( ...)Successful PCR amplification and subcloning of a GC-rich DNA fragment

Petra Majlingov, Paolo Paroncini, Silvia Svegliati, Michele Luchetti, and Armando Gabrielli
Laboratorio di Medicina Molecolare, Istituto di Clinica Medica, Polo Didattico Scientifico, Ancona, Italy

The formation of robust secondary structures that resist denaturation makes GC-rich sequences difficult to amplify by PCR. This article demonstrates how a "difficult" GC-rich sequence was successfully amplified using HotStarTaq DNA Polymerase and Q-Solution. The PCR product was then ligated into the pDrive Cloning Vector, which was used to transform QIAGEN EZ Competent Cells with high efficiency.

Our group has been working for many years on the regulation of expression of the myb family of transcription factors (13). The myb gene family is made up of three genes, c-myb, A-myb, and B-myb, whose pattern of expression is controlled by complex mechanisms. For example, c-myb expression is regulated by a mechanism of transcriptional block in the first intron and alternative splicing.

In order to further investigate the role of the c-myb intron in expression, we attempted to amplify the Myb-INT gene fragment. Myb-INT is a 4500 bp DNA fragment from the first c-myb intron, and has a GC content of about 60%. Sequences that contain a high ratio of guanosine and cytidine base pairs (GC-rich sequences) can form robust secondary structures that resist denaturation and prevent annealing of PCR primers.

PCR was performed using HotStarTaq DNA Polymerase and 4 other commercially available DNA polymerases. Of the 5 polymerases tested, only HotStarTaq DNA Polymerase (in conjunction with Q-Solution) allowed successful amplification. After amplification, the fragment was successfully subcloned into the pDrive Cloning Vector using the QIAGEN PCR Cloning^plus Kit and transformed into QIAGEN EZ Competent Cells.

Materials and methods

Several primer pairs were designed to amplify a 4.5 kb Myb-INT fragment of the c-myb gene. The PCR template was plasmid DNA purified using the QIAGEN Plasmid Maxi Kit. Each PCR contained 300 ng template DNA, 2.5 mM MgCl2, 0.2 mM each dNTP, and 2.5 units DNA polymerase, in 1x PCR Buffer. PCR was carried out in the presence and absence of 1x Q-Solution. PCR products were purified using the MinElute PCR Purification Kit. For the cloning procedure, 225 or 550 ng purified PCR product was added to 50 ng pDrive Cloning Vector in 1x Ligation Master Mix, in a total volume of 10 l. Subsequently, a 3 l aliquot of the ligation reaction was used to transform one tube of QIAGEN EZ Competent Cells, which were then directly plated onto LB agar plates containing X-gal and IPTG for blue/white screening, and ampicillin at 30 g/ml. A second set of ligation reactions were carried out, and 3 l aliquots used to transform electrocompetent DH5-a E. coli. After transformation, bacteria were grown at 37C for 40 minutes before plating as above. White colonies derived from both ligation reactions were picked and grown overnight at 37C in LB medium containing ampicillin at 20 g/ml. Pure plasmid DNA containing the PCR insert was isolated using the QIAprep Spin Miniprep Kit. The presence and correct orientation of the insert was confirmed by restriction digest using ClaI and SacI and by direct sequencing.

Successful PCR Amplification of a GC-Rich Gene Fragment

PCR of a GC-rich c-myb gene fragment using HotStarTaq DNA Polymerase (QIAGEN) or DNA polymerase from other sources (Suppliers WZ) in the presence (+) or absence () of Q-Solution. M: markers.

Proportion of recombinants containing c-myb gene PCR insert after transformation with pDrive Cloning Vector

E.coli strain % white colonies

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QIAGEN EZ Competent Cells 90% DH5-a cells 88%
Results and discussion

The efficiency of amplification of the 4.5 kb PCR product using the five different DNA polymerases is shown in Figure "Successful PCR Amplification of a GC-Rich Gene Fragment". Only the reaction containing HotStarTaq DNA Polymerase and Q-Solution enabled amplification of the GC-rich c-myb gene fragment. Q-Solution facilitates amplification of difficult templates by modifying the melting behavior of DNA.

An aliquot of the PCR product was used to successfully clone the c-myb gene fragment into the pDrive Cloning Vector. Cloning into this vector is based on hybridization of 3'-end A overhangs (added to PCR products by HotStarTaq DNA Polymerase) to U overhangs on each end of the linearized pDrive vector.

Aliquots of the ligation reaction were used to efficiently transform DH5-a and QIAGEN EZ Competent Cells (see Table "Proportion of recombinants containing c-myb gene PCR insert after transformation with pDrive Cloning Vector"). Restriction enzyme analysis and direct sequencing confirmed that all white colonies obtained after plating contained the PCR insert (data not shown).

Conclusions

• Of 5 polymerases tested in PCR, only HotStarTaq DNA Polymerase and Q-Solution enabled successful amplification of a "difficult" GC-rich template.
  
  
• The QIAGEN PCR Cloningplus Kit enabled quick and efficient subcloning of the amplified insert into the pDrive Cloning Vector.
  
  
• After transformation, a high proportion of transformants contained the desired insert. The short ligation incubation time and the ability to immediately plate transformants using the QIAGEN PCR Cloningplus Kit streamlines cloning and screening procedures.

References

1. Scortechini, A.R., et al. (1999) Expression of c-myb and B-myb oncogenes on myelofibrotic marrow fibroblasts. Leuk. Lymphoma, 33, 295.
   
2. Piccinini, G., et al. (1996) c-myb proto-oncogene is expressed by quiescent scleroderma fibroblasts and, unlike B-myb gene, does not correlate with proliferation. J. Invest. Dermatol. 106, 1281.
   
3. Arsura, M., Luchetti, M.M., Erba, E., Golay, J., Rambaldi, A., and Introna, M. (1994) Dissociation between p93B-myb and p75c-myb expression during the proliferation and differentiation of human myeloid cell lines. Blood 83, 1778.

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