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Latest revision as of 20:47, 17 December 2018
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 from each chain (α and β) codify for the extracellular domain of the HLA-DQ that interacts with gluten and non-gluten reactive epitopes.
With this in mind, we designed a robust model to extract the α and β chains of the HLA-DQ from a celiac patient's genomic DNA. A set of primers were designed to conduct 3 different PCRs (that included 10 reactions) to finally 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 expected ones, 252bp and 281bp for the exons that codify the α chain, and 391bp and 328bp for the exons that codify 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 exons 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). For more detailed information see the gene extraction notebook.
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 to prove that the designed protocol was robust. We provided them with all the reagents necessaries to perform it, including primers and the DNA Polymerase (iProof HF). Our designed primers efficiently amplified 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.