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cDNA Library MB206 Angelia 09 1 Angelia 09 2 Making a DNA library Angelia 09 3 Types of Libraries Genomic Library • whole genes w/ promoters & introns (Euk.), operons (bacteria), DNA regulatory elements… cDNA Library • mRNA transcript only w/ 5’ & 3’ untranslated regions (UTRs), no introns, tissue specific. (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. Angelia 09 5 Genomic Libraries cDNA library Genomic DNA mRNA polyA polyA Reverse transcribe cDNA (and more) polyA Genomic DNA library Clone in vector Genomic DNA Digest DNA fragments Angelia 09 6 cDNA 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 7 cDNA Libraries “complementary” DNA [mRNA it is used to create it] 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 Lodish, et al. 1999 Figure 7-14, 7-15 Angelia 09 9 Genomic Libraries “The Details” Angelia 09 10 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? Consideration: Try not to cut the gene or operon of interest V. fischeri LUX operon 9kb Angelia 09 12 Selecting a restriction enzyme What is the average size fragment that any given enzyme will yield? If enzyme recognizes 6bp, statistically any given 6bp sequence will appear randomly every 46 bases (every 4096 bases) Angelia 09 13 How often does the enzymes cut? A ‘four-base cutter’ recognition sequence would occur once every 44 = 256 bp A ‘six-base cutter’ would give you fragments of about 4000 bp An ‘eight-base cutter’ recognition sequence would occur once every 48 or 65,536 bp. Angelia 09 14 How often do enzymes really cut? But also we need to consider G+C content of the genome: V. fischeri G+C content = 40% Sal I recognizes sites that are G+C rich (GTCGAC) it will cut less often in Vibrio genomic DNA. Angelia 09 15 Let’s stop here and think about it! Angelia 09 16 PROBLEM: The genomic DNA is 60% G+C You want a 5000bp fragment Which enzyme would you try first, Why? SmaI (CCCGGG) EcoRI (GAATTC) MseI (TTAA) 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 2. Modified Lambda phage 3. Up to 10kb inserts Up to 20kb inserts/40kbp for cosmids Artificial cloning vectors BAC- Vectors (bacterial artificial chromosome) Up to 100-150kbp 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 mRNA isolation, purification Check the RNA integrity Synthesis of cDNA Treatment of cDNA ends Ligation to vector cDNA libraries 1. 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. 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 specific (differential expression of genes) Angelia 09 26 mRNA isolation Most eukaryotic mRNAs are polyadenylated at their 3’ ends 5’ cap AAAAAAAAAAn • oligo (dT) can be bound to the poly(A) tail and used to recover the mRNA. Angelia 09 27 Check the mRNA integrity Make sure that the mRNA is not degraded. Methods: Translating the mRNA : use cell-free translation system as wheat germ extract or rabbit reticulocyte lysate to see if the mRNAs can be translated Analysis the mRNAs by gel elctrophoresis: use agarose or polyacrylamide gels Angelia 09 28 Cloning the particular mRNAs Is useful especially one is trying to clone a particular 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 transcriptase ,primer( oligo(dT) or hexanucleotides) and dNTPs (Fig 1.1) Second strand synthesis: best way of making 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’ Terminal transferase dCTP mRNA AAAAA-3’ TTTTTP-5’ cDNA cDNA cDNA Alkali (hydrolyaes RNA) Purify DNA oligo(dG) TTTTTP-5’ Klenow polymerase or reverse Transcriotase Four dNTPs -3’ TTTTTP-5’ Duplex cDNA Angelia 09 31 5’-pGGGG 3’-CCCCCCC Duplex cDNA -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 T4 DNA ligase HO-CCGAATTCGGGGGG 3’-GGCTTAAGCCCCCC -3’ TTTTTp-5’ 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 use special acid linkers to create sticky ends for cloning. The process : Move protruding 3’-ends (strand-special nuclease) Fill in missing 3’ nucleotide (klenow fragment of DNA polyI and 4 dNTPs) Ligate the blunt-end and linkers(T4 DNA ligase) Tailing with terminal transferase or using adaptor molecules Restriction enzyme digestion (E.coRI ) 33 Ligation to vector Any vectors with an EcoRI site 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 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 sequence of a protein product of the gene • A DNA fragment/oligo from a related gene of another species Blotting the DNA or RNA on a membrane Hybridize the labeled probe with DNA membrane (Southern) or RNA (Northern) membrane Angelia 09 36 Colony and plaque hybridization Transfer the DNA in the plaque or colony to a Nylon or nitrocellulose membrane Phage DNA bind to the membrane directly Bacterial colonies must be lysed to release DNA on the membrane surface. Hybridization (in a solution (Alkali treatment) Containing Nucleic acid probe) X-ray film(radioactively labeled ) Wash to remove unhybridization probe and visualize Line up the hybridizated region or Angelia 09 repeated hybridization antibody or enzyme (modified nucleotide labeled 37 Transfer to nitrocellulose or nylon membrane Keep master plate Select positive from master plate Denature DNA(NaOH) Bake onto membrane Probe with 32p-labled DNA complementary to gene of interest Expose to film Screening by plaque hybridization Angelia 09 38