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Section I – Gene libraries and screening. Contents. I1 Genomic libraries Representative gene libraries , Size of library , Genomic DNA , Vectors I2 cDNA libraries mRNA isolation, purification and fractionation , Synthesis of cDNA , Treatment of cDNA , Ligation to vector
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Contents I1 Genomic libraries Representative gene libraries, Size of library, Genomic DNA, Vectors I2 cDNA libraries mRNA isolation, purification and fractionation, Synthesis of cDNA, Treatment of cDNA, Ligation to vector I3 Screening procedures Screening, Colony and plaque hybridization, Expression screening, Hybrid arrest and release, Chromosome walking
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 library cDNA libraries (made from cDNA- copy of mRNA)
Too long for the vector used Important consideration: Making a representative library--- Containing all the original sequences Missing original sequence: (1) Lacking restriction sites (2) Does not contain sufficient clones (3) Enrich certain sequences, lack others
I1 Genomic libraries — Size of library Theformula to calculate the number of recombinants: P: desired probability f : the fraction of the genome in one insert
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.
I1 Genomic libraries — Genomic DNA Eukaryotes Purify genomic DNA Correct size for cloning into the chosen vector: Physical shearing and restriction enzyme digestion Prokaryotes Clone the fragments into vectors
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
2. Break DNA into fragments randomly (1) Physical shearing Pipeting, mixing or sonication. The choice of method and time of exposure depend on the size requirement of the chosen vector.
(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’
Endsproduced (sticky or blunt) & the cleaved ends of the vector to be cloned DNA modifications Whether the enzyme is inhibited by DNA modifications (CpG methylation in mammals).
I1 Genomic libraries — Vectors According to genome’s size, select a proper vector to construct a library . VectorsPlasmid phageλ cosmid YAC insert (kb)10 23 45 1000
λ replacement vector 1.Preparation of arms and genomic inserts 2.Ligation 3. Packing with a mixture of the phage coat proteins and phage DNA-processing enzymes 4. Infection and formation of plaques Library constructed
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 libraries 2. Methods to isolate mRNA 3. Check the mRNA integrity 4. Cloning the particular mRNAs
1. Characteristics of cDNA libraries • (a) 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. • (b) cDNA libraries are very useful for eukaryotic gene analysis • cDNAs 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 directly • Tissue or cell type specific (differential expression of genes
2. Methods to isolate mRNA mRNA isolation, purification Check the RNA integrity Fractionate and enrich mRNA Synthesis of cDNA Treatment of cDNA ends Ligation to vector
Most eukaryotic mRNAs are polyadenylated at their 3’ ends. 5’- cap 3’-AAAAAAAAAAn • oligo (dT) can be bound to the poly(A) tail and used to recover the mRNA.
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.
100mM NaCl rRNA and tRNA 10mM Tris, 1mM EDTA, poly(A) and -oligo(dT)
3. Check the mRNA integrity • Make sure that the mRNA is not degraded. • Methods: • Translating the mRNA :use cell-free translation system aswheat germ extractorrabbit reticulocytelysateto see if the mRNAs can be translated • Analysis the mRNAs by gel electrophoresis: Use agarose or polyacrylamide gels
4. 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: make a cDNA library of the mRNA sequences that are induced with a hormone (hybridization ， substrated cDNA library)
I2 cDNA libraries — Synthesis of cDNA • The first strand and Second strand synthesis
I2 cDNA libraries — Treatment of cDNA • Blunt end ligation of large fragments is not efficient, so we have to use special linkers to create sticky ends for cloning. HO-CCGAATTCGGGGGG DNA linker: 3’-GGCTTAAGCCCCCC HO -CGGGGGG DNA adaptor: 3’-TTAAGCCCCCC
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) Restriction enzyme digestion( EcoRI )
I2 cDNA libraries — Ligation to vector Any vectors with an EcoRI site would suitable for cloning the cDNA. The process : Dephosphorylate the vector Ligate vector and cDNA with T4 DNA ligase Plasmid 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.
I3 Screening procedures — Screening • 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
Screening libraries Hybridization to identify the interested DNA or its RNA product. (1) DNA radiolabeled probes which is complementary to a region of the interested gene.
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
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. (Alkali treatment) Hybridization(in a solution containing Nucleic acid probe) X-ray film (radio- actively labeled ) Wash toremoveunhybri- dized probe andvisualize Antibody or enzyme (modified nucleotide labeled) Line upthe hybridizated region or repeated hybridization
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
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 haveone in six possibilities (1/6)to be in both the correct orientation(twopossibilities;)and reading frame(three possibilities).
I3 Screening procedures — Hybrid arrest and release • (1) Hybrid arrested translation • Individual 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 translation • Purify the hybrids and the hybridized mRNAs released from them and translated, it identifies the protein encoded by the cDNA clone
I3 Screening procedures — Chromosome walking Definition:To clone the desired gene by repeated isolating adjacent genomic clones from the library.
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.
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.