Development of Tetra-Primer ARMS-PCR for Hemoglobin E Detection

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Development of Tetra-Primer ARMS-PCR for Hemoglobin E Detection Jatuphol Pholtaisong Biological Science Program Department of Biology, Faculty of Science, Naresuan University Advisor: Dr.Lt.Saisiri Mirasena Co-Advisors: Asst.Prof.Dr.Maliwan Nakkuntod and Dr.Phrutthinun Surit

Hemoglobin E Abnormal Hemoglobin -globin gene Codon 26 (GAG-AAG) Glu-Lys Alternative splicing => reduced mRNA Mildly unstable (sensitive to oxidants) Beta thalassemia/Hemoglobin E => Mild to Severe Lower Northern Part of Thailand (Rawangkran et al.,2013) - Homozygous HbE 5.05% - Heterozygous HbE 42.09%

Hemoglobin E Detections Screening Tests - Dichlorophenol indophenol (DCIP) - High-Performance Liquid Chromatography (HPLC) Confirmation Tests - Reverse Dot-Blot Hybridization - Amplification Refractory Mutation System–PCR (ARMS-PCR)

Amplification Refractory Mutation System–PCR (ARMS-PCR) Reaction 1 Normal Allele Detection Reaction 2 Mutant Allele Detection Forward Primer 5’ 3’ Forward Primer Forward Primer 5’ 3’ 5’ 3’ Reverse Primer (Normal) 3’ C 5’ Reverse Primer (Normal) Reverse Primer (Mutant) Reverse Primer (Mutant) 3’ C 5’ 3’ T 5’ 3’ T 5’

Amplification Refractory Mutation System–PCR (ARMS-PCR) Interpretation 1 (N) 2 (M) 1 (N) 2 (M) 1 (N) 2 (M) Normal Heterozygous HbE Homozygous HbE

Amplification Refractory Mutation System–PCR (ARMS-PCR) Interpretation 1 (N) 2 (M) 1 (N) 2 (M) 1 (N) 2 (M) Internal Control > Normal Heterozygous HbE Homozygous HbE

Tetra-primer ARMS-PCR Principle Ye et al. (2001)

Primer Mismatch at 3’-end 5’ G 3’ 3’C 5’ 5’ G 3’ 3’T 5’ Mismatch

Weak Primer Mismatch at 3’-end 5’ G 3’ 3’C 5’ 5’ G 3’ 3’T 5’ Weak Mismatch

Type of Primer Mismatch Strong Mismatch G/A C/T Medium Mismatch A/A C/C G/G T/T Weak Mismatch C/A G/T > Add secondary mismatch at position –2 from the 3’-terminus to increase specificity Ye et al. (2001)

Study Steps Primer Design PCR Optimization Compare with ARMS-PCR

Tetra-primer ARMS-PCR Principle Ye et al. (2001)

Materials and Methods: Primer Design PRIMER1: primer design for tetra-primer ARMS-PCR (Ye et al., 2001) OligoCalc (Kibbe, 2007) OligoAnalyzer 3.1 (https://sg.idtdna.com/calc/analyzer) GenBank (http://www.ncbi.nlm.nih.gov/genbank/) Primers Sequences TM (ºC) Position Product size HbE-OF CCC TTC CTA TGA CAT GAA CTT AAC CAT A 65.6 5226506-5226533 (NC_000011.10) 691 HbE-OR GGC TGT CAT CAC TTA GAC CTC AC 64.6 5227174-5227196 (NC_000011.10) HbE-IF(Normal) ACCAACCTGCCCAGGGCATC 5226921-5226943 (NC_000011.10) 276 HbE-IR(Mutant) GTGAACGTGGATGAAGTTGGTGTTA 64.1 5226943-5226967(NC_000011.10) 462

Study Steps Primer Design PCR Optimization Compare with ARMS-PCR

PCR Optimization: HbE Samples

PCR Optimization: HbE Samples

PCR Optimization: HbE Samples

PCR Optimization Primer Concentration Annealing Temperature 0.2 µM Each Primer 0.2 µM OF/OR Primer, 0.4 µM IF/IR Primer Annealing Temperature Normal Annealing Temperature (60ºC 35 cycles) Touchdown PCR - 70ºc (-1ºc/cycle) 10 cycles - 60ºc 25 cycles

Results: PCR Optimization 0.2 µM Each Primer Normal Annealing Temperature (60ºC 35 cycles) M: Marker 1: Normal 2: Heterozygous Hb E 3: Homozygous HbE 4: Negative

Results: PCR Optimization 0.2 µM OF/OR Primer, 0.4 µM IF/IR Primer 0.2 µM Each Primer Normal Annealing Temperature (60ºC 35 cycles) M: Marker 1: Normal 2: Heterozygous Hb E 3: Homozygous HbE 4: Negative

Results: PCR Optimization 0.2 µM OF/OR Primer, 0.4 µM IF/IR Primer 0.2 µM Each Primer Normal Annealing Temperature (60ºC 35 cycles) Touchdown PCR - 70ºc (-1ºc/cycle) 10 cycles - 60ºc 25 cycles M: Marker 1: Normal 2: Heterozygous Hb E 3: Homozygous HbE 4: Negative

Results: PCR Optimization 0.2 µM OF/OR Primer, 0.4 µM IF/IR Primer 0.2 µM Each Primer Normal Annealing Temperature (60ºC 35 cycles) Touchdown PCR - 70ºc (-1ºc/cycle) 10 cycles - 60ºc 25 cycles M: Marker 1: Normal 2: Heterozygous Hb E 3: Homozygous HbE 4: Negative

Results: PCR Optimization 0.2 µM OF/OR Primer, 0.4 µM IF/IR Primer 0.2 µM Each Primer Normal Annealing Temperature (60ºC 35 cycles) Touchdown PCR - 70ºc (-1ºc/cycle) 10 cycles - 60ºc 25 cycles M: Marker 1: Normal 2: Heterozygous Hb E 3: Homozygous HbE 4: Negative

Results: PCR Ingredients PCR Ingredients (total volume 20 µL) 1X PCR Buffer (NanoHelix, Korea) 0.2 mM Each DNTPs (NanoHelix, Korea) 1 Unit HelixAmpTM Ab+ Taq DNA polymerase (NanoHelix, Korea) 0.2 µM HbE-OF Primer, 0.2 µM HbE-OR Primer 0.3 µM HbE-IF Primer, 0.3 µM HbE-IR Primer DNA Template 3 µL

Results: PCR Cycles Pre-Denaturation 95ºc 2m Denaturation 97ºc 30s Annealing 70ºc (-1ºc/cycle) 30s Extension 72ºc 1m 25 cycles 60ºc 30s Final Extension 72ºc 5m

Methods: Step by Step Primer Design PCR Optimization Compare with ARMS-PCR

Results: Tetra Primer ARMS-PCR and ARMS-PCR Comparison Known Samples: Normal 20 Samples Heterozygous Hb E 16 Samples Homozygous Hb E 20 Samples E E EE E N N E EE EE E E -

Discussions and Conclusions Tetra-Primer ARMS-PCR for Hemoglobin E Detection was successfully developed. This technique required touchdown PCR and higher inner primer concentrations. When compared with standard technique in 48 known samples, the results were concordant. This technique is efficient, less time-consuming and safe cost for hemoglobin E gene detection.

References Rawangkran, A., Janwithee, N., Wong, P., & Jermnim, N. (2013). Prevalence of Thalassemia Trait from Screening Program in Pregnant Women in the Lower Northern Region of Thailand. Thai Journal of Genetics, S(1), 156-159. Ye, S., Dhillon, S., Ke, X., Collins, A. R., & Day, I. N. (2001). An efficient procedure for genotyping single nucleotide polymorphisms. Nucleic Acids Research, 29(17), E88-88. doi: 10.1093/nar/29.17.e88.

Acknowledgments

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