Presentation on theme: "The Gateway® Cloning System"— Presentation transcript:
1 The Gateway® Cloning System How to generate an entry cloneContentsOptions for entering the Gateway® systemDefining the BP Clonase™ reactionDefining the LR Clonase™ reactionDescription of Ultimate™ ORF collectionDescription of Vector NTI Advance™ software for in silico cloningIn this section, we will focus on the options for entering the Gateway® system.
2 The Gateway® Reactions As we discussed in the introductory section, the entry clone is the door to the Gateway® system. Once you have cloned your DNA fragment into a Gateway® entry vector, you can easily transfer it into a destination vector to generate the expression clone.Invitrogen Proprietary & Confidential
3 Different ways to generate the entry clone PCR Product+TOPO-ActivatedEntry VectorL1L2Gene+attBPCR ProductB2GeneB1Donor VectorP2ccdBP1BP CloningTOPO® CloningBP Clonase™TOPO®L1GeneL2Entry CloneLigaseYou can clone PCR products to make entry clones in three different ways:By using a Gateway® BP cloning reactionBy using directional TOPO® or TOPO® TA CloningBy using restriction enzymes and ligation reactionOrand 5. You can use pre-made or customized entry clones4. Pre-made entry clone5. Custom-made entry cloneRestriction/Ligase Cloning+digested DNA FragmentGeneB1digested Entry VectorL2L1L1ORFL2ORF CollectionInvitrogen Proprietary & Confidential
4 BP Cloning – The Reaction ++BP Clonase™90-99% correct cloneson Kan platesTo perform the BP Cloning reaction, you will combine the PCR product, which is flanked by attB sequences, with a pDONR™ vector and add BP Clonase™ II. The reaction is incubated at room temperature for an hour and then transformed into standard competent E. coli cells, such as DH5α, TOP10 or Mach1™.The next day, you will have >90% correct clones in kanamycin resistant colonies. Efficiency is high because of the specificity of the recombination reaction (i.e., attB1 x attP1, attB2 x attP2), and negative selection is conferred by the presence of ccdB.Let’s take a look at the attB sequences.Invitrogen Proprietary & Confidential
5 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)attB15’ – GGGGACAAGTTTGTACAAAAAAGCAGGCTNNN…attB25’ – GGGGACCACTTTGTACAAGAAAGCTGGGTNNN…Gene SpecificPrimer SequenceThe attB sequences are 21 bp in length. Of the four types of att sites, attB is the shortest. The addition of specific attB sequences to each primer confers directionality to the PCR product. Site-specific changes were made in the attB2 sequence to confer specificity in the recombination reaction. The attB sites were also engineered to eliminate stop codons. The 4G-residues added to 5'-end of each attB sequence improve the efficiency of the recombinase reaction. Reading frame is determined in attB1 by the two lysine-encoding codons (AAA AAA). If you clone in frame with these codons, you will stay in frame in all subsequent reactions. There is a similar rule for the construction of C-terminal fusion proteins: your sequence needs to be in frame with: TTT GTA.Invitrogen Proprietary & Confidential
6 BP Cloning – Some Examples CorrectSize (kb)PCR DNAPCR DNAColonies/lClones/Total(fmol)(ng)TransformationClones Examined15312230.2610/10387.5281515105071.049/503825144715142711.448/50383568315344783.49/10388597615461904.610/1038115195In this example, PCR products ranging from 260 bp to 10.1 kb in size, were combined with a pDONR™ vector at two consistent molar ratios; the amount of the pDONR™ vector was 115 fmol or 300 ng in each reaction.Colonies/µl were normalized to pUC transformation efficiency. The number of clones obtained decreased with increasing PCR product size. However, extending the incubation time of the BP reaction to overnight can improve efficiency.The clones were identified by mini-prep analysis. Cloning efficiency, defined as the frequency of getting the right clone, was 90% or greater.156930 (235)*6.947/503817354 (463)*7.550.516 (112)*10.115/1637.5252.542 (201)**After overnight incubationInvitrogen Proprietary & Confidential
7 BP Cloning – RT-PCR Using attB-Containing Primers TyrosineKinaseTransferrinReceptorTarget Template:EIF4eb-AdaptinMAP4attB-containing primers:Platinum® Taq DNA PolymerasePlatinum® Taq DNA Polymerase High FidelityIn this slide, we are looking at the results obtained using three different polymerases, and five different primer pairs. Overall, the presence of the attB sequences is not detrimental to amplification. In some cases, the yield is either more or less or remains essentially the same; it depends not only on the primer pair, but also on the enzyme used for amplification. Note that all of the polymerase enzymes incorporated Platinum® technology, providing an automatic hot start.Platinum® Pfx DNA PolymeraseTotal RNA isolated from HeLa cells, first strand cDNA synthesized using THERMOSCRIPT™ RT.
8 TOPO® Cloning -TOPO®TA The key to TOPO® Cloning is DNA topoisomerase I. The biological role of the enzyme is to cleave and rejoin DNA during replication. Vaccinia virus topoisomerase I specifically recognizes the pentameric sequence 5´-(C/T)CCTT-3´ and forms a covalent bond with the phosphate group of the 3´ thymidine. It cleaves one DNA strand, enabling the DNA to unwind. The enzyme then re-ligates the ends of the cleaved strand and releases itself from the DNA.To harness the religation activity of topoisomerase, TOPO® vectors are provided linearized with topoisomerase I covalently bound to each 3´ phosphate group. This enables the vectors to readily ligate DNA sequences with compatible ends. Vectors with protruding T- overhangs on both the 5’ and 3’ ends are available to clone PCR products (up to 5 kb) produced by Taq polymerase. This is called TOPO® TA cloning.An example of a TOPO®-adapted entry vector is our pCR8/GW/TOPO® TA plasmid, with attL1 and attL2 flanking sites. This is one of the few entry vectors with a resistance different from kanamycin, making it compatible with a variety of destination vectors bearing ampicillin or kanamycin resistance.Invitrogen Proprietary & Confidential
9 TOPO® Cloning – Directional TOPO® Directional joining of double-stranded DNA using TOPO®-charged oligonucleotides occurs by adding a 3 single-stranded end (overhang) to the incoming DNA. This single-stranded overhang is identical to the 5 end of the TOPO®-charged DNA fragment. At Invitrogen, this idea has been modified by adding a 4 nucleotide overhang sequence to the TOPO®-charged DNA.In this system, PCR products (up to 5 kb) are directionally cloned by adding four bases to the forward primer (CACC). The overhang in the cloning vector (GTGG) invades the 5 end of the PCR product, anneals to the added bases, and stabilizes the PCR product in the correct orientation. Inserts can be cloned in the correct orientation with high efficiency.Invitrogen Proprietary & Confidential
10 Restriction/Ligase cloning Use when there are convenient sites to cut insert out of another plasmidMust cut out ccdB gene by using one of four RE sites flanking the ccdBReading frame of insert must be considered, as well as downstream expression elementsVarious reading frames of pENTR vectors are availableYour restriction strategy must be carefully planned. However, Gateway will make subsequent subcloning much easier.Available vectors are: pENTR™ 1A, pENTR™ 2B, pENTR™ 3C, pENTR™ 4, pENTR™ 11.Corresponding plasmid sequences, multiple cloning regions and restriction enzyme tables for all Invitrogen’s vectors can be found on the web site.Invitrogen Proprietary & Confidential
11 Pre-existing ORF collection Invitrogen’s Ultimate™ ORF collection16,272 human ORFs (Oct 2006 release)Amber stop codonsSequence verifiedReady to use in LR reactionsThe Ultimate™ ORF collection contains over 16,000 human and 2,000 mouse open reading frames (ORFs). Each ORF has been cloned into a Gateway® donor vector, pDONR™221, using the BP recombination method. The ORF contains the particular gene of interest from the start to stop codon. To ensure integrity of the specific gene sequence, each ORF is fully sequenced from start to stop, and is guaranteed down to the amino acid sequence.To express C-terminal tagged proteins in mammalian cells, the amber stop codon can be suppressed using the Tag-On-Demand™ technology.Invitrogen Proprietary & Confidential
12 5. Custom Gene Synthesis Quick and cost-effective No PCR amplification necessary100% accuracy (sequence verified)Optional codon optimization for expressionIf you do not want to clone your own gene, and it is not available within our clone collection, opt for custom gene synthesis. Gene synthesis is also an excellent option for targets that have been difficult to obtain using conventional PCR cloning due to difficult sequences such as GC-rich regions, high secondary structure, or large size.Invitrogen has entered into an exclusive distribution agreement with Blue Heron Biotechnology. Invitrogen’s decision to partner with Blue Heron was based on the company’s market and technological leadership in custom gene synthesis.Blue Heron Bio's GeneMaker® can synthesize any gene regardless of sequence, complexity, or size with 100% accuracy. GeneMaker® is a proprietary, automated, high throughput gene synthesis platform. It utilizes proprietary gene assembly instruments as well as numerous error correction methods to ensure that each synthetic gene is 100% accurate. In addition, GeneMaker®'s Expression Optimization and Codon Optimization service offers the flexibility to design DNA sequences for various expression systems or future subcloning manipulations.As part of the service, the resulting gene is cloned into any vector. As an example, the synthesized ORF is cloned into pDONR™221 to create a Gateway® entry clone that can be used for recombination with a variety of destination vectors.Invitrogen Proprietary & Confidential
13 In silico cloning using Vector NTI AdvanceTM 10.3 DNA of interestPrimers for PCR reactionCloning StrategyVector NTI Advance™ Software allows the generation of entry and expression clones starting from any DNA sequence template and using any of the available configurations. It automatically designs the primers for the generation of the PCR fragments used in the corresponding BP reactions. The program is downloadable from the Vector NTI User Community at Licenses are free for academic and government researchers. Free 30-day trial licenses are available for commercial researchers by ingInvitrogen Proprietary & Confidential
14 Gateway® SummaryA variety of ways to enter the Gateway® system are available, depending on the source of gene sequence.Invitrogen Proprietary & Confidential