Finnzymes' DNA polymerases and cloning

Phusion® DNA polymerases  have an extremely low error rate, which makes them the ideal choice for cloning. Therefore we strongly recommend Phusion DNA polymerases for all cloning applications. Below you can find information about cloning PCR products amplified with Phusion.

However, if you are cloning PCR products amplified with other Finnzymes’ DNA polymerases, you need to consider that different DNA polymerases create different type of ends in the PCR products, which affects the subsequent cloning procedure. Additional information can be found below.

     

PCR product ends

Suitability for cloning

Phusion® Hot Start High-Fidelity DNA Polymerase

blunt

+++

Phusion® High-Fidelity DNA Polymerase

blunt

+++

Phusion® Flash High-Fidelity PCR Master Mix

blunt

+++

Phire™ Hot Start DNA Polymerase

blunt

+

DyNAzyme™ EXT DNA Polymerase

blunt/A-overhang

++

DyNAzyme™ II Hot Start DNA Polymerase

A-overhang

+

DyNAzyme™ II DNA Polymerase

A-overhang

+

DyNAzyme™ I DNA Polymerase

A-overhang

+

Phusion DNA polymerases – the best choice for high-fidelity cloning

Phusion® DNA Polymerases create blunt end DNA products. When cloning fragments amplified with Phusion DNA Polymerases, blunt end cloning is recommended.

If TA cloning is required, it can be performed by adding 3' A overhangs to the blunt PCR product with a different polymerase (e.g.
DyNAzyme™ II DNA Polymerase, Finnzymes).

Procedure for adding 3' A-overhangs (TA cloning)

1. Purify the PCR product (e.g. with a commercial PCR purification kit or phenol extraction and DNA precipitation)
Before adding the overhangs it is very important to remove all the Phusion DNA Polymerase by purifying the PCR product carefully, as the proofreading activity in Phusion DNA Polymerase is very strong. Any remaining Phusion DNA Polymerase will degrade the A overhangs, thus creating blunt ends again.

2. A-addition with DyNAzyme II DNA Polymerase (or Taq DNA polymerase)

Reaction components:

  • Purified PCR product
  • 0.2 mM dATP
  • 1x Optimized DyNAzyme Buffer
  • 1 U DyNAzyme II DNA Polymerase

Incubate 20 min at 72 °C.

3. Proceed to TA cloning. For optimal ligation efficiency, we recommend using fresh PCR products, since 3´A-overhangs will gradually be lost during storage.

Phire Hot Start DNA Polymerase

Phire Hot Start DNA Polymerase creates blunt end PCR products. If you are cloning fragments amplified with Phire Hot Start DNA Polymerase, blunt end cloning is recommended.
If TA cloning is required, it can be performed by adding 3' A overhangs to the blunt PCR product with a different polymerase (e.g.
DyNAzyme™ II DNA Polymerase, Finnzymes). The same protocol that is recommended for Phusion DNA polymerases (see above) can also be applied to Phire DNA Polymerase. Even though Phire only possesses weak proofreading activity, we recommend that the enzyme is removed (step 1 of the protocol) before adding A-overhangs to make sure that blunt ends will not be recreated.

DyNAzyme EXT DNA Polymerase

DyNAzyme EXT can create both blunt ends and A-overhangs to the PCR products. Products amplified with DyNAzyme EXT can be cloned in both blunt and TA vectors.  Blunt ends and A-overhangs  always coexist in the product, but in certain conditions one of them may be more dominant. Some generalization about this can be made as follows:

 

  • If the last nucleotide in the 3’ end of the PCR product is T (the PCR primer starts with A), DyNAzyme EXT prefers  adding A-overhang. This situation favors TA cloning.
  • If the last nucleotide in the 3’ end of the PCR product is  A (the PCR primer starts with T), the addition of a non-templated A-overhang is unlikely, and blunt ends are created. In this case blunt cloning is a better choice.

DyNAzyme II and DyNAzyme I DNA Polymerases

Both DyNAzyme II and DyNAzyme I create A-overhangs in the PCR products. If PCR products amplified with DyNAzyme I or II need to be cloned, TA cloning can be performed.