分子生物学3-sectioni-3

1、Section I Gene libraries and screening,Libraries of DNA Molecules Can Be Created by Cloning,A 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 Const

2、ruction of a DNA library,I 1 Genomic libraries,Representative gene libraries Size of library Genomic DNA,Key notes :,BACK,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 librarie

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

4、ssing original sequence,Too long for the vector used,Back,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:,N =,ln (1-P),ln (1-f),P: des

5、ired 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),Ea

6、sy 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 libraries,Purify genomic DNA Fragmentation of DNA : physical shearing and restriction enzyme digestion,eukaryotes,prokaryotes,Clone the

7、 fragments into vectors,Back,To 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,Back,remove protein, l

8、ipids and other unwanted macro- molecules by protease digestion and phase extraction.,Eukaryotes :prepare cell nuclei,Vectors,According to genomes size,we can select a proper vector to construct a library .,Vectors Plasmid phage cosmid YAC insert (kb) 10 23 45 1000,The most commonly chosen genomic c

9、loning vectors are replacement vectors which must be digested with restriction enzymes to produce the two end fragment or arms between which the genomic DNA will be digested,cos,cos,Long (left) arm,short (right) arm,Exogenous DNA (20-23 kb), phage vector in cloning,cos,cos,Long (left) arm,short (rig

10、ht) arm,Exogenous DNA (20-23 kb),I 2 cDNA libraries,mRNA isolation, purification,Check the RNA integrity,Fractionate and enrich mRNA,Synthesis of cDNA,Treatment of cDNA ends,Ligation to vector,BACK,cDNA libraries,No cDNA library was made from prokaryotic mRNA. Prokaryotic mRNA is very unstable Genom

11、ic libraries of prokaryotes are easier to make and contain all the genome sequences.,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 t

12、o identify new genes Tissue or cell type specific (differential expression of genes),cDNA libraries,Making cDNA libraries-mRNA isolation,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 magne

13、tic 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 gradients,Three methods to isolate mRNA.,Back,Make sure that the mRNA is not degraded. Methods: Translating t

14、he mRNA : use cell-free trans- lation 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,Back,Making cDNA libraries- Check the mRNA integrity,Synthesis of cDNA :,First stand syn

15、thesis: materials as reverse transcriptase ,primer( oligo(dT) or hexanucleotides) and dNTPs Fig2.1,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. Fig2.2,Back,5,mRNA,AAAAA-3,HO-TTTTTP-5,5,Re

16、verse transcriptase Four dNTPs,AAAAA-3,TTTTTP-5,mRNA,mRNA,cDNA,cDNA,cDNA,Duplex cDNA,AAAAACCC-3,TTTTTP-5,TTTTTP-5,3,3-CCCCCCC,Terminal transferase dCTP,Alkali (hydrolyaes RNA) Purify DNA oligo(dG),Klenow polymerase or reverse Transcriptase Four dNTPs,5-pGGGG-OH,5,3-CCCCCCC,5-pGGGG,3-CCCCCCC,TTTTTP-5

17、,-3,Back,5-pGGGG,3-CCCCCCC,HO-CCGAATTCGGGGGG,3-GGCTTAAGCCCCCC,5-pAATTCGGGGGG,TTTTTGGCTTAAGCC-OH,CCGAATTCGG-3,3-CCCC,3-CCCCCCC,3-CCC,5-pGGGG,5-pGGGG,TTTTTp-5,-3,TTTTTp-5,TTTTTp-5,-3,-3,TTTTTGGCTTAAp-5,HO-CCG/AATTCGG-3,3-GGCTTAA/GCC-OH,CCG-3,Duplex cDNA,Single strand-specific nuclease,Klenow polymeras

18、e,treat with EcoRI methylase,Add EcolRI linkers using T4 DNA ligase,EcoRI digestion,Ligate to vector and transform,Back,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 .,Using nucleic acid probe

19、to screen the library based on hybridization with nucleic acids. Analyze the protein product,back,Screening libraries,Hybridization to identify the interested DNA or its RNA product 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 Blotting the DNA or RNA on a membrane Hybridize the labeled probe with DNA membrane (Southern) or RNA (Northern) membrane,Searching the genes of