2 Contents I1 Genomic libraries I2 cDNA libraries Representative gene libraries, Size of library, Genomic DNA, VectorsI2 cDNA librariesmRNA isolation, purification and fractionation, Synthesis of cDNA, Treatment of cDNA, Ligation to vectorI3 Screening proceduresScreening, Colony and plaque hybridization, Expression screening, Hybrid arrest and release, Chromosome walking
3 I1 Genomic libraries — Representative gene libraries Genomic 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)
4 Important consideration: Making a representative library--- Containing all the original sequencesMissing original sequence:(1) Lacking restriction sitesToo long for the vector used(2) Does not contain sufficient clones(3) Enrich certain sequences, lack others
5 I1 Genomic libraries — Size of library The formula to calculate the number of recombinants:P: desired probabilityf : the fraction of the genome in one insert
6 For a probability of 0.99 with insert sizes of 20kb these values for the E. coli (4.6×106 bp) and human (3×109 bp) genomes are :Easy 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.
7 I1 Genomic libraries — Genomic DNA EukaryotesPurify genomic DNACorrect size for cloning into the chosen vector:Physical shearing and restriction enzyme digestionProkaryotesClone the fragments into vectors
8 1. Purification of genomic DNA Eukaryotes: Prepare cell nuclei (fractionation, reduce contamination from organelle DNA)Remove protein, lipids and other unwanted macro-molecules by protease digestion and phase extraction（Phenol-chloroform）.Prokaryotes: Extracted DNA directly from cells
9 2. Break DNA into fragments randomly (1) Physical shearingPipeting, mixing or sonication. The choice of method and time of exposure depend on the size requirement of the chosen vector.
10 (2)Restriction enzyme digestion Partial digestion:Get a greater lengths of DNA fragments. Time of digestion and ration of restriction enzyme to DNA are dependent on the desired insert size range, the DNA is not digested at every recognition sequence that is present.Sau3A: 5’-/GATC-3’,BamH1: 5’-G/GATCC-3’
11 Ends produced (sticky or blunt) & the cleaved ends of the vector to be cloned DNA modificationsWhether the enzyme is inhibited by DNA modifications (CpG methylation in mammals).
12 I1 Genomic libraries — Vectors According to genome’s size, select a proper vector to construct a library .Vectors Plasmid phageλ cosmid YACinsert (kb)
13 2.Ligation λ replacement vector Library constructed 1.Preparation of arms and genomic inserts2.Ligation3. Packing with a mixture of the phage coat proteins and phage DNA-processing enzymes4. Infection and formation of plaquesLibrary constructed
14 I2 cDNA libraries — mRNA isolation, purification and fractionation The most commonly chosen genomic cloning vectors are λ replacement 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 ligated.1. Characteristics of cDNA libraries2. Methods to isolate mRNA3. Check the mRNA integrity4. Cloning the particular mRNAs
15 1. Characteristics of cDNA libraries (a) No 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.(b) cDNA libraries are very useful for eukaryotic gene analysiscDNAs represent the transcribed parts of the genome (i.e. the genes rather than the nontranscribed DNA). cDNAs have no introns genes can be expressed in E. coli directlyTissue or cell type specific (differential expression of genes
16 2. Methods to isolate mRNA mRNA isolation, purificationCheck the RNA integrityFractionate and enrich mRNASynthesis of cDNATreatment of cDNA endsLigation to vector
17 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.3’-AAAAAAAAAAn5’- cap
18 Three methods:Traditional method was done by pass a preparation of total RNA down a column of oligo (dT)-cellulose.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.Lying cells and then preparing mRNA-ribosome complexes on sucrose gradients.
19 100mM NaClrRNA and tRNA10mM Tris, 1mM EDTA, poly(A) and -oligo(dT)
20 3. 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 translatedAnalysis the mRNAs by gel electrophoresis: Use agarose or polyacrylamide gels
21 4. Cloning the particular mRNAs Is useful especially one is trying to clone a particular gene rather to make a complete cDNA libraryFractionate on the gel: Performed on the basis of size, mRNAs of the interested sizes are recovered from agarose gelsEnrichment: Carried out by hybridization.Example: make a cDNA library of the mRNA sequences that are induced with a hormone (hybridization ， substrated cDNA library)
22 I2 cDNA libraries — Synthesis of cDNA The first strand and Second strand synthesis
23 I2 cDNA libraries — Treatment of cDNA HO-CCGAATTCGGGGGGDNA linker:3’-GGCTTAAGCCCCCCHO - CGGGGGGDNA adaptor:3’-TTAAGCCCCCCBlunt end ligation of large fragments is not efficient, so we have to use special linkers to create sticky ends for cloning.
25 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)Restriction enzyme digestion( EcoRI )
26 I2 cDNA libraries — Ligation to vector Any vectors with an EcoRI site would suitablefor cloning the cDNA.The process :Dephosphorylate the vectorLigate vector and cDNA with T4 DNA ligasePlasmid or l phage vector, short, plasmid vector;cDNA libraries, λ phage vector;λgt11 has EcoRI site placed near the C terminus of its lacZ gene, enabling expression of the cDNA as part of a large β-galactosidase fusion protein.
27 I3 Screening procedures — Screening Screening: The process of identifying one particular clone containing the gene of interest from among thevery 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
28 Screening librariesHybridization to identify the interested DNA or its RNA product.(1) DNA radiolabeled probes which is complementary to a region of the interested gene.
29 DNA sequence information: An oligonucleotide derived from the sequence of a protein product of the gene.A DNA fragment/oligo from a related gene of another species.Preparation methods:Automated chemical synthesis (short probes)PCR
30 I3 Screening procedures — Colony and plaque hybridization Transfer the DNA in the plaque or colony to a Nylon ornitrocellulose membranePhage DNA bind tothe membrane directlyBacterial colonies must be lysed torelease DNA on the membrane surface.(Alkali treatment)Hybridization (in a solutioncontaining Nucleic acid probe)X-ray film (radio-actively labeled )Wash to remove unhybri-dized probe and visualizeAntibody or enzyme(modified nucleotide labeled)Line up the hybridizated region orrepeated hybridization
31 Transfer to nitrocellulose or nylon membrane Keep masterPlateSelect positivefrom master plateDenature DNA (NaOH). Bake onto membraneProbe with 32p-labled DNAcomplementary togene of interestExpose to filmScreening by plaque hybridization
33 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.Example: the EcoRI site of lgt11 vector. The inserted genes have one in six possibilities (1/6) to be in both the correct orientation (two possibilities; ) and reading frame (three possibilities).
34 I3 Screening procedures — Hybrid arrest and release (1) Hybrid arrested translationIndividual cDNA clones or pools of clones can be used to hybridize to mRNA preparation.Translate the mRNA population directly, and the inhibition of translation of some products detected.（2）Hybrid release translationPurify the hybrids and the hybridized mRNAs released from them and translated, it identifies the protein encoded by the cDNA clone
35 I3 Screening procedures — Chromosome walking Definition: To clone the desired gene by repeated isolating adjacent genomic clones from the library.
37 Multiple choice questions 1. Which two of the following statements about genomic libraries are false?A genomic libraries are made from cDNA.B genomic libraries must be representative if they are to contain all the genes in an organism.C genomic libraries must contain a minimum number of recombinants if they are to contain all the genes In an orgamsm.D the DNA must be fragmented to an appropriate size for the vector that is used.E genomic libraries made from eukaryotic DNA usually use plasmid vectors.2. Which statement correctly describes sequential steps in cDNA cloning?A reverse transcription of Mrna second strand synthesis cDNA end modification ligation to vector.B mRNA preparation cDNA synthesis using reverse transcriptase second strand synthesis using terminal transferase, ligation to vector.C mRNA synthesis using RNA polymerase reverse transcription of mRNA, second strand synthesis, ligation to vector.D double stranded cDNA synthesis restriction enzyme digestion addition of linkers ligation to vector.
38 3. Which one of the following is not a valid method of screening a library? A hybridization of colony / plaque-lifted DNA using a nucleic acid probe.B using antibodies raised against the protein of interest to screen an expression library.C screening pools of clones from an expression library for biological activity.D hybridization of colony/plaque-lifted DNA using an antibody probe.