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%
2
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)
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Amplification Refractory Mutation System–PCR
(ARMS-PCR)
Forward Primer
5’
3’
Reverse Primer (Normal)
3’ C
5’
Reverse Primer (Mutant)
3’ T
5’
Reaction 1
Reaction 2
Normal Allele Detection
Mutant Allele Detection
Forward Primer
5’
3’
Forward Primer
5’
3’
Reverse Primer (Normal)
3’ C
5’
Reverse Primer (Mutant)
3’ T
5’
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Amplification Refractory Mutation System–PCR
(ARMS-PCR) Interpretation
1 (N)
2 (M)
Normal
1 (N)
2 (M)
Heterozygous HbE
1 (N)
2 (M)
Homozygous HbE
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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
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Tetra-primer ARMS-PCR Principle
Ye et al. (2001)
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Primer Mismatch at 3’-end
5’
G
3’C
5’
G
3’T
Mismatch
5’
5’
3’
3’
8
Weak Primer Mismatch at 3’-end
5’
G
3’C
5’
G
3’T
Weak Mismatch
5’
5’
3’
3’
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Type of Primer Mismatch
Strong Mismatch
Medium Mismatch
Weak Mismatch
G/A C/T
A/A C/C G/G T/T
C/A G/T
> Add secondary mismatch at position –2 from the 3’-terminus to increase specificity
Ye et al. (2001)
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Study Steps
Primer
Design
PCR
Optimization
Compare with
ARMS-PCR
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Tetra-primer ARMS-PCR Principle
Ye et al. (2001)
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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)
64.6 5226921-5226943 (NC_000011.10)
276
ACCAACCTGCCCAGGGCATC
HbE-IR(Mutant)
GTGAACGTGGATGAAGTTGGTGTTA
64.1
5226943-5226967(NC_000011.10)
462
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Study Steps
Primer
Design
PCR
Optimization
Compare with
ARMS-PCR
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PCR Optimization: HbE Samples
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PCR Optimization: HbE Samples
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PCR Optimization: HbE Samples
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PCR Optimization
 Primer Concentration
 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
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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
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Results: PCR Optimization
0.2 µM Each Primer
0.2 µM OF/OR Primer,
0.4 µM IF/IR Primer
Normal Annealing
Temperature
(60ºC 35 cycles)
M: Marker
1: Normal
2: Heterozygous Hb E
3: Homozygous HbE
4: Negative
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Results: PCR Optimization
0.2 µM Each Primer
0.2 µM OF/OR Primer,
0.4 µM IF/IR 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
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Results: PCR Optimization
0.2 µM Each Primer
0.2 µM OF/OR Primer,
0.4 µM IF/IR 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
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Results: PCR Optimization
0.2 µM Each Primer
0.2 µM OF/OR Primer,
0.4 µM IF/IR 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
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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
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Results: PCR Cycles
Pre-Denaturation
Denaturation
Annealing
Extension
Denaturation
Annealing
Extension
Final Extension
95ºc 2m
97ºc 30s
70ºc (-1ºc/cycle) 30s
10 cycles
72ºc 1m
97ºc 30s
60ºc 30s
25 cycles
72ºc 1m
72ºc 5m
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Methods: Step by Step
Primer
Design
PCR
Optimization
Compare with
ARMS-PCR
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Results: Tetra Primer ARMS-PCR and ARMSPCR 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 -
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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.
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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.
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Acknowledgments
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T
G
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