基因克隆表達(dá)及功能研究-1

1、Gene cloning, expression and functional study基因克隆，表達(dá)及功能研究,vectors,Cloning vectors: 克隆載體to clone a gene in a vectorExpression vectors: 表達(dá)載體to express a gene from a vectorIntegration vectors: 整合載體to integrate a

2、 gene in a genome through a vector,Cloning vectors,1 Plasmid vecters2 Bacteriophage vectors3 Cosmids & BACs 4 Eukaryotic vectors,Cloning vectors: allowing the exogenous DNA to be inserted, stored, and manipulated

3、mainly at DNA level.,expression vectors: allowing the exogenous DNA to be inserted, stored, and expressed.,Contains an origin of replication, allowing for replication independent of host’s genome.Contains Selective mark

4、ers: Selection of cells containing a plasmid twin antibiotic resistanceblue-white screeningContains a multiple cloning site (MCS)Easy to be isolated from the host cell.,A plasmid vector for cloning,Ampicillin resis

5、tant? yes yesTetracycline resistant? No yes,,,,,,,,,,,,,,,,,B X B,,,,B,B,X,Ampr,ori,Ampr,Tcr,ori,-Screening by insertional inactivation of a resistance gene,,Twin

6、antibiotic resistance screening,,,Replica plating: transfer of the colonies from one plate to another using absorbent pad or Velvet (絨布).,transfer of colonies,+ampicillin,+ ampicillin+ tetracycline,these colonies have b

7、acteria with recombinant plasmid,Blue white screening,,Ampr,ori,pUC18(3 kb),,,,,,,,MCS (Multiple cloning sites,多克隆位點(diǎn)）,Lac promoter,,lacZ’,Screening by insertional inactivation of the lacZ gene,The insertion of a DNA f

8、ragment interrupts the ORF of lacZ’ gene, resulting in non-functional gene product that can not digest its substrate x-gal.,Recreated vector: blue transformantsRecombinant plasmid containing inserted DNA: white transfor

9、mants,,,,,,,,,,,,,,,,Recreated vector (no insert),,Recombinant plasmid (contain insert),back,Multiple cloning sites,Multiple restriction sites enable the convenient insertion of target DNA into a vector,A plasmid vector

10、for gene expression,Expression vectors: allowing the exogenous DNA to be inserted, stored and expressed.,Promoter and terminator for RNA transcription are required.Intact ORF and ribosomal binding sites (RBS) are requir

11、ed for translation.Include：(1) bacterial expression vectors, (2) yeast expression vectors, (3) mammalian expression vector,T7 promoter,RBS,Start codon,MCS,Transcription terminator,Ampr,ori,T7 expression vector,An bacter

12、ial expression vector,A yeast expression vector,Bacteriophage vector,,Two examples:λ phage bacteriophageλλ replacement vector M13 phageM13 phage vectorCloning in M13Hybrid plasmid-M13 vectors,viruses that can infe

13、ct bacteria. 48.5 kb in lengthLinear or circular genome (cos ends)Lytic phase (Replicate and release)Lysogenic phase (integrate into host genome),λ phage,,,Analysis of eukaryotic genes and the genome organization of

14、 eukaryotes requires vectors with a larger capacity for cloned DNA than plasmids or phage ?.Human genome (3 x 109 bp): large genome and large gene demand vectors with a large size capacity.,Cloning large DNA fragments,(

15、Eukaryotic Genome project),Genomic library VS cDNA library,Cosmid vectors,Utilizing the properties of the phage l cos sites in a plasmid vector.A combination of the plasmid vector and the COS site which allows the targ

16、et DNA to be inserted into the l head.The insert can be 37-52 kb,C) Packaging and infect,Formation of a cosmid clone,YAC vectors,Accommodates genomic DNA fragments of more than 1 Mb, and can be used to clone the entire

17、 human genome, but not good in mapping and analysis.,(yeast artificial chromosome),Essential components of YAC vectors : Centromers (CEN), telomeres (TEL) and autonomous replicating sequence (ARS) for proliferation in

18、the host cell. ampr for selective amplification and markers such as TRP1 and URA3 for identifying cells containing the YAC vector in yeast cells. Recognition sites of restriction enzymes (e.g., EcoRI and BamHI),YAC

19、 Cloning,BAC vectors 細(xì)菌人工染色體1. More stable than YAC2. Capacity is 300-350 kb3. One to two copies in each cell4. Easy to handle5. More popular in genomic mapping,I1 Genomic libraries,I1-1 Representative gene li

20、braries I1-2 Size of library I1-3 Genomic DNA I1-4 Vectors,Gene libraries and screening,,,Gene library: a collection of different DNA sequence from an organism, each of which has been cloned into a vector for

21、ease of purification, storage and analysis.,,Genomic libraries,cDNA libraries,,Gene library,(made from genomic DNA),(made from cDNA- copy of mRNA),I1 Genomic libraries,I1-1 Representative gene libraries,--- Contain all t

22、he original sequences,Certain sequences have not been cloned.Example: repetitive sequences lacking restriction sites,2. Library does not contain sufficient clones,Missing original sequence,,,,Too long for the vector u

23、sed,I1 Genomic libraries,,I1-2 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 sequence,The formula to calculate th

24、e number of recombinants:,N =,ln (1-P),ln (1-f),,P: desired probability f : the fraction of the genome in one insert,I1 Genomic libraries,For example :for a probability of 0.99 with insert sizes of 20 kb these val

25、ues for the E.coli (4.6×106 bp) and human (3×109 bp) genomes are :N E.coli= = 1.1 ×103,,ln( 1-0.99),ln[1-(2×104/4.6×106)],Nhuman=

26、 = 6.9 ×105,,ln(1-0.99),ln[1-(2 ×104/3 ×109)],These values explain why it is possible to make good genomic libraries from prokaryotes in plasmids where the insert size is 5-1

27、0kb ,as only a few thousand recombinants will be needed.,I1 Genomic libraries,I1-3 Genomic DNA libraries,Purify genomic DNA Fragment this DNA : physical shearing and restriction enzyme digestion,eukaryotes,prokaryot

28、es,,,,,,Clone the fragments into vectors,I1 Genomic libraries,,To make a representative genomic libraries ,genomic DNA must be purified and then broken randomly into fragments that are correct in size for cloning int

29、o the chosen vector.,,Purification of genomic DNA :,,Prokaryotes :extracted DNA directly from cells,remove protein, lipids and other unwanted macro-molecules by protease digestion and phase extraction.,Eukaryotes :prep

30、are cell nuclei,I1 Genomic libraries,,Break DNA into fragments randomly:,Physical shearing : pipeting, mixing or sonicaion,Restriction enzyme digestion: partial digestion is preferred to get a greater length