Angelia 09 30 5’ 5’ 3’ 5’ 3’-CCCCCCC 5’-pGGGG-OH 3’-CCCCCCC 5’-pGGGG 3’-CCCCCCC mRNA AAAAA-3’ HO-TTTTTP-5’ Reverse transcriptase Four dNTPs mRNA AAAAA-3’ TTTTTP-5’ cDNA Terminal transferase dCTP mRNA AAAAA-3’ TTTTTP-5’ cDNA Alkali (hydrolyaes RNA) Purify DNA oligo(dG) TTTTTP-5’ cDNA Klenow... would suitable for cloning the cDNA The process : Dephosphorylate the vector with alkaline phosphatase Ligate vector and cDNA with T4 DNA ligase (plasmid or λ phage vector) 34 Screening The process of identifying one particular clone containing the gene of interest from among the very large number of others in the gene library 1 Using nucleic acid probe to screen the library based on hybridization... is very unstable • Genomic libraries of prokaryotes are easier to make and contain all the genome sequences Angelia 09 25 cDNA libraries 2 .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... Terminal transferase dCTP mRNA AAAAA-3’ TTTTTP-5’ cDNA Alkali (hydrolyaes RNA) Purify DNA oligo(dG) TTTTTP-5’ cDNA Klenow polymerase or reverse Transcriotase Four dNTPs -3’ TTTTTP-5’ Duplex cDNA Angelia 09 31 Duplex cDNA 5’-pGGGG 3’-CCCCCCC -3’ TTTTTp-5’ Single strand-specific nuclease 5’-pGGGG 3’-CCC -3’ TTTTTp-5’ Klenow polymerase treat with E.coRI methylase 5’-pGGGG 3’-CCCC Add E.colRI linkers using... SacI (GAGCTC) Angelia 09 Take your time! 17 Genomic Libraries Genomic DNA library Clone in vector Genomic DNA Digest DNA fragments Angelia 09 18 Choosing a Vector  Usually you select a vector (plasmid, λ, other) depending on how big you want your DNA fragments to be & the capacity of the vector Angelia 09 19 Common vectors used in library construction 1 Plasmids  1 Modified Lambda phage  1 Up to 10kb... HO-CCG/AATTCGG-3’ 3’-GGCTTAA/GCC-OH CCGAATTCGG-3’ TTTTTGGCTTAAGCC-OH E.colRI digestion 5’-pAATTCGGGGGG 3’-CCCCCCC Fig2.1 CCG-3’ TTTTTGGCTTAAp-5’ Ligate to vector and transfom Second strand synthesis 32 Treatment of cDNA ends Blunt and ligation of large fragment is not efficient, so we have to Blunt and ligation of large fragment is not efficient, so we have to use special acid linkers to create sticky ends for cloning... library 1 Using nucleic acid probe to screen the library based on hybridization with nucleic acids 2 Analyze the protein product Angelia 09 35 Screening libraries Searching the genes of interest in a DNA library Hybridization to identify the interested DNA or its RNA product 1 2 3 Radiolabeled probes which is complementary to a region of the interested gene Probes: • An oligonucleotide derived from the . Libraries polyA polyA polyA Genomic DNA Genomic DNA mRNA Reverse transcribe (and more) Digest DNA fragments cDNA Clone in vector cDNA library Genomic DNA library Angelia 09 7 cDNA. fragments Clone in vector polyA polyA polyA mRNA Genomic DNA Reverse transcribe (and more) cDNA cDNA library Genomic DNA library Angelia 09 6 cDNA Libraries polyA polyA polyA Genomic