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Gene engineering & Gene expression in vitro

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Genentech Founders 1973,Stanley Cohen and Herbert Boyer DNA recombination pSC101 RSF1010 pSC109 E coli C600

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DNA recombination concept: DNA molecule from different scources covalently ligated with phosphodieaster bond to produce a noval DNA molecule

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Basic concepts zgenetic engineering are terms that apply to the direct manipulation of an organism's genes. It uses the techniques of molecular cloning and transformation to alter the structure and characteristics of genes directly.organismgenesmolecular cloningtransformation Molecular cloning Molecular cloning refers to the procedure of isolating a defined DNA sequence and obtaining multiple copies of it in vivo. Cloning is frequently employed to amplify DNA fragments containing genes, but it can be used to amplify any DNA sequence such as promoters, non-coding sequences, chemically synthesised oligonucleotides and randomly fragmented DNA.Its core technique is DNA recombination refers to the procedure of isolating a defined DNA sequence and obtaining multiple copies of it in vivo. Cloning is frequently employed to amplify DNA fragments containing genes, but it can be used to amplify any DNA sequence such as promoters, non-coding sequences, chemically synthesised oligonucleotides and randomly fragmented DNA.Its core technique is DNA recombinationin vivogenespromotersoligonucleotidesin vivogenespromotersoligonucleotides clone In genetics, an exact replica of all or part of a macromolecule (e.g. DNA) In cell biology, a group of identical cells naturally derived from a common mother cell; of significance in vertebrate physiology, and concepts related to immunology and cancer biology

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Section 1 Tool enzymes zDNA recombination technique is hghly dependent on tool enzymes and various vectors zTool enzymes: incision, synthesis, ligation and modification

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restriction enzyme zan enzyme that cuts double-stranded or single stranded DNA at specific recognition nucleotide sequences known as restriction sites. enzymeDNAnucleotiderestriction sitesenzymeDNAnucleotiderestriction sites zSuch enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses. Inside a bacterial host, the restriction enzymes selectively cut up foreign DNA in a process called restriction; host DNA is methylated by a modification enzyme (a methylase) to protect it from the restriction enzymes activity. bacteria archaeaviruses methylatedmethylasebacteria archaeaviruses methylatedmethylase zthese two processes form the restriction modification system. restriction modification system restriction modification system

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1. Classification of restriction enzyme zTypical type II restriction enzymes differ from type I restriction enzymes in several ways. They are composed of only one subunit, their recognition sites are usually undivided and palindromic and 4-8 nucleotides in length, they recognize and cleave DNA at the same site, and they do not use ATP or AdoMet for their activity they usually require only Mg2+ as a cofactor.In the 1990s and early 2000s, new enzymes from this family were discovered that did not follow all the classical criteria of this enzyme class, and new subfamily nomenclature was developed to divide this large family into subcategories based on deviations from typical characteristics of type II enzymes.[15] These subgroups are defined using a letter suffix.Type II of restriction enzymes are widely used nomenclature[15] nomenclature[15]

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2. Nomenclature of restriction enzyme Each enzyme is named after the bacterium from which it was isolated using a naming system based on bacterial genus, species and strain.genus speciesstrain

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3. Characterics of type II restriction enzyme zRecognization site undivided and palindromic and 4-8 nt in length Rsa I EcoR I Not I Rsa I EcoR I Not I GTAC GAATTC GCGGCCGC GTAC GAATTC GCGGCCGC CATG CTTAAG CGCCGGCG CATG CTTAAG CGCCGGCG z6nt palindromic sequence is widely used the frequency of cutting site: 4 6 4096 bp the frequency of cutting site: 4 6 4096 bp zSpecial recognization site BstE II EcoR II BstE II EcoR II GGTNACC CC GG GGTNACC CC GG AT

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zCutting modes Sticky end 5 overhang 3 overhang 3 overhang Blunt end zDNA molecule with same end can be recognized and ligated

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isoschizomer and isoaudamer isoschizomer and isoaudamer zIsoschizomers are pairs of restriction enzymes specific to the same recognition sequence. restriction enzymesrecognition sequencerestriction enzymesrecognition sequence BamH I and BstI G GATCC BamH I and BstI G GATCC Xho I and PaeR7 C TCGAG Xho I and PaeR7 C TCGAG zisoaudamer An enzyme that recognizes slightly different sequence, but produces the same ends BamH I Sau3A I BamH I Sau3A I GGATCC NGATCN GGATCC NGATCN CCTAGG NCTAGN CCTAGG NCTAGN

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4. Application of restriction enzyme zEssential tool for DNA recombination yDNA cutting yMapping the physical map of genome yDetection of DNA mutation (RFLP) zReaction for restriction enzyme yMg 2+ ypH neutral condition yIonic strength with different concentration of Na+ or Ka+

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Restriction digestion of lamda bateriaophage DNA

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modifcation enzyme z 1) DNA polymerase I MW 109KD 5, 3, polymerizarion 5, 3, polymerizarion y 3, 5, exonuclease activity 5, 3, exonuclease activity 5, 3, exonuclease activity yapplication nickle translation the second strand synthesis of cDNA, DNA 3end labeling nickle translation the second strand synthesis of cDNA, DNA 3end labeling DNA pol I 53

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zKlenow fragment 76KD) z5, 3, polymerization and 3, 5, exonuclease activity yBlunting the 3end yLabeling the 3end ySynthesis of the second strand of cDNA yDNA sequencing zTaq DNA polymerase yCloned from Thermus aquaticus yNo proof reading activity and the optimal temperature is 72 yUsed in PCR Klenow

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2 reverse transcriptase zReverse transcriptase RNA-dependent DNA polymerase, is a DNA polymerase enzyme that transcribes single-stranded RNA into single-stranded DNA. It also helps in the formation of a double helix DNA once the RNA has been reverse transcribed into a single strand cDNA. Normal transcription involves the synthesis of RNA from DNA; hence, reverse transcription is the reverse of this DNA polymeraseenzymetranscribesRNADNA polymeraseenzymetranscribesRNADNA ztypes AMV and MMLV zactivity zreverse transcriptase RNase H DNA pol RNase H DNA pol zRNase H deleted zPrimer is needed for RT

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3 DNA ligase zDNA ligase is a special type of ligase that can link together two DNA strands that have double-strand break (a break in both complementary strands of DNA). The alternative, a single-strand break, is fixed by a different type of DNA ligase using the complementary DNA as a template but still requires DNA ligase to create the final phosphodieaster bond to fully repair the DNA ligaseDNAligaseDNA zLigase mechanism The mechanism of DNA ligase is to form two covalent phosphodieaster bond between 3 hydroxyl end of one nucleotide with the 5 phosphate end of another. ATP is required for the ligase reaction. The mechanism of DNA ligase is to form two covalent phosphodieaster bond between 3 hydroxyl end of one nucleotide with the 5 phosphate end of another. ATP is required for the ligase reaction. zLigase works both with blunt and sticky ends of DNA. T4 DNA Ligase T4 DNA Ligase Ecoli DNA ligase Ecoli DNA ligase

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4 alkaline phosphatase zAlkaline phosphatase is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. hydrolaseenzymephosphatenucleotidesproteinsalkaloidshydrolaseenzymephosphatenucleotidesproteinsalkaloids zCalf intestinal alkaline phosphatase (CIAP) Application: prevent vector self-ligation 5end labeling 5end labeling enzyme-linked developing enzyme-linked developing

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5 Terminal deoxynucleotidyl transferase(TdT) zTdT catalyses the addition of nucleotides to the 3' terminus of a DNA molecule. Unlike most DNA polymerases it does not require a template. The preferred substrate of this enzyme is a 3'-overhang, but it can also add nucleotides to blunt or recessed 3' ends. catalysesnucleotides3' terminus DNA 3'-overhangcatalysesnucleotides3' terminus DNA 3'-overhang zHighest activity with 3end overhang zLabeling of 3end Adding homo-polytail Adding homo-polytail cpnstructing artifical sticky end cpnstructing artifical sticky end

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Section 2 vector zVector:vehicle used to transfer genetic material to a target cell zCharacterics of vectors yorgin ySelection marker yMultiple cloning site yCapacity

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Vectors which host is Ecoli plasmid bacteriophage cosmid M13 phage

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plasmid zA plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms (e.g., the 2-micrometre-ring in Saccharomyces cerevisiae DNAchromosomal DNA Saccharomyces cerevisiaeDNAchromosomal DNA Saccharomyces cerevisiae

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Natural plasmid classification yFertility-F-plasmids, which contain tra-genes. They are capable of conjugation (transfer of genetic material between bacteria which are touching). F-plasmidsconjugationF-plasmidsconjugation yResistance-(R)plasmids, which contain genes that can build a resistance against antibiotics or poisons and help bacteria produce pili. Historically known as R-factors, before the nature of plasmids was understood. antibioticspoisonsantibioticspoisons yCol-plasmids, which contain genes that code for (determine the production of) bacteriocins, proteins that can kill other bacteria. bacteriocinsproteins bacteriocinsproteins Features of Plasmid used in molecular biology yRelativel small molecula weight yDrug resistance gene yMultiple cloning site yOrgin site for replication

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zApplication of plasmids cloning cloning Sequencing Sequencing In vitro transcription In vitro transcription Gene expression Gene expression

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Main types of plasmids in molecular biology zpBR322 ymost commonly used E. coli cloning vectors. E. colicloningvectorsE. colicloningvectors ythe first artificial plasmid. Created in 1977, it was named eponymously after its Mexican creators, p standing for plasmid, and BR for Bolivar and Rodriguez. y4363 base pairs in length and contains a replicon region (source plasmid pMB1), the ampR gene, and the tetR gene,(source plasmid pSC101). pSC101 yThe plasmid has unique restriction sites for more than forty restriction enzymes. 11 of these 40 sites lie within the tetR gene. There are 2 sites for HindIII and within the promoter of the tetR gene. There are 6 key restriction sites inside the ampR gene. The origin of replication or ori site in this plasmid is pMB1 (a close relative of ColE1)[2]. The ori encodes two RNAs (RNAI and RNAII) and one protein (called Rom or Rop). HindIII restriction sitesori[2]HindIII restriction sitesori[2]

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zpUC18 and pUC19 yHigh copies in cell yContaining LacZ to faciliate the recombinant selection yMCS containing more enzyme recognization site zpUC118/119 yRepilcation origin based on pUC18/19 based on pUC18/19

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bacteriophage zThe virus particle consists of a head and a tail that can have tail fibres. The head contains 48,490 base pairs of double-stranded, linear DNA flanked by 12-base-pair, single- stranded segments that make up the two strands of the cos site. In its circular form in the host cytoplasm, the phage genome therefore is 48,502 base pairs in length. The prophage exists as a linear section of DNA inserted into the host chromosome zLytic or lysogenic

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Charterictics of phage zBig genome,packing into virus particle to infect cell zCos site is needed for packaging, recombinant size ranging between 75 -105% of genome z40 of genome can be replaced. The longest insert is 23kb

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Types of phage zTwo types : yInsetion vector one restricition enzyme recognization site theoritical capacity 0 13.5 kb theoritical capacity 0 13.5 kb yReplacement vector two RE recognization site theoritical capacity 9 22.5 kb theoritical capacity 9 22.5 kb

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Types of phage DNA zCharon 40 replacement, insert 9.2 24.2 kb zEMBL 3 replacement, insert 7 22 kb Spi selection Spi selection zgt 10 insertion insert 0 7.6 kb zgt 11 insertion insert 0 7.2 kb LacZ gene selection LacZ gene selection zDASH replacement insert 9 22 kb Spi selection T3 and T7 promoter Spi selection T3 and T7 promoter zZAP insertion insert less than10 kb ColE1 replicon convert to plasmid ColE1 replicon convert to plasmid

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cosmid zA cosmid, first described by Collins and Hohn in 1978, is a type of hybrid plasmid (often used as a cloning vector) that contains cos sequences, DNA sequences originally from the lambda phage. Cosmids can be used to build genomic library. Insert 40-45 kb

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Features of cosmid zadvantages yAmp r or Tet r yCos site in vitro packaging yOne or more RE recognization site yHigh capacity especially for genomic library construct yNon-recombinant cosmid is small, can not be in vitro packaged, easily for selection zdisadvantages The length of insert affect the amplification efficiency

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Filamentous phage vector zPhage particle is filamentous, circular single-strand DNA genome, the length is 6.5kb M13, f1,fd M13, f1,fd zM13 only infect the Ecoli with F factor zM13mp18/M13mp19 yContaining LacZ and MCS yLimited packing capacity used in sequencing

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Other vectors YAC capacity 0.5-2MB

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BAC capacity 0.1-0.4MB

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expressing vector zAn expression vector, otherwise known as an expression construct, is generally a plasmid that is used to introduce a specific gene into a target cell. Once the expression vector is inside the cell, the protein that is encoded by the gene is produced by the cellular-transcription and translation machinery.plasmidgeneprotein encodedtranscription translation zExpression vector is frequently engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector. The goal of a well-designed expression vector is the production of large amounts of stable messenger RNAenhancerpromotermessenger RNA zHost cell prokaryote and eukaryote

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expression vector in Ecoli promoter SD sequence terminator Expression element

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Commonly used prokaryotic promoter zTac promoter heterogenous promoter with Trp (-35), artifical synthesised -10 regions and Lac operator. Containing LacI heterogenous promoter with Trp (-35), artifical synthesised -10 regions and Lac operator. Containing LacI very strong promoter can be induced by IPTG very strong promoter can be induced by IPTG zT7 phage promoter highly specific promoter only expressed in T7 RNA polymerase-containing host cell. Lac operator in the downstream of T7 promoter, and can be induced by IPTG Lac operator in the downstream of T7 promoter, and can be induced by IPTG zpL promoter of controlled by temperature-sensitive inhibitors cIts857 repress transcription at low temprature and ddoes not work at high temparature

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S D sequence zLocated between transcription starting site and initiation zThe distance between SD sequence and initiation codon is very important to transcription activiety

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transcription termination sequence zlength to 800bp zcharacterics strong termination for RNA ploymerase, A/T rich and G/C rich Palindromic structure

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expression vector in mammalian zBasic element needed ori, resistance gene, MCS ori, resistance gene, MCS zElement needed for expression yPromoter and enhancer from virus, strong activity yPoly A tailing AAUAAA ySplicing signal provided by insert ySelection marker tk and neo r

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Section 3 Process of gene cloning

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preparation of target DNA first and very important step for cloning first and very important step for cloning Strategy for gene cloning Strategy for gene cloning positional clonging: RE digestion positional clonging: RE digestion directional cloning directional cloning Sequence-based cloning: PCR Sequence-based cloning: PCR chemical synthesis chemical synthesis capture from library capture from library Function-based cloning immuno selection

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preparation of genomic DNA zgenomic library: a population of host bacteria, each of which carries a DNA molecule that was inserted into a cloning vector, such that the collection of cloned DNA molecule represents the entire genome of the source organism. This term also represents the collection of all of the vector molecules, each carrying a piece of the chrosomal DNA of the organism, prior to the insertion of these molecules into the host cells. populationDNApopulationDNA zVectors: plasmid, phage, YAC zshotgun zUse: Sequencing for whole genome

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preparation of cDNA zA cDNA library is a collection of cloned cDNA fragments inserted into a collection of host cells, which together constitute some portion of the transcriptome of the organism. cDNA z cDNA is produced from fully transcribed mRNA found in the nucleus and therefore contains only the expressed genes of an organism. mRNA

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Full length cDNA synthesis zSMART Switch mechanism at RNA terminal

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Characterics of cDNA zExpressed library zLack of intron, and can be expressed in prokaryote ztissue specific cDNA libraries zLow abundant mRNA is difficult to get from cDNA library

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PCR zPCR polymerase chain reaction zThe simplest way to obtain the specific DNA

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Chemical synthesis zOligonucleotide by chemical synthesis (poly">

Application of SDM zLong-term insulin chain B Thr27Arg chain A Asn21Gly C-terminal NH2 result pI 5.4 pI6.8 zRapid absorbe insulin monomer>polymer zIncreasing the affinity of receptor chain B His27Asp