Amplification and Cloning of a Low-expression Antibody Heavy-chain Variable Region Gene (IGHV4-59) Using Megaprimer-mediated Domain Swap
PrimeSTAR GXL DNA Polymerase
Data kindly provided by: Xuchu Que, University of California, San Diego
Megaprimer-mediated domain swapping allows construction of clones using PCR to synthesize DNA fragments, then using those fragments as megaprimers for a second PCR. This second PCR may be designed to allow domain swapping and synthesis of entire plasmids. Therefore, megaprimer PCR is an alternative to conventional restriction enzyme digestion and ligation for construction of individual clones or a series of chimeric clones.
The objective of this experiment was to amplify a low-expression antibody heavy-chain variable region gene (IGHV4- 59) from human B cells and clone it into an expression vector using a domain-swap megaprimer PCR technique. This approach required a high-fidelity PCR polymerase to re-amplify a larger plasmid vector with insert cDNA.
In brief, total RNA from human PBMC cells was reverse transcribed to synthesize cDNA. This cDNA was used as template for amplification of the IGHV4-59 gene by PCR with PrimeSTAR GXL DNA Polymerase. To clone IGHV4-59 into a larger expression vector (approximately 9 kb), IGHV4-59 was re-amplified and purified for use as a megaprimer. IGHV4-59 was then mixed with the expression vector DNA for a domain swap quick-change PCR. This domain swap PCR was designed to insert the IGHV gene into the vector by PCR using PrimeSTAR GXL DNA Polymerase and two overlapping primers.
DNA sequencing verified that the IGHV4-59 gene was inserted successfully into the large plasmid vector using high fidelity PrimeSTAR GXL DNA Polymerase, but not by the conventional method of restriction digestion, cloning and ligation.
Figure 1. Amplification of a human IGHV gene using PrimeStar DNA polymerase (Al) and cloning into a TOPO vector for quality control sequencing (B).
Figure 2. DNA sequencing showed that the cloned insert amplified by PrimeSTAR GXL DNA Polymerase was a 100% match to the human germline V, D, J gene. No mutations were found.
Figure 3. DNA corresponding to the IGHV4-59 gene was amplified with PrimeSTAR DNA Polymerase and cloned into pLiv7 vector. Domain-swap PCR was used to amplify the large (approximately 9 kb) expression vector.
Figure 4. Sequencing chromatograms verified the joint region sequences of inserted gene cloned into the expression vector by domain-swap megaprimer PCR.
Under the conditions tested, PrimeSTAR GXL DNA polymerase could be used to amplify low-expression genes from cDNA products with high fidelity and sensitivity, and to amplify a large plasmid (approximately 10 kb) vector containing a domain-swapped insert. No nucleotide mutations were found, confirming the accuracy of the PCR amplification.
The reverse transcription reaction included 1 µg total RNA purified from human PBMC B cells. This total RNA was reverse transcribed to cDNA using RNA to cDNA EcoDry Premix.
PCR Amplification of IGHV
The composition of the 50 µl PCR performed to amplify IGHV gene was as follows:
Cycling conditions for touch-down PCR were: 95°C, 3 min (hot start); 5 cycles of 94°C 20 sec, 72°C 2 min; 30 cycles of 94°C 20 sec, 68°C 20 sec, 72°C 1 min; hold at 10°C.
After amplification, products were cloned into the plasmid and sequenced (see Figure 1 and Figure 2).
A domain-swap PCR was performed with vector DNA to insert IGHV4-59 gene into expression vector. The composition of the reaction to amplify the megaprimer was as follows:
The megaprimer amplification PCR cycling conditions were: 30 cycles of 95°C 20 sec, 70°C 20 sec, 72°C 20 sec. The DNA fragment was gel-purified.
The composition of the domain-swap PCR was as follows:
Conditions for touch-down PCR were: 95°C 3 min hot start; 5 cycles of 94°C 20 sec, 72°C 5 min; 25 cycles of 94°C 15 sec, 68°C 15 sec, 72°C 2 min; 72°C 15 min.
E. coli Transformation
Dpn I (1 µl) was used to digest the template, and 5 µl of the PCR product was used to transform XL10-Gold E. coli competent cells. Recombinant plasmids were purified and subjected to sequencing (see Figures 3 and 4).
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