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                         <h1 class="mb-5">Dry Lab | PCR Standardization</h1>
 
                         <h1 class="mb-5">Dry Lab | PCR Standardization</h1>
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Revision as of 21:40, 7 December 2018

<!DOCTYPE html> BIO IQS

Dry Lab | PCR Standardization

Have a look!

First steps

The first step to develop our sensor is to amplify, express and effectively obtain the HLA-DQ heterodimer (chains α and β) from the patient's DNA, regardless of the CD-haplotype that she/he could have (either DQ2 or DQ8).

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 PCRS (including 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) has been 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 and so this is the only primer that needs special attention to.

Here are presented the Hidden Markov Models for all primers. For α chain dominant bases corresponds to DQ2 and for β corresponds to DQ8.