Team:BioIQS-Barcelona/Basic Part
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Wet Lab | Personalization (PCRs)
Have a look!The strategy
One of the objectives of our project is to obtain the HLA-DQ protein from scratch. Based on former studies, only the exons 2 and 3 form each chain (α and β) codify for the extracellular domain of the HLA-DQ that interacts with gluten epitopes.
With this in mind, we designed a robust model for the extraction of the α and β chains of the HLA-DQ from the genomic DNA of a celiac patient. A set of primers were designed to conduct 3 different PCRS (including 10 reactions) to obtain the α and β chains flanked with restriction sites for further cloning.
PCR 1
In the PCR1, amplification of the exons 2 and 3 is carried out by using primers P1-P2/P1’-P2’ and P3-P4/P3’-P4’ respectively in 4 separated reactions, called PCR_E2A/PCR_E2B and PCR_E3A/PCR_E3B. The product size correspond to the ones expected, 253bp and 281bp for the exons that codify for the α chain, and 391-328bp for the exons that codify for the β chain.
PCR 2
The products of the beforementioned reactions are to be the template of the following PCR2. In this step, the restriction sites NdeI at 5' (in the case of exon 2) and SalI at 3' (for exon 3) are introduced for further cloning in the vector pET22b(+). Besides, a complementary region between exon 2 (3' end) and 3 (5' end) for further assembling is also introduced. To accomplish this, 4 separated reactions called PCR_HE2A/PCR_HE2B and PCR_HE3A/PCR_HE3B are carried out using primers P1*-P2*/P1’-P2’ and P3*-P4*/P3’-P4’ respectively. The resulting products with the restriction sites and overhangs should be of 264 and 292 bp for the α chain and 289 and 298 bp for the β chain.
PCR 3
Finally, in the PCR3, assembling of the exons 2 and 3 is done in one single reaction (Final PCRα/Final PCRβ), using a mix of the products from PCR_HE2A and PCR_HE3A (to obtain the α chain) and PCR_HE2B and PCR_H3B (to obtain the β chain) and the flanking primers P1*-P4*/ P1’-P4’. The expected product size are 533 bp (for the α chain) and 566 bp for the β chain).
UPF CRG Barcelona Results
The UPF iGEM conducted 3 PCRs for the obtention of the β chain of the HLA-DQ. They used a different DNA sample for demonstrating that the designed protocol was robust. We provided them with all the reagents necessaries to do it, including primers and the iPRoof HF. The primers designed efficiently hybridized with the exons 2 and 3 of the β chain.
The resulting product flanked with the restriction sites is ready to be cloned in a pET22b vector!
Congratulations UPF CRG Barcelona!
Note that P1,P2,P3,P4,P1*,P2*,P3* and P4* refer to the primers used to obtain the α chain, whereas P1',P2',P3’,P4’,P1’,P2’,P3’* and P4’* were used to obtain the β chain.