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Revision as of 23:42, 10 December 2018
Dry Lab | PCR Standardization
Have a look!Based on former studies, only the exons 2 and 3 from each chain codify for the extracellular domain of the HLA-DQ that interacts with the reactive 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 PCR steps (in a total of 10 reactions) to obtain the α- and β-chains flanked with restriction sites for further cloning.
Therefore, we designed 8 specific primers (forward and reverse) to amplify the exons 2 and 3 of each chain. The nomenclature is as follows: P1 to P4 for the α-chain and P1’ to P4’ for the β-chain.
| Primer | Chain | Exon | Genomic Location* | Length (bp)** |
|---|---|---|---|---|
| P1 | A | 2 (5’->3’) | 4643-4664 bp | 22 |
| P2 | A | 2 (3’->5’) | 4867-4889 bp | 23 |
| P3 | A | 3 (5’->3’) | 5302-5326 bp | 25 |
| P4 | A | 3 (3’->5’) | 5561-5583 bp | 23 |
| P1' | B | 2 (5’->3’) | 2072-2097 bp | 26 |
| P2' | B | 2 (3’->5’) | 2436-2456 bp | 21 |
| P3' | B | 3 (5’->3’) | 5199-5226 bp | 28 |
| P4' | B | 3 (3’->5’) | 5504-5527 bp | 24 |
* All genomic locations are referred to the HLA DQA1*05:01:01:01 and DQB1*02:01:01, chain A and B respectively.
** Complementary primer overhangs are not taken into account for the analysis.
Since we started the project focusing on expressing HLA-DQ from Roger’s DNA sample, primers were designed accordingly to his HLA-DQ haplotype, which is DQ2 (HLA DQA1*05:01:01:01 and DQB1*02:01:01). Remember that, for celiac disease, the haplotype nomenclature is as follows:
DQ2-positive
(HLA-DQA1*05:01 or *05:05 and HLA-DQB1*02:01 or *02:02)
Half DQ2-positive
(HLA-DQA1*05:01 or 05:05 or HLA-DQB1*02:01 or 02:02)
DQ8-positive
(HLA-DQA1*03 and HLA-DQB1*03:02)
In order to elucidate if the designed primers are capable of amplifying every CD-associated genotype, a multiple sequence alignment (MSA) was performed to evaluate genetic variability at primer regions.
Genomic HLA-DQ sequences (DQA1_gen.fasta and DQB1_gen.fasta) were downloaded from IPD-IMGT/HLA (Directory hosted at the European Bioinformatics Institute) version 3.33.0. Then, the genomic sequences were curated for DQ2 and DQ8 haplotypes.
| Download Sequences | DQ2 | DQ8 | |
|---|---|---|---|
| DQA1 | 73 | 14 | 7 |
| DQB1 | 198 | 6 | 13 |
Genomic MSA surprisingly resulted on a perfect alignment for sequences sharing CD-DQ haplotypes. This means that, for primer regions, all DQ2-associated sequences match perfectly and the same happens on DQ8. Moreover, for P2, P3, P1’ and P4’, primer region alignment is perfect and so, there is no need to edit the standard primers.
* Hidden Markov Model of P1 genomic primer region curated for DQ2 and DQ8 genotypes. In this case, there are only 3 differing bases among primer region. Since 14 DQ2 and 6 DQ8 sequences were aligned, dominant residues are those corresponding to DQ2 genotypes. DQ2 sequences presents C-C-T and DQ8 presents T-T-C.
| Primer | Exon | Total seq. | DQ2 | DQ8 |
|---|---|---|---|---|
| P1 | 2 (5’->3’) | 21 | GCTGACCACGTCGCCTCTTATG | GCTGACCATGTTGCCTCTTACG |
| P2 | 2 (3’->5’) | 21 | CATTGGTAGCAGCGGTAGAGTTG | CATTGGTAGCAGCGGTAGAGTTG |
| P3 | 3 (5’->3’) | 21 | AGGTTCCTGAGGTCACAGTGTTTTC | AGGTTCCTGAGGTCACAGTGTTTTC |
| P4 | 3 (3’->5’) | 21 | CCCAGTGTTTCAGAAGAGGCTTG | CCCAGTGTTTCAGAAGAGGCTCA |
| P1' | 2 (5’->3’) | 19 | GAGGATTTCGTGTACCAGTTTAAGGG | GAGGATTTCGTGTACCAGTTTAAGGG |
| P2' | 2 (3’->5’) | 19 | TCCTCTGGGGTGGAACAAACG | TCAGCCGGGGTGGAACGAACA |
| P3' | 3 (5’->3’) | 19 | CCTATATCTTTCCCTGTCTGTTACTGCC | CC--TATCTTTCCCTGTCTGTTACTGCC |
| P4' | 3 (3’->5’) | 19 | CAATATCCCCTTACGCCACTCCAC | CAATATCCCCTTACGCCACTCCAC |
For P3’ primer, a two-base deletion is reported as refSNP Cluster Report: rs756895762. Therfore, this is the only primer that needs special attention, as it could compromise the capability to amplify the HLA-DQ genes by PCR of all possible celiac disease involved HLA-DQ genotypes.
This are the Hidden Markov Models that represent the sequence conservation for the region complementary to each primer. For the α chain, the dominant bases correspond to DQ2, while for the β chain, the dominant bases correspond to DQ8.