TOPO Tools Elements for in vitro Transcription/Translation - for rapid, directional TOPO Joining of PCR products to elements containing T7 or T3 polymerase binding sites

TOPO Tools technology uses the unique properties of DNA topoisomerase I to allow rapid, directional topoisomerase I-mediated joining (“TOPO Joining”) of PCR products to a choice of 5′ and 3′ elements. You will choose the 5′ and 3′ elements depending on the application you wish to perform. Once your PCR product and the 5′ and 3′ elements are TOPO Joined, the resulting linear DNA template is amplified using the element specific primers to generate the linear DNA construct. This linear DNA construct can be used directly in the appropriate application without the need for additional cloning or transformation steps.

To use the TOPO Tools technology, you will need:

• Your PCR product(s) of interest containing the appropriate ends to allow efficient topoisomerase I-mediated joining
• TOPO-adapted 5′ and/or 3′ elements specific for the application of interest

For more details on the TOPO Tools technology, refer to the TOPO Tools Technology manual.

The TOPO Tools Elements for in vitro Transcription/Translation are designed for directional topoisomerase I mediated joining of your PCR products to a choice of 5′ or 3′ element to create a linear DNA construct. The resulting linear DNA construct is used to generate RNA transcripts or protein from your PCR product. The important features of the TOPO Tools Elements for in vitro Transcription/ Translation are listed below:

• T7 or T3 promoter sequences for transcription of your PCR product using the T7 or T3 RNA polymerase
• Topoisomerase binding site (CCCTT) for directional topoisomerase I-mediated joining of your PCR product to the 5′ or 3′ element of choice
• Polyadenylation (pA) sequence for stabilization of mRNA transcripts
• Priming site for secondary amplification of the linear DNA template to generate a linear DNA construct

Depending on the desired downstream application, seven types of TOPO Tools Elements for in vitro Transcription/Translation Kits are available to allow you to generate a linear DNA construct of your choice

Once you have created a linear DNA construct containing your PCR product linked to a choice of elements, you can use the linear DNA construct for the following downstream applications:

• In vitro transcription
• Coupled in vitro transcription/translation
• RNA probe production
• Anti-sense RNA production

Using the TOPO Tools Technology to perform in vitro transcription or coupled in vitro transcription/translation of your PCR product provides the following advantages:

• Choice of a polymerase promoter and the direction for linking your gene of interest to the polymerase promoter
• Directional linking of your PCR product to a choice of TOPO-adapted elements
• Transcript or protein generation from PCR products without the need for subcloning or adding additional promoter sequences to the PCR primer
• Adaptable to high throughput format

To create a linear DNA construct and use the linear DNA construct for appropriate downstream applications such as in vitro transcription or coupled in vitro transcription/translation, you will need instructions from this webpage and the TOPO Tools Technology manual.

This webpage provides the following information:

• Sequences of the 5′ and 3′ TOPO-adapted elements
• Designing PCR primers
• Guidelines to perform in vitro transcription or coupled in vitro transcription/translation

For information on the TOPO Tools technology, please refer to the TOPO Tools Technology manual. You may download the manual from our web site at www.thermofisher.com/cloning or contact Technical Service. The TOPO Tools manual includes the information to:

• Design PCR primers
• Amplify your sequence of interest
• TOPO-Join your PCR product to the appropriate TOPO-adapted 5′ and 3′ elements to create a linear DNA template
• Amplify the linear DNA template to generate a linear DNA construct
• Perform the control reaction using the CAT control PCR templateControl Reaction

Using an Appropriate Element

Seven types of elements are available to create a linear DNA construct of your choice. Use the appropriate element based on your specific application.

T7 5' or T3 5' Element for in vitro transcription of your gene of interest in the sense orientation using the T7 or T3 RNA polymerase.

T7 3' or T3 3' Element for in vitro transcription of your gene of interest in the anti-sense orientation using the T7 or T3 RNA polymerase.

T7 Kozak 5' Element and the pA 3' Element for coupled in vitro transcription/translation of your gene of interest using the T7 RNA polymerase. This element contains the Kozak consensus sequence for optimal translation of your gene of interest.

Note: If your gene of interest contains an ATG with a Kozak consensus sequence, you may use the T7 5′ or T3 5′ Element and the pA 3′ Element for coupled in vitro transcription/translation of your gene of interest.

T7 5′ and T7 3′ Elements or T3 5' and T3 3' Elements for generating double stranded RNA from your gene of interest using T7 or T3 RNA polymerase.

pA 5' or pA 3' Element with T7 5', T7 3', T3 5', or T3 3' Elements to generate stable mRNA.

Introduction

The table below outlines the experimental steps necessary to perform in vitro transcription or coupled in vitro transcription/translation using TOPO Tools T7 or T3 elements and your gene of interest. Please refer to the indicated manual for more details on each step. We recommend that you read the entire TOPO Tools Technology manual to familiarize yourself with the technology and the various steps necessary to generate a linear DNA construct containing your gene of interest and a choice of TOPO-adapted element.

High-Throughput Application

If you need to perform the TOPO Tools technology in a high-throughput format, please refer to the TOPO Tools Technology manual for more guidelines. You can amplify your gene of interest, perform the TOPO Joining reaction, secondary amplification reaction, and in vitro transcription or translation in a high-throughput format.

Introduction

This section provides guidelines to design the PCR primers for TOPO Joining.

Requirements for Directional TOPO Joining

Guidelines are provided below for designing PCR primers to amplify your gene of interest. The PCR products must contain appropriate sequences to allow directional TOPO Joining. The 5′ ends of your forward and reverse PCR primers MUST contain an 11 base pair sequence consisting of the following:

• A six base pair sequence which base pairs with the overhang sequence on the TOPO-adapted 5′ or 3′ element. The six base pair sequence facilitates directional TOPO Joining and differs for the forward and reverse primer (see below)
• A five base pair sequence (AAGGG, shown in bold in the sequences below) which is complementary to the topoisomerase I recognition site (CCCTT) to increase the efficiency of topoisomerase I-mediated joining of the PCR product to the 5′ and 3′ elements

Forward Primer: 5′-CGGAACAAGGG-3′
Reverse Primer: 5′-TGAGTCAAGGG-3′

For more details and an example of designing PCR primers for proper TOPO Joining and generation of the linear DNA construct, please refer to the TOPO Tools Technology manual.

• To obtain consistent and efficient results in the TOPO Joining reaction, we highly recommend that you use HPLC-purified oligonucleotides to produce your PCR products. Using a mixture of full-length and non full-length primers to produce your PCR products can reduce the efficiency of TOPO Joining and result in poor yield of linear DNA construct after secondary amplification. Once you have obtained your HPLC-purified oligonucleotides, we also recommend that you use polyacrylamide gel electrophoresis to verify the length of your oligonucleotides.
• Do not add 5´ phosphates to your primers for PCR. This will prevent TOPO Joining.
• Be sure to include a stop codon in the reverse primer or design the reverse primer to hybridize downstream of the native stop codon, if you wish to translate your gene of interest.

Introduction

Sequence information and important features of the various TOPO-adapted elements are described in this section.

T7 Elements

The sequences of the T7 elements are provided below. The elements contain the following features:

• T7 promoter sequences for transcription of your gene of interest using the T7 RNA polymerase
• Topoisomerase binding site (CCCTT) for directional topoisomerase I-mediated joining of your PCR product to the 5′ or 3′ element of choice.
• Priming site for secondary amplification of the linear DNA template using the T7 Promoter Primer #2.

T3 Elements

The sequences of the T3 elements are provided below. The elements contain the following features:

• T3 promoter sequences for transcription of your gene of interest using the T3 RNA polymerase
• Topoisomerase binding site (CCCTT) for directional topoisomerase I-mediated joining of your PCR product to the 5′ or 3′ element of choice.
• Priming site for secondary amplification of the linear DNA template using the T3 Promoter Primer #2.

T7 Kozak 5′ Element

The sequence of the T7 Kozak 5′ Element is provided below. The element contains the following features:

• T7 promoter sequences for transcription of your gene of interest using the T7 RNA polymerase
• Topoisomerase binding site (CCCTT) for directional topoisomerase I-mediated joining of your PCR product to the 5′ or 3′ element of choice.
• Kozak translation initiation sequence (shown in bold in the sequence) with an ATG initiation codon for proper initiation of translation (Kozak, 1987; Kozak, 1991; Kozak, 1990).
• T7 Kozak priming site for secondary amplification of the linear DNA template

pA Elements

The sequences of the pA elements are provided below. The elements contain the following features:

• Topoisomerase binding site (CCCTT) for directional topoisomerase I-mediated joining of your PCR product to the 5′ or 3′ element of choice.
• Poly A tail for stable mRNA production and efficient translation of your gene of interest.
• pA priming site for secondary amplification of the linear DNA template.

Introduction

Once you have generated a linear DNA construct containing your gene of interest and a TOPO Tools element of choice, you are ready to use the linear DNA construct for your downstream application such as in vitro transcriptio  or coupled in vitro transcription/translation. You may use the protocol of choice or commercially available kits to perform in vitro transcription or coupled in vitro transcription/translation reaction. If you are using commercially available kits, please refer to the manufacturer’s recommendations.

For general information and protocols on in vitro transcription or coupled in vitro transcription/translation, please refer to Current Protocols in Molecular Biology (Ausubel et al., 1994).

Before Starting

Before performing in vitro transcription or coupled in vitro transcription/translation, be sure to have the following:

• 100-200 ng of your linear DNA construct that you created by TOPO Joining the PCR product with a choice of T7 or T3 Elements.
• A single discrete band corresponding in size to the linked linear DNA construct of sufficient yield after the secondary amplification. If you need to purify the PCR product, you may use the protocol provided in the Appendix section of the TOPO Tools Technology manual. Other protocols are suitable.

General Handling of RNA

Please note that the transcription reaction produces RNA. When working with RNA, be sure to follow the tips listed below to prevent RNA degradation:

• Use disposable, individually wrapped, sterile plasticware.
• Use only sterile, new pipette tips and microcentrifuge tubes.
• Wear latex gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin.
• Use proper microbiological aseptic technique when working with RNA.

1. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1994). Current Protocols in Molecular Biology (New York: Greene Publishing Associates and Wiley-Interscience).
2. Kozak, M. (1987). An Analysis of 5´-Noncoding Sequences from 699 Vertebrate Messenger RNAs. Nucleic Acids Res. 15, 8125-8148.
3. Kozak, M. (1991). An Analysis of Vertebrate mRNA Sequences: Intimations of Translational Control. J. Cell Biology 115, 887-903.
4. Kozak, M. (1990). Downstream Secondary Structure Facilitates Recognition of Initiator Codons by Eukaryotic Ribosomes. Proc. Natl. Acad. Sci. USA 87, 8301-8305.