Vectors. Plasmids as Vectors. Vectors : the DNA carriers. Plasmids as Vectors. Plasmids as Vectors

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1 Vectors Vectors DNA carriers to allow replication of recombinant DNAs ( 재조합 DNA 의복제를위한 DNA 운반자, 매개체 ) Typical experiment uses 1 vector plus a piece of foreign DNA Foreign DNA depends on the vector for its replication ( 외부 DNA 를벡터를이용복제 ) There are 2 major classes of vectors: Vectors : the DNA carriers Vector 가갖추어야하는점 origin of replication ( 복제기점 ) High transformation efficiency ( 높은형질전환율 ) Selection marker ( 선별표식자 ) Ability to receive larger amounts of foreign DNA Easy to treat 4-1 Plasmids as Vectors Plasmids as Vectors Plasmid Extrachromosomal DNA The first cloning experiment in vitro Boyer & Cohen Plasmid: psc101 and RSF1010 EcoRI restriction enzyme DNA ligase Origin of replication ( 복제기점 ) Plasmids as Vectors Antibiotic-resistant genes ( 선별 Marker) Multiple cloning sites ( 다중클로닝부위 ) 1

2 Plasmids as Vectors pbr plasmids were developed early but are rarely used today Meaning of pbr322 p : plasmid BR : 벡터를처음으로만든사람 Boliver, Rodriguez 322 : 플라스미드를만든수 : pbr325, pbr327, pbr328.. Characteristics of pbr322 Size : 4,363bp - 10kb 이하 Selection marker : ampicillin, tetracycline 저항성유전자 Origin of replication between the 2 resistance genes High copy number ( 높은사본수 ) Multiple cloning site 를가짐 Only 1 site for several restriction enzymes Bacterial Transformation Traditional method involves incubating bacterial cells in concentrated calcium salt solution The solution makes the cell membrane leaky, permeable to the plasmid DNA Newer e method uses high voltage to drive the DNA into the cells in process called electroporation Later in detail pbr Screening Transformants Transformation produces bacteria with: Religated plasmid ( 재결합된플라스미드 ) Religated insert ( 재결합된유전자 ) Recombinants ( 재조합 ; plasmid + insert) Screening Transformants Identify the recombinants using the antibiotic resistance ( 재조합된유전자선별 ) Screening with replica plating ( 복사판제조 ) puc and β-galactosidase puc series : Typical Plasmid for cloning to Bacteria similar to pbr : Based on pbr322 Ampicillin resistance gene Creation of a polylinker or multiple cloning site(mcs; 다중클로닝부위 )

3 puc and β-galactosidase lacz (LacZ 유전자 ) Coding for the amino terminal portion of β-galactosidase X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) IPTG (isopropyl-thiogalactoside) : β-galactosidase 활성유도물질 puc and β-galactosidase Newer puc plasmids have: One step screening : Amp r + color (white) Multiple cloning site inserted into the gene lacz coding for the enzyme β-galactosidase Later in detail 4-14 Directional Cloning Newer puc plasmids have: Directional cloning ( 방향성클로닝 ) : Cloned with 2 different restriction enzymes and Insert DNA is placed in one orientation Phages As Vectors Bacteriophages (phages) are natural vectors that transduce bacterial DNA from one cell to another Phage : A virus for a bacterial cell (bacteria eating) Clones are plaques ( 플라크 ), a clearing of the bacterial lawn due to phages -peptide or N-terminus of - galactosidase Multiple cloning sites Phage Vector Advantages High efficiency of infection than plasmid transformation ( 고효율 ) Used as natural vectors that transduce DNA from one bacterial cell to another Phage vectors can receive larger amounts of foreign DNA ( 긴 DNA 삽입가능 ) Phage vectors require a minimum size foreign DNA piece (12 kb) inserted to package into a phage particle Bacteriophage 에의한 bacteria 의감염양식

4 Phage Vectors First phage vectors were constructed by Fred Blattner and colleagues Retained genes needed for phage replication Could replace removed phage genes with foreign DNA Cloning Using a Phage Vector Phage Vectors 1/3 of genome (48.5 Kb) is non essential The DNA is packaged into phage particles Can only fit Kb of DNA, But take up much more DNA than plasmid Have an in vitro packaging system ( 세포외조립 ) Originally named Charon phage ( 샤론파지 ) More general term, replacement vectors ( 대체벡터, Charon 4) Bacteriophage 의용원성감염주기 Genomic Libraries Used for constructing genomic libraries ( 유전체라이브러리 ) Restriction fragments of a genome can be packaged into phage using about kb per fragment Once a library is established, it can be used to search for any gene of interest Plaque Hybridization Searching a genomic library requires probe showing which clone contains desired gene ( 유전자라이브러리에서관심유전자탐색 ) Ideal probe ( 탐침 ) : labeled nucleic acid with sequence matching the gene of interest 4-21 Later in this lecture Plaque hybridization ( 플라크잡종화 ): Selection of positive genomic clones 4-22 M13 Phage Vectors Long, thin, filamentous phage M13 (M13 파지벡터 ) Contains: Gene fragment with -galactosidase Multiple cloning site ( 다중제한효소부위 ) like the puc family Advantage This phage s genome is single-stranded DNA easy to site directed

5 M13 Cloning to Recover Single-stranded DNA Product After infecting E. coli cells, single-stranded phage DNA is converted to double-stranded replicative form ( 이중가닥의복제형으로변화 ) Recombinant DNA infects host cells resulting in single-stranded recombinant DNA ( 단일가닥형성 ) Cosmids Cosmids ( 코스미드 ) are designed for cloning large DNA fragments Behave as plasmid and phage Contain cos sites, cohesive ends of phage DNA that allow the DNA to be packaged into a phage head Plasmid origin of replication permitting replication as plasmid in bacteria DNA 의선형과원형 Cosmids Nearly all genome removed so there is room for large inserts (40-50 kb) So little phage DNA can t replicate, but they are infectious carrying recombinant DNA into bacterial cells Preparation of recombinant cosmid - replication as plasmid in E.coli. Phagemids Phagemids ( 파지미드 ) are also vectors Like cosmids have aspects of both phages and plasmids Has a MCS inserted into lacz gene to screen blue/white colonies Has origin of replication of single-stranded phage f1 to permit recovery of single-stranded recombinant DNA (fi 복제원점 : 단일가닥생성 ) MCS has 2 phage RNA polymerase promoters (T3, T7), 1 on each side of MCS COS COS pbluescript (pbs) lacz, MCS, origin of replication of the single strand phage f1 Two phage RNA polymerase promoter : T3, T Eukaryotic Vectors and Very High Capacity Vectors There are vectors designed for cloning genes into eukaryotic cells Yeast artificial chromosomes (YAC, 효모의인공염색체 ) and bacterial artificial chromosomes (BAC, 세균의인공염색체 ) are used for cloning huge pieces of DNA Other vectors are based on the Ti plasmid to carry genes into plant cells

6 Yeast Artificial Chromosomes (YAC; 효모인공염색체 ) Origin of replication Selectable markers Very High Capacity Vectors CEN (centromere sequence) for proper segregation Telomere sequences ( 말단소체서열 ) Suitable for very large DNAs : used for human genome project Bacterial Artificial Chromosomes (BAC; 세균인공염색체 ) YAC 의단점극복 Very High Capacity Vectors 100 to 300 Kb in size Have selectable markers Stable origin of replication ( 복제기점 ) Size of inserts is ~100 KB Uses electroporation ( 전기천공법 ) 인간유전체프로젝트의후반부에사용 Identifying a Specific Clone With a Specific Probe Probes ( 탐침 ) are used to identify a desired clone from among the thousands of irrelevant ones Two types are widely used Polynucleotides (oligonucleotides) Antibodies Polynucleotide Probes Looking for gene; using homologous gene from another organism ( 상동유전자이용 ) If already cloned Hope enough sequence similarity to permit hybridization ( 염기서열유사도- 잡종화 ) Need to lower stringency ( 충실성 ) of hybridization conditions to tolerate t some mismatches Control of Hybridization Stringency Factors that promote separation of two strands in a DNA double helix (2 중나선구조의변성 ) High temperature High organic solvent concentration Low salt concentration Adjust conditions until only perfectly matched DNA strands form a duplex Lowering these conditions lowers stringency until DNA strands with a few mismatches can hybridize Protein-based Polynucleotide Probes No homologous DNA from another organism? If amino acid sequence is known, deduce a set of nucleotide sequences to code for these amino acids Construct these nucleotide sequences chemically using the synthetic probes ( 탐침의합성 )

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