TOPO Cloning Technology Fast, efficient, and simple cloning

Get the cloning results you can count on When Invitrogen™ TOPO™ cloning technology was launched, it sparked a revolution. Over the past 18 years, TOPO technology has become the most widely used cloning method in the world. TOPO cloning technology is: • Efficient—up to 95% of clones contain desired insert • Fast—5-minute, room temperature reaction • Easy—simple 3-step procedure • Proven—over 20,000 citations • Flexible—available with or without competent cells, in multiple reaction sizes Whether you’re performing general subcloning, sequencing, TA or blunt-end cloning, long-fragment cloning, expression vector cloning, directional cloning, or using the Gateway™ system, there’s a TOPO cloning solution that’s right for you. Fast, reliable, and direct, TOPO cloning helps you get the right clone sooner, freeing up your time to answer more important questions.

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TOPO cloning

Contents The technology behind TOPO cloning .......................4

TOPO TA cloning ......................................................5

Blunt-end TOPO cloning ...........................................6

TOPO long-fragment cloning.....................................7

Directional TOPO cloning ..........................................8

TOPO expression vector cloning ...............................9

Entry into the Gateway system ................................10

Custom TOPO Adaptation Service .......................... 11

thermofisher.com/topo

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The technology behind TOPO cloning TOPO cloning is as easy as 1, 2, 3 Perform PCR with Taq or a proofreading polymerase.

A

CACC

PCR product PCR product

A

OR

1. Add 1 µL of PCR reaction to 1 µL of TOPO cloning vector.

2. Incubate 5 min at room temperature.

3. Transform the competent E. coli provided.

PCR product

TOPO TOPO

TOPO cloning vector

55

0

5 10

50 45

15 20

40 35

30

25

Figure 1. TOPO PCR cloning requires just three easy steps. Simply combine your PCR product and a TOPO cloning vector in the provided reaction buffer, wait 5 minutes, then transform E. coli. With TOPO cloning, the additional time, steps, and reagents required for ligase-mediated cloning are eliminated.

The key to TOPO cloning is the enzyme DNA topoisomerase I, which functions both as a restriction enzyme and as a ligase. Its biological role 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 attached to the 3´ thymidine. It cleaves one DNA strand, enabling the DNA to unwind. The enzyme then religates the ends of the cleaved strand and is released from the DNA.

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TOPO cloning

To harness the religating activity of topoisomerase, TOPO™ vectors are provided linearized with topoisomerase I covalently bound to the 3´ phosphate on each end. This enables the vectors to readily ligate DNA sequences with compatible ends. The ligation is complete in only 5 minutes (Figure 1).

TOPO TA cloning Invitrogen™ TOPO™ TA cloning kits are designed for cloning PCR products amplified by Taq DNA polymerase, which leaves an adenine at the 3´ end of the product, creating overhangs or sticky ends (Figure 2). The TOPO™ TA vectors include 3´-thymine overhangs for fast, effective, and direct cloning of Taq-amplified PCR products. Figure 3. The pCR®2.1®-TOPO® TA vector.

Applications

EcoR I EcoR V BstX I Not I Xho I Nsi I Xba I Apa I

EcoR I EcoR V BstX I Not I Xho I Nsi I Xba I Apa I

T Figure 4. The pCR®4-TOPO® TA Vector. T

Blunt-end PCR product

TOPO pCR2.1TOPO

33 bp

T3

Am

• Ampicillin and kanamycin resistance genes and a lacZα–ccdB gene fusion for positive selection

3.9 kb

Taq-amplified PCR product

P

CCCTT GGGA

3´ phosphate

+

A

PCR product

A

TOPO

PCR product

Spe I Sse8387 I Pme I EcoR I

A AGGG

T TCCC

M13

TOPO lacZα –cc dB

l

Topoisomerase I is released

pCR4-TOPO 4.0 kb

Ligation complete

yc in

Taq-amplified PCR product

T

60 bp

T7

Kana m

TOPO TA cloning vector

33 bp

T

c

TOPO

TOPO

TOPO

33 bp

Pla

CCCT T GGGA A

A

ri

P

5 min at room temperature

T3

p UC o

AGGG TTCCC

M13

pCR®4Blunt-TOPO®

Figure 3. The pCR2.1TOPO TA Vector.

TOPO

73 bp

TOPO

M13

n Ka

pi c il lin

A

Topoisomerase I recognition sites

60 bp

T7

yci

M13

33 bp

n

73 bp

T7

TOPO

lacZα

am

• Easy blue⁄white colony screening for selection of recombinants

M13

TOPO

EcoR I Not I

• Ampicillin and kanamycin resistance genes for your choice of selection in E. coli

M13 reverse priming site

EcoR I Not I

• Minimal multiple cloning site to shorten the distance between sequencing primer sites and the insert site to as little as 33 bp

TOPO

Spe I Sse8387 I Pme I EcoR I

• 15 convenient and validated restriction sites flanking the insert for easy, directional subcloning

®

pCR®2.1-TOPO®

pUC or i

• EcoRI sites flanking the PCR product insertion site for easy excision of inserts

®

Nsi I Hind III Kpn I Sac I BamH I Spe I BstX I EcoR I

• T7 promoter and M13 forward and reverse primer sites for in vitro transcription and sequencing

• Flanking EcoRI sites for simplified excision of pCR II-TOPO cloned PCR products and a unique Sse8387I TOPO site for generation of nested deletions prior to T SP6 T7 sequencing T

Hind III Kpn I Sac I BamH I Spe I BstX I EcoR I

TOPO™ TA sequencing kits utilize our pCR™4TOPO™ TA Vector, which allows you to directly select recombinants by disrupting the lethal E. coli gene, ccdB. The vector contains the ccdB gene fused to the C-terminus of the lacZα gene (Figure 4). Ligation of a PCR product disrupts expression of the lacZα–ccdB gene fusion, permitting growth of only positive recombinants upon transformation. Competent cells that contain nonrecombinant vector are killed upon plating, so blue/white screening is not required. The pCR4-TOPO TA Vector features:

i

TOPO™ TA subcloning kits utilize our pCR™2.1-TOPO™ TA Vector (Figure 3), which features:

or

Sequencing

f1

Subcloning

Am

Figure 2. TOPO TA cloning of Taq-amplified DNA.

pici

ll in

Figure 4. The pCR4TOPO TA Vector.

Topoisomerase I recognition sites

TOPO

AAGGG TTCCC

5 min at room temperature

TOPO

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Blunt-end TOPO cloning Invitrogen™ Zero Blunt™ TOPO™ PCR cloning kits are designed for cloning PCR products amplified with thermostable proofreading polymerases. These polymerases have extensive 3´ to 5´ exonuclease activity, and do not leave 3´-A overhangs. High-fidelity polymerases such as Invitrogen™ Platinum™ Pfx DNA Polymerase have this exonuclease activity, leaving blunt-ended PCR products. Therefore, the vectors supplied in our Zero Blunt TOPO PCR cloning kits have blunt ends as well for effective ligation (Figure 5).

All Zero Blunt TOPO vectors contain the lethal ccdB gene. With this positive selection method, only clones with an insert will grow as colonies on your plate, giving you the confidence you need in your blunt-end cloning results.

Applications Subcloning

Sequencing

Zero Blunt™ TOPO™ subcloning kits utilize our pCR™-Blunt II-TOPO™ Vector (Figure 6). It features:

Zero Blunt™ TOPO™ sequencing kits utilize our pCR™4Blunt-TOPO™ Vector (Figure 7). It features:

• The ccdB gene for positive selection, only permitting growth of plasmid vectors with recombinants

• Minimal multiple cloning site to shorten the distance between sequencing primer sites and the insert site to as little as 33 bp

• Kanamycin and Zeocin™ antibiotic resistance genes for your choice of selection in E. coli

• The ccdB gene for positive selection, only permitting growth of plasmid vectors with recombinants

T3

33 bp

P

CCCTT GGGAA

3´ phosphate

+

Topoisomerase I is released

pCR 4-TOPO Figure 6. The pCRBlunt II-TOPO Vector.

M13

pCR4Blunt-TOPO

®

SpeI Sse8387 I PmeI EcoR I

lac Z

ac

cc

TOPO dB

pCR4BluntTOPO 4.0 kb

Ligation complete

60 bp

T7

yc in

TOPO cloning

Pl

33 bp

Kana m

Blunt-end PCR product

Figure 5. Zero Blunt TOPO cloning of blunt-end DNA.

6

T3

AAGGG TTCCC

TOPO

TOPO

Zero Blunt TOPO vector

PCR product

M13

®

T7 T TOPO

T TOPO

33 bp

TOPO

CCCTT GGGAA

PCR product

T

60 bp

Blunt-end PCR Product

M13

5 min at room temperature

33 bp

3.5 T kb Ze ocin

ri

P

AAGGG TTCCC

A

EcoR I Not I

M13

Taq-amplified PCR Product

A

73 bp

p UC o

TOPO

ccd

pCR-Blunt IITOPO TOPO

73 bp

Topoisomerase I recognition sites

EcoR I Pst I EcoR V Not I Xho I Nsi I Xba I Dra II Apa I

Nsi I Hind III Asp718 I Kpn I Ecl136 II Sac I BamH I Spe I EcoR I

• Ampicillin and kanamycin resistance genes for 5 min at TOPO your choice of selection in E. coli room temperature

CCCT T AGGG PCR product • EcoRI sites flanking the PCR ATproduct insertion GGGA A TCCC site for easy excision of inserts and aTopoisomerase unique I GGGA is released TOPO Sse8387I site for generation of nested deletions • M13 forwardTOPO and reverse primer sites for sequencing PCR screening Taq-amplified PCR product prior to sequencingLigation complete TOPO TAor cloning vector

TOPO

la cZα

c

T7

B Pla Figure 7. The pCR®-4Blunt II-TOPO® Vector.

EcoR I Not I

• SP6 promoter/primer site for in vitro transcription + A PCR product A P P 3´ phosphate and sequencing CCCTT

TOPO

SP6

pU C o r i

AGGG TTCCC

TOPO

• M13 forward, M13 reverse, T7, and T3 priming sites for sequencing

Kanamycin

• EcoRI sites flanking the PCR product insertion site for easy excision of inserts

• T7 and T3 promoters for in vitro transcription

SpeI Sse8387 I PmeI EcoR I

Topoisomerase I recognition sites

Figure 6. The pCR®-Blunt II-TOPO® Vector.

Am

p i ci

lliin

Figure 7. The pCR4Blunt-TOPO Vector.

TOPO long-fragment cloning Invitrogen™ TOPO™ XL PCR Cloning Kit combines TOPO cloning, the ccdB gene for positive selection, and a unique gel purification step to enhance cloning of PCR products from 3 to 10 kb. Long PCR often yields multiple products, making gel purification necessary prior to cloning. However, gel purification requires exposure to ethidium bromide and UV light, which can nick and damage DNA. To help protect against nicking, the TOPO XL PCR Cloning Kit uses crystal violet to enable visualization of DNA bands in an agarose gel under ambient light. This eliminates the need for ethidium bromide and UV light exposure, helping to ensure safe gel purification. This results in significantly more colonies and a greater percentage of recombinants than using ethidium bromide and UV light (Figure 8). Features of the pCR™-XL-TOPO™ Vector (Figure 9) include: • ccdB gene to help eliminate background of nonrecombinant clones • Kanamycin and Zeocin antibiotic resistance genes for your choice of selection in E. coli • T7 promoter for in vitro transcription and sequencing

• M13 forward (–20) and reverse priming sites for sequencing or PCR screening Figure 9. The pCR®-XL-TOPO® Vector. • Selection of competent cells for routine cloning, high-speed cloning, or electroporation

300

M13

T T

T7

200 c

Pla

la cZα

ccd B

TOPO

150

50

0 Crystal violet

Ethidium bromide

Figure 8. Crystal violet enhances TOPO cloning of large fragments. A 7 kb ampicilin resistance gene sequence was PCR-amplified, and PCR products were loaded onto one gel containing crystal violet and another gel containing ethidium bromide. PCR products were gel purified and cloned into the pCR-XL-TOPO Vector. The number of recombinants was determined by plating 125 µL of each transformation on LB plates containing either kanamycin or kanamycin and ampicillin. The crystal violet– stained gel allowed for 94% positive recombinants, while the ethidium bromide–stained gel allowed only 60% positive recombinants.

pCR-XLTOPO 3.5 kb

Kanamyci n

100

p UC o r i

Number of colonies

Colonies with insert (KanR, AmpR)

EcoR I Pst I EcoR V Not I Xho I Nsi I Xba I Dra II Apa I

250

Mlu I Nsi I Hind III Kpn I Ecl136 II Sac I BamH I Spe I EcoR I

TOPO Total colonies (KanR)

Ze ocin

Figure 9. The pCR-XL-TOPO Vector.

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Directional TOPO cloning Directional TOPO cloning enables cloning of blunt-ended PCR products directly into an expression vector in a single orientation. With a 5-minute ligation reaction, this technology eliminates subcloning steps and saves you time. Directional TOPO cloning vectors have a single-stranded GTGG overhang at one end and a blunt end at the other (Figure 10). The GTGG overhang invades the doublestranded DNA of the PCR product and anneals to the CACC sequence that you place in your primer. Topoisomerase I then ligates the PCR product in the correct orientation for expression. With Directional TOPO Cloning Expression Kits, you can: • Save time—TOPO cloning of your PCR product takes just 5 minutes • Obtain efficient cloning results—more than 90% of recombinant clones will be in the correct orientation for expression • Achieve high-level expression—vectors carry powerful promoters for expression in E. coli or mammalian cells

Topoisomerase I recognition sites

TOPO

P

5 min at room temperature

AAGGG TTCCC P

CCCTT GGGAAGTGG

+

3´ phosphate

CACC GTGG

Directional TOPO cloning vector

TOPO

Blunt-end PCR product (designed with CACC at one end, no modification at the other)

Figure 10. Directional TOPO cloning of blunt-end DNA.

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TOPO cloning

CCCTT CACC GGGAAGTGG PCR product

PCR product

TOPO

TOPO

AA GGG TT CCC

GTGG

Extra GTGG removed after transformation

Ligation complete

Topoisomerase I is released

TOPO expression vector cloning TOPO cloning for E. coli expression Invitrogen™ Champion™ pET Directional TOPO™ vectors are powerful E. coli expression vectors that use the highly efficient T7 RNA polymerase to achieve high protein yields. T7 RNA polymerase is expressed by host E. coli under the control of the IPTG-inducible lacUV5 promoter. This allows you to regulate transcription with IPTG. The additional lacO element found in the T7 lac promoter used in these vectors further reduces basal expression levels while enabling strong transcriptional activity upon induction with IPTG. Reported yields of recombinant proteins from pET vectors are typically in the range of tens to hundreds of milligrams per liter of culture.

TOPO cloning for mammalian expression

ViraPower HiPerform Lentiviral Expression Systems

For constitutive mammalian expression, the pcDNA™ mammalian expression vector is one of the most popular expression vectors available today. The newest versions are the pcDNA™ 3.3-TOPO™ TA Vector and pcDNA™ 3.4TOPO™ TA Vector, which enable expression of exceptionally high levels of recombinant protein in mammalian cells and are ideal for use with our Invitrogen™ Expi293™ and FreeStyle™ Expression Systems. These vectors offer:

Invitrogen™ ViraPower™ HiPerform™ Lentiviral Expression Systems are designed to provide stable gene expression and reproducible delivery to both dividing and nondividing cells. This system offers:

• Two- to five-fold higher protein yields compared to other expression vectors

• A choice of Gateway™ or TOPO TA cloning vectors

• Generation of native (or tagged) proteins without extraneous amino acids—ideal for antibody production and structural biology • Full-length human cytomegalovirus (CMV) immediate-early promoter/enhancer for highlevel gene expression in a wide range of mammalian cells • TOPO cloning site for rapid and efficient (>85%) cloning of Taq-amplified PCR products • Neomycin resistance gene for selection of stable cell lines with Geneticin™ antibiotic • pUC origin for high copy number and maintenance of the plasmid in E. coli

• Greater than four-fold increase in protein expression compared to other lentiviral vectors • Efficient gene delivery into cells that are virtually impossible to transfect • Accurate and fast 2-day titer determination of functional lentivirus

ViraPower™ expression systems enable high levels of stable gene expression necessary for valid results in virtually any cell line, especially in primary or difficult-to-transfect cells.

We also carry TOPO™ expression vectors for yeast, algae, and insect cells. Find the right vector for your research at thermofisher.com/vectors

Additionally, the pcDNA 3.4-TOPO TA Vector includes the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) downstream of the cloning site to enhance transcript expression and yields. thermofisher.com/topo

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Entry into the Gateway system The Gateway™ system is a powerful cloning technology that offers a rapid and highly efficient route to multiple expression systems (Figure 11). There are many ways to clone a gene, but only Gateway technology lets you rapidly transfer your gene into different expression vectors with minimal cloning effort. Take the first step towards accessing multiple expression vectors by simply cloning your gene into a Gateway™ entry vector. Whether you prefer TOPO or restriction enzyme cloning with PCR products or Invitrogen™ GeneArt™ Strings™ DNA Fragments, we have a Gateway entry vector for you. Enter the Gateway system via: TOPO cloning Restriction enzyme cloning BP Clonase™ enzyme reaction

Looking for an alternative to PCR for your cloning? GeneArt Strings DNA Fragments are custom-made, uncloned, doublestranded linear DNA fragments up to 3,000 bp in length, assembled from synthetic oligonucleotides using the same high-quality process developed for Invitrogen™ GeneArt™ Gene Synthesis. Strings DNA Fragments are delivered dried and ready for resuspension, cloning, and screening to enable identification of the correct clone. • Affordable—Strings DNA Fragments are a cost-effective alternative to complete gene synthesis • Flexible—full gene design and cloning flexibility with no template required; clone with any downstream method of choice, including TOPO TA cloning and Zero Blunt TOPO cloning

DNA insert sources: PCR GeneArt Strings DNA Fragments Ultimate™ ORF Collection

• Streamlined—enter your gene sequence and directly edit, optimize, and order through our online GeneArt™ portal Mammalian

Viral

Gene

Gene

att

att

att

• Fast—at least 200 ng of Strings DNA Fragments are produced within 5 (for up to 1,000 bp) or 8 (for 1,000–3,000 bp) business days att

Find out more at thermofisher.com/strings Entry clone

E. coli Gene att

Your vector

Gene att

att

Gene att

att

Baculovirus

Two-hybrid screening vector

Gene

Gene

att

att

att

att

Figure 11. The flexibility of Gateway technology. This powerful technology is designed to simplify cloning and provide a rapid and highly efficient route to multiple expression and functional analysis options.

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att

We offer several TOPO cloning kits that allow direct access to the Gateway system. Go to thermofisher.com/gateway to learn more about Gateway technology.

Custom TOPO Adaptation Service The development of gene-based therapeutic and diagnostic products requires the rapid analysis of a vast number of gene sequences. When screening gene targets that are of commercial importance, being the first to identify, clone, express, and validate these genes is crucial. Our Invitrogen™ Custom TOPO™ Adaptation Service puts the power of TOPO cloning into your vector. With your own vector adapted with TOPO technology, you can: • Save time—the TOPO cloning reaction only takes 5 minutes and is 95% efficient • Maintain your current experimental strategy—no need to change downstream assays or experimental parameters

Flexible solutions For your convenience and to help maximize your success, many of our TOPO cloning vectors: • Are now available with or without competent cells • Now include 25% more reactions for the same price

Cloning just got simpler—to learn more about TOPO cloning and select the best kit for your research, visit thermofisher.com/topo

thermofisher.com/topo

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Find out more at thermofisher.com/cloning For Research Use Only. Not for use in diagnostic procedures. © 2015 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. CO36607 0715