PCR Success Story #17: High-Fidelity PCR Amplification of repetitive sequences and PCR Overlap Assembly of 3 FragmentsPCR Success Story #17
High-Fidelity PCR Amplification of repetitive sequences and PCR Overlap Assembly of 3 Fragments
This project originated from a customer referral who learned about our problem-solving abilities. The researcher’s lab is located at the Centre de Recherche du Centre Hospitalier de l’Université Laval (CR-CHUL) in Québec city. They challenged us to amplify DNA sequences from confidential genomic DNA and from DNA plasmids containing other sequences of interest. Their goal was to assemble these 3 fragments by PCR overlap extension.
They tried to perform this using Phusion DNA Polymerase but were unsuccessful in the steps detailed below:
- The first challenge was to obtain specific bands by high-fidelity PCR.
- Then, assemble everything together by PCR overlap and extension.
Note: we had to redesign some of their primers because one of them had 2 binding sites, thus the PCR amplification could not be specific.
Competitor: Phusion® High-Fidelity DNA Polymerase (NEB version)
Phusion® is a trademark of ThermoFisher Scientific
Desired DNA Fragment Assembly DesignSo the goal and strategy of the design for PCR overlap and extension is depicted in this image. We have to produce 3 DNA fragments by high-fidelity PCR and join them together by high-fidelity PCR forward primer A and reverse primer F.
Problem: primer C has 2 binding sites…
Original primer C :
5′ end of MIDDLE fragment (primer C binding sites in bold and underlined):
A second binding site was identified for primer C containing a single mismatch in its extreme 5′ (A). Therefore we redesigned this primer to: GTTCTCCTTCCCCTCTCTCTCCTCTCTCCTCTCTCCAGGAG
PCR amplification of AB, CD and EF fragments
The AB fragment was amplified using TransStart KD Plus (Ultra-HiFi) DNA Polymerase.
CD and EF fragment were amplified by PCR using TransStart FastPfu (High-Fielity) DNA Polymerase.
The arrows point to the specific amplicons that were then extracted ad purified using the Favorprep GEL/PCR Purification Kit.
Successful PCR Overlap Assembly with FastPfu1 ul of purified LEFT, MIDDLE and RIGHT fragments were mixed with primer A and F in a standard FastPfu PCR reaction. The correct amplicon corresponding to the full assembly of LEFT-MIDDLE-RIGHT is shown by the arrow, along with the corresponding fragments. Likely due to the nature of fragment LEFT and MIDDLE fragments to multimerize, we can also see some non-desired bands above the fully assembled amplicon.
2×TransStart FastPfu FLY (Ultra-HiFi) PCR SuperMix – AS231111.00$ – 444.00$ CAD
2x TransStart FastPfu PCR SuperMix (-dye) – AS22187.00$ – 372.00$ CAD
2x TransStart® KD Plus PCR SuperMix – AS30193.00$ – 396.00$ CAD
5x FastPfu FLY Buffer (with Mg2+) – GK22130.00$ CAD
CUSTOM KIT – EasyTaq + High-Fidelity DNA PolymerasesFrom:
CUSTOM KIT – High-Fidelity DNA Polymerase + Competent Cells + Cloning KitFrom: 189.00$ CAD