1Graduate School of Systems Life Sciences, Kyushu University, Hakozaki, Fukuoka 812-8581, Japan and Research Fellow of the Japan Society for the Promotion of Science, Japan and 2Department of Biology, Juntendo University School of Medicine, 1-1 Hiraga Gakuendai, Inzai, Chiba 270-1695, Japan
Traditional cloning procedures with restriction enzymes have limitations and lack efficiency when constructing recombinant molecules using multiple fragments. SA-cloning can construct an expression plasmid over 10kb with multiple fragments at one time by the self-assembly of complementary staggered overhangs on PCR-amplified fragments.
⇒ATTENTION:The following procedure is a typical example of SA-cloning to insert one fragment into a vector via short sequences. You can apply this procedure to multiple fragment assembly or in-frame seamless cloning with appropriate primer sets.
Amplification of the target gene and cloning vector
In SA-cloning, two PCR reactions should be prepared for one insert (Fig. 1, Tubes A and B); one contains a tailed forward primer (Fig. 1, primer 1') and an untailed gene specific reverse primer (Fig. 1, primer 2) and the other has an untailed gene specific forward primer (Fig. 1, primer 1') and a tailed reverse primer (Fig. 1, primer 2'). Additionally, two PCR reactions should be prepared for the vector (Fig. 1, Tube C and D).
1.Design and synthesize the gene specific PCR primer sets for an insert and cloning vector (Fig. 1, primer 1, 2, 3, and 4).
2. Design and synthesize tailed PCR primers with complementary overhangs required for self-assembly (Fig. 1, primer 1', 2', 3', and 4').
For cloning multiple fragments at one time, you must design each overhang sequence specifically to avoid undesired assembly.
To perform seamless cloning of a fusion gene with multiple fragments, the sequences of overhang regions need to be designed so that the corresponding fragments can assemble in frame.
3. Amplify the target by PCR using sets of tailed and untailed primers (Table 3 and Fig. 1, Tube A to D).
⇒ATTENTION:You must use a type DNA polymerase having 3'-5'exonuclease activity because the PCR fragment used in SA-cloning procedure must NOT have single 3' adenine overhangs.
*HINT:In order to minimize PCR error, you should use a high fidelity DNA polymerase like PrimeSTAR® Max DNA polymerase (Takara) in this protocol.
4. After amplification, add 1µL DpnI (20 units/µL; NEB) and incubate at 37°C for 60 minutes to completely digest the template DNA (Table 4).
5. Using 3µL of the PCR reaction, visualize each PCR fragment by gel electrophoresis to verify the quality and quantity.
⇒ATTENTION:If you find nonspecific bands, adjust the PCR conditions and repeat the PCR step.
⇒ATTENTION:If you find that the template DNA remains undigested, digest the template further with an additional 1µL of DpnI. Step 5 is quite important for successful cloning.
6. Mix two sibling amplicons (Fig. 1, Tube A + B, and Tube C + D) and purify the pooled PCR fragments with the QIAquick Gel Extraction Kit (QIAGEN) (Fig. 1, Tube E and F).
*HINT:You can purify the PCR fragments manually or with another kit.
7. Quantify the concentration of purified fragments (Fig. 1 Tube E and F) by spectrophotometer.
⇒ATTENTION:For multiple fragment assembly, higher concentrations are better.
Denaturation, Re-annealing, and Self-assembly
Denaturation and re-annealing are performed in thin-wall PCR tubes (Fig. 1, Tube G and H) to produce fragments with single-stranded overhangs, one tailed on the 5' end and the other on the 3' end, using 500 ng of pooled PCR fragments. The self-assembly of fragments to make a desired circular plasmid is also performed in thin-wall PCR tubes (Fig. 1, Tube I).
8. Prepare Master Mixes (Table 5) in PCR tubes (Fig. 1, Tube G and H).
9. Set Tube G and Tube H in the thermal cycler.
10. Denature at 95°C for 3 minutes.
11. Re-anneal by gradually cooling the tubes to 25°C. (It usually takes 3 minutes.)
⇒ATTENTION:To avoid misannealing, you must not rapidly cool the tubes.
12. Self-assembly step: Mix equal amounts of two reactions (Fig. 1 Tube G + H) into Tube I, and then keep at 25°C for 5 minutes (Fig. 1 Tube I)
*HINT:Instead of using 10x PCR buffer supplied in TaKaRa Ex Taq (Takara), you can manually prepare the Master Mix so DNA fragments can be denatured and re-annealed.
Regarding the transformation steps, follow the instruction manual of competent cells you use. We usually use One Shot® TOP10 Chemically Competent E. coli (Invitrogen) by the following procedure.
13. Keep the vial of competent E. coli on ice.
14. After thawing, add 7µL reaction mixture (Fig. 1, Tube I) into a vial and mix gently.
15. Incubate on ice for 5 to 30 minutes.
16. Heat-shock the cells for 30 sec. at 42°C without shaking.
17. Immediately transfer the tube to ice.
18. Add 250 to 300µL of 37°C SOC medium (Invitrogen).
19. Shake the tube at 250 rpm at 37°C for 60 minutes.
20. Spread the full volume on a pre-warmed selective plate with appropriate antibiotics.
✋RESTIncubate overnight at 37°C.
21. Pick up several colonies and check the vector sequence by PCR or sequencing.
*HINT:Instead of One Shot® TOP10 Chemically Competent E. coli, other competent cells can be used.
⇒ATTENTION:Almost all colonies usually have a positive plasmid as far as our experience goes, however we strongly recommend that you check several colonies with PCR and/or sequencing analysis.
Non-specific bands in PCR
Raise temperature at annealing step in PCR.
Reduce the amount of a template DNA.
Re-design the overhang sequences.
Prepare a new primer that anneals to a different site.
If there is no improvement with the changes described above, purify the desired fragment after gel electrophoresis.
Be sure to check the quality and quantity of the PCR reaction in Tube A, B, C and D by gel electrophoresis.
Be sure to troubleshoot as described above for any non-specific bands seen in the gel following PCR.
For undigested template DNA, add 1µL of DpnI into the reaction and completely digest it.
Do not rapidly cool down the reaction after the denaturation step.
Check whether the sequences of overhang regions are unique. Although you need to design overhang regions that are similar to each other, for example to establish a repetitive sequence, we strongly recommend that the sequence homology between overhang regions be less than 75%.
Lengthen the overhang region. The minimum length of the overhang region for successful self-assembly is six base pairs.
Check the quality and quantity of the PCR reaction in Tube A, B, C and D by gel electrophoresis. See above.
Increase the concentration of PCR fragments in the denaturation and re-annealing steps (Fig. 1 Tube G and H).
Check whether each pair of overhang sequences is perfectly complementary. Even a single mismatch in an overhang region drastically reduces the cloning efficiency in SA-cloning.
Lengthen the overhang region. The minimum length of overhangs for successful self-assembly is six base pairs.
PrimeSTAR® Max DNA polymerase (Takara Bio, Tokyo, Japan)
DpnI (20 units/µL; New England Biolabs, Ipswich, MA, USA)
QIAquick Gel Extraction Kit (QIAGEN KK, Tokyo, Japan)
10x PCR buffer supplied in TaKaRa Ex Taq® (Takara Bio, Tokyo, Japan)
One Shot® TOP10 Chemically Competent E.coli [1 × 109 colony formation units (cfu)/g pUC19] (Invitrogen Japan, Tokyo, Japan]
T-Personal Thermal Cycler 48 (Biometra GmbH, Göttingen, Germany)
DU70 spectrophotometer (Beckmann, München, Germany)
Mupid-2Plus submarine electrophoresis system (Advance, Tokyo, Japan)