Knight:TOPO TA cloning

Purpose

Efficient cloning of PCR products.

Materials

• PCR product generated with Taq polymerase (for 3' A tails)
• TOPO cloning kit

Procedure

Reshma's procedure

(Note that the kit details a slightly different procedure which may give superior efficiencies.)

1. Mix 1 μL PCR product (amplified with Platinum PCR supermix) with 0.5 or 1μL TOPO vector (0.5μL should be sufficient.)
2. Incubate 5 mins on the bench top
3. Place on ice
4. Transform

Notes

• Tom Knight claims you can cut the reaction volume in half and it will still work fine.
• Use fresh PCR product for best results. (The A overhang tends to get chewed back over time ... even using day old PCR product can reduce efficiency.)
• Your primers should NOT have 5' phosphates. They interfere with topoisomerase I.
• From Tom Knight: As this article discusses, the 5' end of the PCR primer has a strong effect on the likelihood of 3' A addition to the PCR product when using Taq or Taq mixtures as enzymes during PCR. Since it is often necessary to add such an overhang to PCR products for cutting of an added cloning site, and since for such applications the sequence is immaterial, we should probably standardize that sequence to the A tail favoring sequence, GTTTCT for all PCR primers we make. This will favor a 3' A overhang on the PCR products, and allow TA cloning, or TOPO TA cloning of these products. Blunt end cloning could still be done by using Pfu or Phusion enzymes. I can't make any sense from the result about extending the primer sequence another base which is not specified. It seems to me this can not have any effect.
  • The 3' A overhang efficiency can be promoted even further by increasing the sequence length to GTTTCTT.
• Reshma Shetty was generating some PCR fragments via annealing and primer extension and trying to cut with BioBricks enzymes and clone but the reactions were failing. Switching to TOPO TA cloning solved the problem.
• In order for your part to be in BioBricks format after TOPO TA cloning, you must have the full BioBricks ends on your primers.
• Austin Che observed anecdotally that he had a greater percentage of correct clones when he screened colonies from Kan plates rather than Amp plates.
• Also see Knight:Addition of 3' A-overhangs.

Resources

• Website
• Manual
• Product notes
• Brochure
• pCR4-TOPO

References

1. Brownstein MJ, Carpten JD, and Smith JR. Modulation of non-templated nucleotide addition by Taq DNA polymerase: primer modifications that facilitate genotyping. Biotechniques. 1996 Jun;20(6):1004-6, 1008-10. DOI:10.2144/96206st01 |
2. Shuman S. Recombination mediated by vaccinia virus DNA topoisomerase I in Escherichia coli is sequence specific. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):10104-8. DOI:10.1073/pnas.88.22.10104 |
3. Cheng C and Shuman S. DNA strand transfer catalyzed by vaccinia topoisomerase: ligation of DNAs containing a 3' mononucleotide overhang. Nucleic Acids Res. 2000 May 1;28(9):1893-8. DOI:10.1093/nar/28.9.1893 |
4. Shuman S. Novel approach to molecular cloning and polynucleotide synthesis using vaccinia DNA topoisomerase. J Biol Chem. 1994 Dec 23;269(51):32678-84.
5. Heyman JA, Cornthwaite J, Foncerrada L, Gilmore JR, Gontang E, Hartman KJ, Hernandez CL, Hood R, Hull HM, Lee WY, Marcil R, Marsh EJ, Mudd KM, Patino MJ, Purcell TJ, Rowland JJ, Sindici ML, and Hoeffler JP. Genome-scale cloning and expression of individual open reading frames using topoisomerase I-mediated ligation. Genome Res. 1999 Apr;9(4):383-92.

Patents

1. US Patent 6,916,632; Methods and reagents for molecular cloning. (On topoisomerase-mediated cloning using a 5' overhang and blunt end.) pdf html
2. US Patent 5,766,891; Method for molecular cloning and polynucleotide synthesis using vaccinia topoisomerase pdf html
3. US Patent 6,653,106; Topoisomerase based ligation and cloning methods pdf
4. US Patent 6,548,277; Method for molecular cloning and polynucleotide synthesis using vaccinia topoisomerase pdf html
5. US Patent 5,487,993; Direct cloning of PCR amplified nucleic acids pdf html
6. US Patent 5,827,657; Direct cloning of PCR amplified nucleic acids pdf html