1. 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

2. 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%

3. 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)

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

6. 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

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

8. Primer Mismatch at 3’-end5’ G 3’
3’C ’ 5’ G 3’
3’T ’
Mismatch

9. Weak Primer Mismatch at 3’-end5’ G 3’
3’C ’ 5’ G 3’
3’T ’
Weak Mismatch

10. 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)

11. Study Steps
Primer Design
PCR Optimization
Compare with ARMS-PCR

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

13. Materials and Methods: Primer Design
PRIMER1: primer design for tetra-primer ARMS-PCR (Ye et al., 2001)
OligoCalc (Kibbe, 2007)
OligoAnalyzer 3.1 (
GenBank (
Primers
Sequences
TM (ºC)
Position
Product size
HbE-OF
CCC TTC CTA TGA CAT GAA CTT AAC CAT A
65.6
(NC_ )
691
HbE-OR
GGC TGT CAT CAC TTA GAC CTC AC
64.6
(NC_ )
HbE-IF(Normal)
ACCAACCTGCCCAGGGCATC
(NC_ )
276
HbE-IR(Mutant)
GTGAACGTGGATGAAGTTGGTGTTA
64.1
(NC_ )
462

14. Study Steps
Primer Design
PCR Optimization
Compare with ARMS-PCR

15. PCR Optimization: HbE Samples

16. PCR Optimization: HbE Samples

17. PCR Optimization: HbE Samples

18. 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

19. 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

20. 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

21. 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

22. 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

23. 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

24. 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

25. 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

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

27. 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 -

28. 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.

29. 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), 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), E doi: /nar/29.17.e88.

30. Acknowledgments

31. T G