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Dna library lecture-Gene libraries and screening

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Dna library lecture-Gene libraries and screening

  1. 1. Gene libraries and screening Genomic libraries cDNA libraries Screening procedures
  2. 2. Introduction • The use of genetic information is a powerful tool that today is becoming more readily available to scientists. • In order to use this powerful tool it necessary to know how to navigate throughout the entire genome. The human genome is about 3 x 10E9 bp. • In humans this project is known as Human Genome Project.
  3. 3. I1 Genomic libraries Gene library: a collection of different DNA sequence from an organism, each of which has been cloned into a vector for ease of purification, storage and analysis. Genomic libraries Gene library (made from genomic DNA) cDNA libraries (made from cDNA- copy of mRNA)
  4. 4. I1 Genomic libraries Size of library (ensure enough clones) must contain a certain number of recombinants for there to be a high probability of it containing any particular sequence The formula to calculate the number of recombinants: ln (1-P) N= ln (1-f) P: desired probability f : the fraction of the genome in one insert
  5. 5. I1 Genomic libraries For example :for a probability of 0.99 with insert sizes of 20 kb these values for the E.coli (4.6×106 bp) and human (3×109 bp) genomes are : N E.coli= Nhuman= ln( 1-0.99) ln[1-(2×104/4.6×106)] ln(1-0.99) = 1.1 ×103 = 6.9 ×105 ln[1-(2 ×104/3 ×109)] These values explain why it is possible to make good genomic libraries from prokaryotes in plasmids where the insert size is 5-10kb ,as only a few thousand recombinants will be needed.
  6. 6. I1 Genomic libraries Genomic DNA libraries eukaryotes Purify genomic DNA prokaryotes Fragment this DNA : physical shearing and restriction enzyme digestion Clone the fragments into vectors
  7. 7. I1 Genomic libraries To make a representative genomic libraries , genomic DNA must be purified and then broken randomly into fragments that are correct in size for cloning into the chosen vector. Purification of genomic DNA : Eukaryotes :prepare cell nuclei remove protein, lipids and other unwanted macromolecules by protease digestion and phase extraction. Prokaryotes :extracted DNA directly from cells
  8. 8. I1 Genomic libraries Break DNA into fragments randomly: Physical shearing : pipeting, mixing Restriction enzyme digestion: partial digestion is preferred to get a greater lengths of DNA fragments.
  9. 9. I1 Genomic libraries Selection of restriction enzyme 1. Ends produced (sticky or blunt) & The cleaved ends of the vector to be cloned Sau3A: 5’-/GATC-3’, less selectivity BamH1: 5’-G/GATCC 2. Whether the enzyme is inhibited by DNA modifications (CpG methylation in mammals 3. Time of digestion and ratio of restriction enzyme to DNA is dependent on the desired insert size range.
  10. 10. Generating A Genomic Library • • • • λ-phage is treated with restriction enzymes that produce λ arms with sticky end. These arms contain all the lytic genetic information that is needed for replication and produces room for insertion of new genetic information. DNA sequence is obtain from the cell of interest. It is cleaved with restriction enzymes that produce 20kb fragments that have complementary sticky ends. Both are mixed in equal amounts and are treated with a DNA ligase that cleaves them together. Afterward the entire combined sequence is packed to the phage head.
  11. 11. λ-phage as a Vector • The genomic library is generated by using λ-phage for the following reasons. 1. A large number of λ phage can be screened simultaneously (5 x 10E4 phage plagues). λ phage as a higher transformation efficiency about 1000 times higher compared to a plasmid. 2. • • The vector as to maintain its lytic growth. Lysogenic pathway and other viral genes that are not important are replaced with the DNA to be cloned.
  12. 12. λ-phage as a Vector (Cont.) • • • An infected E.Coli will produce what are know as concatomers (which is the viral genome) on either site of the concatomers there is a site called COS Site. Two proteins recognize this site A protein and Nu protein, which will lead to the insertion of the λ DNA into the phage head. The chromosomal DNA that lacks the COS sites will not enter the phage head. Once the genetic information is inserted the tail will assemble. A 50kb can be inserted into the phage.
  13. 13. Packaging of the Recombinant DNA • • • • • • • To prepare the phage an E.coli cell is infected with a mutant λ-phage that as a defective “A-protein” (which is one of two genes that are responsible for packaging genetic information). Therefore the E.Coli accumulates empty heads and also preassembled tails. Once enough heads and tails are assembled we lysate the E.Coli cells. To the mixture of heads and tail we add isolated A protein (obtained from E.Coli infected with λ-phage). In the next step we add the recombinant DNA that has the λ-phage genetic information (which also includes COS sites). At this point we have a mixture containing mutant λ-phage heads and tails. There is isolated A protein and recombinant DNA containing λphage genetic information with COS sites. Therefore we have all the components necessary to package the recombinant DNA into the λ-phage head. Once the information is inserted the tail assembles and we have an infectious phage that contains the recombinant DNA sequence.
  14. 14. I1 Genomic libraries Vectors According to genome’s size,we can select a proper vector to construct a library . Vectors Plasmid insert (kb) 5 phageλ 23 cosmid 45 YAC 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
  15. 15. λ phage vector in cloning Long (left) arm cos short (right) arm Exogenous DNA (~20-23 kb) Long (left) arm cos short (right) arm cos cos Exogenous DNA (~20-23 kb)
  16. 16. 0.preparation of arm and genomic inserts 1. Ligation λ replacement vector cloning 2. Packing with a mixture of the phage coat proteins and phage DNAprocessing enzymes 3. Infection and formation of plaques Library constructed
  17. 17. Gene libraries and screening I cDNA libraries mRNA isolation, purification Check theRNA integrity Fractionate and enrich mRNA Synthesis of cDNA Treatment of cDNA ends Ligation to vector
  18. 18. I 2 cDNA libraries 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.
  19. 19. I 2 cDNA libraries 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)
  20. 20. I 2 cDNA libraries 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.
  21. 21. I 2 cDNA libraries
  22. 22. I2 cDNA libraries Three methods to isolate mRNA. 1.Traditionally method was done by pass a preparation of total RNA down a column of oligo (dT)-cellulose 2.More rapid procedure is to add oligo(dT) linked to magnetic beads directly to a cell lysate and ‘pulling out’ the mRNA using a strong magnet 3.Alternative route of isolating mRNA is lysing cells and then preparing mRNAribosome complexes on sucrose gradients
  23. 23. I2 cDNA libraries 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
  24. 24. I2 cDNA libraries 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)
  25. 25. I2 cDNA libraries Synthesis of cDNA : First stand synthesis: materials as reverse transcriptase ,primer( oligo(dT) or hexanucleotides) and dNTPs 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.
  26. 26. I2 cDNA libraries 5’ 5’ 3’ 5’ 3’-CCCCCCC 5’-pGGGG-OH 3’-CCCCCCC 5’-pGGGG 3’-CCCCCCC mRNA Reverse transcriptase Four dNTPs mRNA AAAAA-3’ HO-TTTTTP-5’ cDNA AAAAA-3’ TTTTTP-5’ cDNA AAAAA-3’ TTTTTP-5’ Terminal transferase dCTP mRNA Alkali (hydrolyaes RNA) Purify DNA oligo(dG) TTTTTP-5’ Klenow polymerase or reverse Transcriotase Four dNTPs -3’ TTTTTP-5’ Duplex cDNA cDNA The first strand synthesis
  27. 27. 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 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 CCG-3’ TTTTTGGCTTAAp-5’ Ligate to vector and transfom Second strand synthesis
  28. 28. I2 cDNA libraries 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 )
  29. 29. I2 cDNA libraries Ligation to vector Any vectors with an E.coRI 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)
  30. 30. Gene libraries and screening Screening procedures Screening Colony and plaque hybridization Expression screening Hybrid arrest and release Chromosome walking (repeat screening)
  31. 31. I3 Screening procedures 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.
  32. 32. I3 Screening procedures Screening libraries Searching the genes of interest in a DNA library Hybridization to identify the interested DNA or its RNA product 1. 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 2. Blotting the DNA or RNA on a membrane 3. Hybridize the labeled probe with DNA membrane (Southern) or RNA (Northern) membrane
  33. 33. I3 Screening procedures 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 repeated hybridization antibody or enzyme (modified nucleotide labeled
  34. 34. I3 Screening procedures Expression screening Identify the protein product of an interested gene 1. Protein activity 2. Western blotting using a specific antibody
  35. 35. I3 Screening procedures Expression screening If the inserts are cloned into an expression sites, it may be expressed. Therefore, we can screen for the expressed proteins. However, this screening may miss the right clone
  36. 36. I3 Screening procedures Expression screening Antibodies can be used to screen the expression library. The procedure ‘Plaque lift’ ( taken by placing a membrane on the dish of plaque) Immersed in a solution of the antibody Detected by other antibodies Repeat cycles of screening to isolate pure plaques
  37. 37. I3 Screening procedures Hybrid arrest and screen Individual cDNA clones or pools of clones can be used to hybridize to mRNA preparation Hybrid arrest :translate the mRNA population directly, and the inhibition of translation of some products detected. Hybrid release translation : purify the hybrids and the hybridized mRNAs released from them and translated, it identifies the protein encoded by the cDNA clone
  38. 38. I3 Screening procedures I3-5 Chromosome walking Definition: To clone the desired gene by repeated isolating adjacent genomic clones from the library. to obtain overlapping genomic clones that represent progressively longer parts of a particular chromosome .
  39. 39. I3 Screening procedures Process: 1. Prepare a probe from the end insert . 2.The probe are used to re-screen the library by colony or plaque hybridization 3.Analyzed the new isolate clones and posited them relative to the starting clone. some will be overlapping. 4. Repeated the whole process using a probe from the distal end of the second clone.
  40. 40. Vector arm } } Restriction Genomic clone insert Vector arm Prepare probe from ends of insert Re-screen genomic library Restriction map new genomic clones } } Prepare new probes from distal ends of least overlapping insert. Re-screen genomic library . Restriction map new genomic clones Chromosome walking
  41. 41. Thanks