4 (made from cDNA- copy of mRNA) I1 Genomic librariesGene 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(made from genomic DNA)Gene librarycDNA libraries(made from cDNA- copy of mRNA)
5 --- Contain all the original sequences I1 Genomic librariesI1-1 Representative gene libraries--- Contain all the original sequencesMissing original sequenceCertain sequences have not been cloned.Example: repetitive sequences lacking restriction sitesToo long for the vector used2. Library does not contain sufficient clones
6 I1-2 Size of library (ensure enough clones) I1 Genomic librariesI1-2 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 sequenceThe formula to calculate the number of recombinants:ln (1-P)N =ln (1-f)P: desired probabilityf : the fraction of the genome in one insert
7 I1 Genomic librariesFor 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= = 1.1 ×103ln( )ln[1-(2×104/4.6×106)]ln(1-0.99)Nhuman= = ×105ln[1-(2 ×104/3 ×109)]These values explain why it is possible to make goodgenomic libraries from prokaryotes in plasmids wherethe insert size is 5-10kb ,as only a few thousandrecombinants will be needed.
8 I1-3 Genomic DNA libraries I1 Genomic librariesI1-3 Genomic DNA librarieseukaryotesPurify genomic DNAFragment this DNA : physical shearing and restriction enzyme digestionprokaryotesClone the fragments into vectors
9 Eukaryotes :prepare cell nuclei I1 Genomic librariesTo make a representative genomic libraries ,genomic DNA must be purified and thenbroken randomly into fragments that arecorrect in size for cloning into the chosen vector.Purification of genomic DNA :Eukaryotes :prepare cell nucleiremove protein, lipids and other unwanted macro-molecules by protease digestion and phase extraction.Prokaryotes :extracted DNA directly from cells
10 pipeting, mixing or sonicaion I1 Genomic librariesBreak DNA into fragments randomly:Physical shearing :pipeting, mixing or sonicaionRestriction enzyme digestion:partial digestion is preferred to get a greater lengths of DNA fragments.
11 Selection of restriction enzyme I1 Genomic librariesSelection of restriction enzymeEnds produced (sticky or blunt) &The cleaved ends of the vector to be clonedSau3A: 5’-/GATC-3’, less selectivityBamH1: 5’-G/GATCCWhether the enzyme is inhibited by DNA modifications (CpG methylation in mammalsTime of digestion and ratio of restriction enzyme to DNA is dependent on the desired insert size range.
12 I1-4 Vectors Vectors Plasmid phageλ cosmid YAC I1 Genomic libraries According to genome’s size,we can select a proper vector to construct a library .Vectors Plasmid phageλ cosmid YACinsert (kb)The most commonly chosen genomic cloning vectorsare λ relacement vectors which must be digested withrestriction enzymes to produce the two λ end fragmentor λ arms between which the genomic DNA will bedigested
13 λ phage vector in cloning Long (left)armshort (right)armcosExogenous DNA(~20-23 kb)cosLong (left)armshort (right)armcoscosExogenous DNA(~20-23 kb)
14 λ replacement vector cloning 0.preparation of arm and genomic inserts2. Packing with a mixture of the phage coat proteins and phage DNA-processing enzymesLigation3. Infection and formation of plaquesLibrary constructed
15 I 2 cDNA libraries Gene libraries and screening I2-1 mRNA isolation, purificationI2-2 Check theRNA integrityI2-3 Fractionate and enrich mRNAI2-4 Synthesis of cDNAI2-5 Treatment of cDNA endsI2-6 Ligation to vector
16 No cDNA library was made from prokaryotic mRNA. I 2 cDNA librariescDNA librariesNo cDNA library was made from prokaryotic mRNA.Prokaryotic mRNA is very unstableGenomic libraries of prokaryotes are easier to make and contain all the genome sequences.
17 cDNA libraries are very useful for eukaryotic gene analysis I 2 cDNA librariescDNA librariescDNA libraries are very useful for eukaryotic gene analysisCondensed protein encoded gene libraries, have much less junk sequences.cDNAs have no introns genes can be expressed in E. coli directlyAre very useful to identify new genesTissue or cell type specific (differential expression of genes)
18 I 2 cDNA librariesI2-1 mRNA isolationMost eukaryotic mRNAs are polyadenylated at their 3’ endsoligo (dT) can be bound to the poly(A) tail and used to recover the mRNA.AAAAAAAAAAn5’ cap
20 Three methods to isolate mRNA. I2 cDNA librariesThree methods to isolate mRNA.1.Traditionally method was done by pass a preparation of total RNA down a column of oligo (dT)-cellulose2.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 magnet3.Alternative route of isolating mRNA is lysing cells and then preparing mRNA-ribosome complexes on sucrose gradients
21 I2-2 Check the mRNA integrity I2 cDNA librariesI2-2 Check the mRNA integrityMake 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 translatedAnalysis the mRNAs by gel elctrophoresis: use agarose or polyacrylamide gels
22 Enrichment: carried out by hybridization I2 cDNA librariesI2-3 Cloning the particular mRNAsIs 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 gelsEnrichment: carried out by hybridizationExample: clone the hormone induced mRNAs (substrated cDNA library)
23 Second strand synthesis: best way of I2 cDNA librariesI2-4 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 ofmaking full-length cDNA is to ‘tail’ the 3’-end of the first strand and then use a complementary primerto make the second. (Fig2.1)
24 I2 cDNA libraries Fig 1.1 The first strand synthesis mRNA 5’ AAAAA-3’ HO-TTTTTP-5’Reverse transcriptaseFour dNTPsmRNA5’AAAAA-3’3’TTTTTP-5’cDNATerminal transferasedCTPmRNA5’AAAAA-3’3’-CCCCCCCTTTTTP-5’cDNAAlkali (hydrolyaes RNA)Purify DNA oligo(dG)5’-pGGGG-OH3’-CCCCCCCTTTTTP-5’cDNAKlenow polymerase or reverseTranscriotase Four dNTPs5’-pGGGG-3’3’-CCCCCCCTTTTTP-5’Duplex cDNAFig The first strand synthesis
25 Single strand-specific nuclease Duplex cDNA5’-pGGGG-3’3’-CCCCCCCTTTTTp-5’Single strand-specific nuclease5’-pGGGG-3’3’-CCCTTTTTp-5’Klenow polymerasetreat with E.coRI methylase5’-pGGGG-3’3’-CCCCTTTTTp-5’Add E.colRI linkersusing T4 DNA ligaseHO-CCG/AATTCGG-3’3’-GGCTTAA/GCC-OHHO-CCGAATTCGGGGGGCCGAATTCGG-3’3’-GGCTTAAGCCCCCCTTTTTGGCTTAAGCC-OHE.colRI digestion5’-pAATTCGGGGGGCCG-3’3’-CCCCCCCTTTTTGGCTTAAp-5’Ligate to vector and transfomFig Second strand synthesis
26 I2-5 Treatment of cDNA ends I2 cDNA librariesI2-5 Treatment of cDNA endsBlunt 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 ofDNA polyI and 4 dNTPs)Ligate the blunt-end and linkers(T4 DNA ligase)Tailing with terminal transferase or using adaptor moleculesRestriction enzyme digestion (E.coRI )
27 I2-6 Ligation to vector The process : I2 cDNA libraries Any vectors with an E.coRI site would suitablefor cloning the cDNA.The process :Dephosphorylate the vector with alkalinephosphataseLigate vector and cDNA with T4 DNA ligase(plasmid or λ phage vector)
28 I3 Screening procedures Gene libraries and screeningI3 Screening proceduresI3-1 ScreeningI3-2 Colony and plaque hybridizationI3-3 Expression screeningI3-4 Hybrid arrest and releaseI3-5 Chromosome walking (repeat screening)
29 I3-1 Screening I3 Screening procedures The process of identifying one particular clone containing the gene of interest from among the very large number of others in the gene library .Using nucleic acid probe to screen the library based on hybridization with nucleic acids.Analyze the protein product.
30 I3 Screening procedures Screening librariesSearching the genes of interest in a DNA libraryHybridization to identify the interested DNA or its RNA productRadiolabeled probes which is complementary to a region of the interested geneProbes:An oligonucleotide derived from the sequence of a protein product of the geneA DNA fragment/oligo from a related gene of another speciesBlotting the DNA or RNA on a membraneHybridize the labeled probe with DNA membrane (Southern) or RNA (Northern) membrane
31 I3-2 Colony and plaque hybridization I3 Screening proceduresI3-2 Colony and plaque hybridizationTransfer the DNA in the plaque or colony to aNylon or nitrocellulose membranePhage DNA bind tothe membrane directlyBacterial colonies must be lysed torelease DNA on the membrane surface.Hybridization (in a solutionContaining Nucleic acid probe)(Alkali treatment)X-ray film(radio-actively labeled )antibody or enzyme(modified nucleotide labeledWash to remove unhybri-dization probe and visualizeLine up the hybridizated region orrepeated hybridization
32 I3 Screening procedures Transfer to nitrocelluloseor nylon membraneKeep masterplateSelect positivefrom master plateDenature DNA(NaOH)Bake onto membraneProbe with 32p-labled DNAcomplementary togene of interestExpose to filmScreening by plaque hybridization
33 I3-3 Expression screening I3 Screening proceduresI3-3 Expression screeningIdentify the protein product of an interested geneProtein activityWestern blotting using a specific antibody
34 Expression screening (1) I3 Screening proceduresExpression screening (1)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 cloneExample: the EcoRI site of lgt11 vector. The inserted genes have one in six change (1/6) to be in both the correct orientation (2 possibilities; ) and reading frame (three possibilities; three nucleotide code XXX).
35 Expression screening (2) I3 Screening proceduresExpression screening (2)Antibodies can be used to screen the expression library.The procedure has similarities to the plaquehybridization protocol.‘Plaque lift’ ( taken by placing amembrane on the dish of plaque)Immersed in a solution of the antibodyDetected by other antibodiesRepeat cycles of screeningto isolate pure plaques
36 I3-4 Hybrid arrest and screen I3 Screening proceduresI3-4 Hybrid arrest and screenIndividual cDNA clones or pools of clones can be used to hybridize to mRNA preparationHybrid arrest :translate the mRNA populationdirectly, and the inhibition of translation ofsome products detected.Hybrid release translation : purify thehybrids and the hybridized mRNAs releasedfrom them and translated, it identifies theprotein encoded by the cDNA clone
37 I3-5 Chromosome walking I3 Screening procedures Definition: To clone the desired gene byrepeated isolating adjacentgenomic clones from the library.to obtain overlapping genomic clonesthat represent progressively longerparts of a particular chromosome .
38 Process: I3 Screening procedures 1. Prepare a probe from the end insert .2.The probe are used to re-screen the libraryby colony or plaque hybridization3.Analyzed the new isolate clones and positedthem relative to the starting clone.some will be overlapping.4. Repeated the whole process using a probefrom the distal end of the second clone.
39 Chromosome walking Prepare probe from Restriction ends of insert Vector arm Genomic clone insert Vector arm}}Prepare probe fromends of insertRestrictionRe-screen genomiclibraryRestriction 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