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METODI DI CLONAGGIO: Polymerase Incomplete Primer Extension (PIPE) Method Non richiede digestioni con enzimi di restrizione Non necessita reazioni di ligasi.

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Presentation on theme: "METODI DI CLONAGGIO: Polymerase Incomplete Primer Extension (PIPE) Method Non richiede digestioni con enzimi di restrizione Non necessita reazioni di ligasi."— Presentation transcript:

1 METODI DI CLONAGGIO: Polymerase Incomplete Primer Extension (PIPE) Method Non richiede digestioni con enzimi di restrizione Non necessita reazioni di ligasi Veloce Utilizzabile per clonaggio di frammenti di DNA in plasmidi e per mutagenesi

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4 METODI DI CLONAGGIO: Polymerase Incomplete Primer Extension (PIPE) Protocollo: V-PIPE PCR: amplificazione del vettore I-PIPE PCR: amplificazione dell’inserto Mix prodotti di PCR V-PIPE e I-PIPE Trasformazione di cellule competenti Gene ccdB is lethal to most strains of E. coli. 'Empty' destination vectors are therefore selected against upon transformation of E. coli cells with the recombination reaction

5 I-PIPE PCR product V-PIPE PCR product 5’ Mix Annealing intermolecolare trasformazione Le cellule vengono trasformate solamente dal plasmide chiuso (ricombinato) 5’ 3’ METODI DI CLONAGGIO: Polymerase Incomplete Primer Extension (PIPE) CLONAGGIO

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7 METODI DI CLONAGGIO: Polymerase Incomplete Primer Extension (PIPE) MUTAGENESI

8 METODI DI CLONAGGIO: Polymerase Incomplete Primer Extension (PIPE) MUTAGENESI

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10 This cloning technology is based on the nature of site-specific recombination: the integrative and excisive recombination reactions between chromosomes of Bacteriophage lambda and E. coli bacterium. These biochemical reaction processes, performed in vitro, are the basis of the Gateway® Cloning Technology. Gateway® Cloning Technology

11 The integrative recombination is catalyzed by Int (integrase) and IHF (Integration Host Factor). The recombination between attB and attP sites results in attL and attR sites that flank the integrated lambda DNA. Three proteins (IHF, Int and Xis) are required for the excisive recombination. The attL and attR sites located at both sides of the inserted phage genome DNA recombine site- specifically during the excision event, reforming the attP site in lambda and the attB site in the E. coli chromosome.

12 The Gateway® Reactions LR Clonase= enzyme mix of Int, IHF, Xis catalyising in vitro recombination between an Entry clone (containing a gene of interest flanked by attL sites) and a Destination vector (containing attR sites) to generate your expresion clone. BP Clonase= enzyme mix of Int and IHP catalyising the in vitro recombination of PCR products or Dna segments from clones (containing attB sites) and a Donor vector (containing attP sites) to generate the Entry clones

13 Different ways to generate the entry clone 2.TOPO® Cloning TOPO®BP Clonase™ 1.BP Cloning PCR Product + TOPO-Activated Entry Vector L1L2 Gene + attB PCR Product B2GeneB1 Donor Vector P2 ccdB P1 Entry Clone L2 Gene L1 + digested DNA Fragment GeneB1 digested Entry Vector L2L1 4. Pre-made entry clone 5. Custom-made entry clone Ligase 3.Restriction/Ligase Cloning ORF Collection L2 ORF L1

14 3.Restriction/Ligase cloning Use when there are convenient sites to cut insert out of another plasmid Must cut out ccdB gene by using one of four RE sites flanking the ccdB Reading frame of insert must be considered, as well as downstream expression elements Various reading frames of pENTR vectors are available

15 4.Pre-existing ORF collection 16,272 human ORFs (Oct 2006 release) Amber stop codons Sequence verified Ready to use in LR reactions Invitrogen’s Ultimate™ ORF collection

16 1.BP Cloning – The Reaction 90-99% correct clones on Kan plates ++ BP Clonase™

17 pDESTTM17

18 1.BP Cloning - Primer Design for PCR GGGG and the attB1 sequence must be added to the 5’-primer (sense) GGGG and the attB2 sequence must be added to the 3’-primer (antisense) attB1 5’ – GGGGACAAGTTTGTACAAAAAAGCAGGCTNNN… attB2 5’ – GGGGACCACTTTGTACAAGAAAGCTGGGTNNN… Gene Specific Primer Sequence

19 PRIMERS PER RICOMBINAZIONE NEL pDONOR FORWARD: a) Per esprimere la proteina nativa o con tag C-terminale b) Per esprimere la proteina con tag N-terminale REVERSE: a) Per esprimere la proteina nativa o con tag N-terminale b) Per esprimere la proteina con tag C-terminale

20 The vector pDEST17 allows the addition of a polyhistidine tag to the target gene sequence. An inherent result of the cloning technology used is the presence of 21 supplementary amino acids at the N-terminus of the recombinant protein compared to the native protein. The majority of these additional amino acids are due to the recombination sequence attB1 and the histidine tag. A target sequence for the TEV protease is therefore added systematically to our current constructions, between the ORF and the attB1 recombination site. The elimination of 20 out of the 21 supplementary amino acids after digestion of the recombinant protein.

21 6-His GB1 Z-Tag Trx GST MBP NusA Target TEV site : Glu-Asn-Leu-Tyr-Phe-Gln / Gly pDEST17 pTH34 pTH30 pETG20A pETG30A pDESTHis-MBP pETG60A PROTEINE DI FUSIONE CON TAG N-TERMINALI Plasmide di espressione

22 pDEST: Plasmidi di espressione (pET-derived) Possibilità di tag N- o C- terminali

23 TOPO Adapted Gateway Entry Vector seminar.html All entry clones have attL's on both sides of their gene of interest. Necessary in the Gateway system because L's cut to form sticky ends by the Gateway recombination proteins. These sticky ends will then match up with the sticky ends on a destination vector which contains attR restriction sites.

24 2.TOPO® Cloning – Directional TOPO®

25 METODI DI CLONAGGIO: TOPO CLONING_____________________ Mappa e sequenza del polilynker del plasmide pENTR/SD/D-TOPO

26 CACC facilitate directional incorporation into the pENTR/D-TOPO vector (obtained from Invitrogen). topoisomerase molecules (TOPO) catalyze ligation of target and vector sequences Once flanked by attL recombination sites, the sequence can be recombined with attR sites using the LR clonase reaction mix (Invitrogen). This reaction transfers the target sequence into a desired destination vector Destination vectors contain a gene (ccdB) that is lethal to most strains of E. coli. 'Empty' destination vectors are therefore selected against upon transformation of E. coli cells with the recombination reaction

27 The Gateway® Technology is a universal cloning method based on the site- specific recombination properties of bacteriophage lambda and provides a rapid and highly efficient way to move DNA sequences into multiple vector systems for functional analysis and protein expression.

28 Primers and PCR products Gateway cloning Entry vectors (pDONR) E. coliP. pastoris Insect cellsSFV mammalian Mammalian cells pDest14 pDest17 pDest24 pDest42 …. pPICZ  C-GW pPIC3.5K-Dest1 pPIC9K-Dest1 pDest8 pDest10 pDest-SFV1pDest12.2 pDest26 pDest27 Gateway technology

29 Gateway: LR Reaction

30 The Gateway

31 Gateway technology Advantages 1) No restriction analysis of ORF prior to cloning. 2) No restriction digestion of the vector. 3) Fast, parallel sub-cloning into different expression vectors. 4) ~100% sub-cloning efficiency (no background). 5) Flexibility, automation. 6) Recombination sites may serve as linkers. Disadvantages 1) Number of available expression vectors. 2) Mandatory recombination sites. 3) Very long primers containing the attB sites and also other sequences (TAGs) can be inefficient for the PCR reaction.

32 Bressanone, 2004 Gateway technology Comments on disadvantages 1) number of available expression vectors : a) Invitrogen provides a conversion cassette to make your favorite expression vector(s) a Gateway expression vector. b) as a consequence, there is a growing list of new vectors. 2) mandatory recombination sites: attb1/2-encoded peptide would hamper crystallogenesis: a) protease cleavage site (Tev…) can be added at primer synthesis. b) short tags (His6) can be encoded by primers.


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