The Gateway® Cloning System How to generate an entry clone Contents Options for entering the Gateway® system Defining the BP Clonase™ reaction Defining the LR Clonase™ reaction Description of Ultimate™ ORF collection Description of Vector NTI Advance™ software for in silico cloning In this section, we will focus on the options for entering the Gateway® system.

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

Different ways to generate the entry clone PCR Product + TOPO-Activated Entry Vector L1 L2 Gene + attB PCR Product B2 Gene B1 Donor Vector P2 ccdB P1 BP Cloning TOPO® Cloning BP Clonase™ TOPO® L1 Gene L2 Entry Clone Ligase You can clone PCR products to make entry clones in three different ways: By using a Gateway® BP cloning reaction By using directional TOPO® or TOPO® TA Cloning By using restriction enzymes and ligation reaction Or and 5. You can use pre-made or customized entry clones 4. Pre-made entry clone 5. Custom-made entry clone Restriction/Ligase Cloning + digested DNA Fragment Gene B1 digested Entry Vector L2 L1 L1 ORF L2 ORF Collection Invitrogen Proprietary & Confidential

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

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 The 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

BP Cloning – Some Examples Correct Size (kb) PCR DNA PCR DNA Colonies/l Clones/Total (fmol) (ng) Transformation Clones Examined 15 3 1223 0.26 10/10 38 7.5 2815 15 10 507 1.0 49/50 38 25 1447 15 14 271 1.4 48/50 38 35 683 15 34 478 3.4 9/10 38 85 976 15 46 190 4.6 10/10 38 115 195 In 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. 15 69 30 (235)* 6.9 47/50 38 173 54 (463)* 7.5 50.5 16 (112)* 10.1 15/16 37.5 252.5 42 (201)* *After overnight incubation Invitrogen Proprietary & Confidential

BP Cloning – RT-PCR Using attB-Containing Primers Tyrosine Kinase Transferrin Receptor Target Template: EIF4e b-Adaptin MAP4 attB-containing primers: + + - - + + - - + + - - + + - - + + - - Platinum® Taq DNA Polymerase Platinum® Taq DNA Polymerase High Fidelity In 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 Polymerase Total RNA isolated from HeLa cells, first strand cDNA synthesized using THERMOSCRIPT™ RT.

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

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

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 Your 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

Pre-existing ORF collection Invitrogen’s Ultimate™ ORF collection 16,272 human ORFs (Oct 2006 release) Amber stop codons Sequence verified Ready to use in LR reactions The 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. http://orf.invitrogen.com/cgi-bin/ORF_Browser Invitrogen Proprietary & Confidential

5. Custom Gene Synthesis Quick and cost-effective No PCR amplification necessary 100% accuracy (sequence verified) Optional codon optimization for expression If 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

In silico cloning using Vector NTI AdvanceTM 10.3 DNA of interest Primers for PCR reaction Cloning Strategy Vector 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 www.invitrogen/VectorNTICommunity. Licenses are free for academic and government researchers. Free 30-day trial licenses are available for commercial researchers by emailing bioinfotrial@invitrogen.com. Invitrogen Proprietary & Confidential

Gateway® Summary A variety of ways to enter the Gateway® system are available, depending on the source of gene sequence. Invitrogen Proprietary & Confidential