分子生物学教学课件：Section IGene libraries and screening

1、 Section I Gene libraries and screening Libraries of DNA Molecules Can Be Created by CloningA DNA library(文库)is a population of identical vectors that each contains a different DNA insert.Genomic libraries(基因组文库) derived from total genomic DNA cleaved with a restriction enzyme.返回Figure 20-8 Construc

2、tion of a DNA libraryI 1 Genomic libraries nRepresentative gene libraries nSize of library nGenomic DNA Key notes :Key notes :BACKGene 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 l

3、ibraries cDNA libraries Gene library (made from genomic DNA) (made from cDNA- copy of mRNA)Making a representative library - Containing all the original sequences1. Certain sequences have not been cloned.Example: repetitive sequences lacking restriction sites 2.Library does not contain sufficient cl

4、onesMissing original sequenceToo long for the vector usedBack 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:N = ln (1-P) ln (1-f) P: d

5、esired probability f : the fraction of the genome in one insert For example :for a probability of 0.99 with insert sizes of 20kb these values for the E. coli (4.6106 bp) and human (3109 bp) genomes are :N E.coli= =1.1 103 ln( 1-0.99) ln1-(2104/4.6106)Nhuman= = 6.9 105 ln(1-0.99) ln1-(2 104/3 109)Eas

6、y 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. Back Genomic DNA librariesPurify genomic DNA Fragmentation of DNA : physical shearing and restriction enzyme digestion eukaryotes prokaryotesClone the fr

7、agments into vectorsBackTo make a representative genomic libraries ,genomic DNA must be purified and then broken randomly into fragments that are correct size for cloning into the chosen vector. Purification of genomic DNA : Prokaryotes: extracted DNA directly from cells Backremove protein, lipids a

8、nd other unwanted macro-molecules by protease digestion and phase extraction.Eukaryotes :prepare cell nucleiBreak DNA into fragments randomly:Physical shearing : pipeting, mixing or sonicaion Restriction enzyme digestion: partial digestion is preferred to get a greater lengths of DNA fragments.Sau3A

9、: 5-/GATC-3, less selectivityBamH1: 5-G/GATCCSelection of restriction enzyme1. Ends produced (sticky or blunt) &The cleaved ends of the vector to be cloned2. Whether the enzyme is inhibited by DNA modifications (CpG methylation in mammals3. Time of digestion and ration of restriction enzyme to D

10、NA is dependent on the desired insert size range.BackVectors According to genomes size,we can select a proper vector to construct a library .Vectors Plasmid phage cosmid YAC insert (kb) 10 23 45 1000The most commonly chosen genomic cloning vectors are replacement vectors which must be digested with

11、restriction enzymes to produce the two end fragment or arms between which the genomic DNA will be digestedcoscosLong (left)armshort (right)armExogenous DNA(20-23 kb) phage vector in cloning coscosLong (left)armshort (right)armExogenous DNA(20-23 kb) I I 2 2 cDNA librariescDNA librariesmRNA isolation

12、, purificationCheck the RNA integrity Fractionate and enrich mRNA Synthesis of cDNA Treatment of cDNA ends Ligation to vectorBACKcDNA libraries1. 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

13、genome sequences.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 e

14、xpression of genes) cDNA libraries Making cDNA libraries-mRNA isolation 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.AAAAAAAAAAn5 cap1.Traditionally method was done by pass a preparation of total RNA down a column o

15、f 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 magnet 3.Alternative route of isolating mRNA is lysing cells and then preparing mRNA-ribosome complexes on sucrose gradientsThree methods to iso

16、late mRNA.Back Make sure that the mRNA is not degraded. Methods:Translating the mRNA : use cell-free trans- lation 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 Back Making

17、cDNA libraries- Check the mRNA integritySynthesis of cDNA :First stand synthesis: materials as reverse transcriptase ,primer( oligo(dT) or hexanucleotides) and dNTPs Fig2.1Second strand synthesis: best way of making full-length cDNA is to tail the 3-end of the first strand and then use a complementa

18、ry primer to make the second. Fig2.2Back 5 mRNA AAAAA-3 HO-TTTTTP-55Reverse transcriptaseFour dNTPsAAAAA-3TTTTTP-5 mRNA mRNA cDNAcDNAcDNADuplex cDNAAAAAACCC-3TTTTTP-5 TTTTTP-533-CCCCCCCTerminal transferasedCTPAlkali (hydrolyaes RNA)Purify DNA oligo(dG)Klenow polymerase or reverseTranscriptase Four d

19、NTPs5-pGGGG-OH53-CCCCCCC5-pGGGG3-CCCCCCCTTTTTP-5-3Back5-pGGGG3-CCCCCCCHO-CCGAATTCGGGGGG 3-GGCTTAAGCCCCCC 5-pAATTCGGGGGG TTTTTGGCTTAAGCC-OH CCGAATTCGG-3 3-CCCC 3-CCCCCCC 3-CCC 5-pGGGG 5-pGGGGTTTTTp-5 -3TTTTTp-5TTTTTp-5 -3 -3 TTTTTGGCTTAAp-5 HO-CCG/AATTCGG-3 3-GGCTTAA/GCC-OH CCG-3Duplex cDNASingle str

20、and-specific nucleaseKlenow polymerasetreat with EcoRI methylaseAdd EcolRI linkers using T4 DNA ligaseEcoRI digestionLigate to vector and transformBackScreening Screening The process of identifying one particular clone containing the gene of interest from among the very large number of others in the

21、 gene library . 1. Using nucleic acid probe to screen the library based on hybridization with nucleic acids. 2. Analyze the protein productbackScreening libraries Hybridization to identify the interested DNA or its RNA product1. Radiolabeled 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 species 2. Blotting the DNA or RNA on a membrane 3. Hybridize the labeled probe with DNA