Original Article

Yonago Acta Medica 2017;60:24–30

Optimization of PCR Condition: The First Study of High Resolution Melting
Technique for Screening of APOA1 Variance
Hesty Wahyuningsih,* Ferdy K Cayami,* Udin Bahrudin,*† Mochamad A Sobirin,†‡ Farmaditya EP Mundhofir,*
Sultana MH Faradz* and Ichiro Hisatome§*Center for Biomedical Research, Diponegoro University Faculty of Medicine, Semarang, Indonesia, †Department of Cardiology and
Vascular Medicine, Diponegoro University Faculty of Medicine, Semarang, Indonesia, ‡Department of Pharmacology and Therapeutic,
Diponegoro University Faculty of Medicine, Semarang, Indonesia, §Division of Regenerative Medicine and Therapeutics, Departmentof Genetic Medicine and Regenerative Therapeutics, Institute of Regenerative Medicine and Biofunction, Tottori University, Yonago
683-8503, Japan

ABSTRACTan estimated PCR product size, the data of this study
showed that appropriate annealing temperature and PCR
cycles were important factors in optimization of HRM
technique for variant screening in APOA1.

Background High resolution melting (HRM) is a post-

PCR technique for variant screening and genotyping
based on the different melting points of DNA fragments.
The advantages of this technique are that it is fast, simple, and efficient and has a high output, particularly forscreening of a large number of samples. APOA1 encodes
apolipoprotein A1 (apoA1) which is a major component of high density lipoprotein cholesterol (HDL-C).
This study aimed to obtain an optimal quantitative
polymerase chain reaction (qPCR)-HRM condition forscreening of APOA1 variance.
Methods Genomic DNA was isolated from a peripheral blood sample using the salting out method. APOA1
was amplified using the RotorGeneQ 5Plex HRM. The
PCR product was visualized with the HRM amplification curve and confirmed using gel electrophoresis. The
melting profile was confirmed by looking at the melting
curve.
Results Five sets of primers covering the translated region of APOA1 exons were designed with expected PCR
product size of 100–400 bps. The amplified segments of
DNA were amplicons 2, 3, 4A, 4B, and 4C. Amplicons
2, 3 and 4B were optimized at an annealing temperatureof 60 °C at 40 PCR cycles. Amplicon 4A was optimizedat an annealing temperature of 62 °C at 45 PCR cycles.
Amplicon 4C was optimized at an annealing temperature of 63 °C at 50 PCR cycles.
Conclusion In addition to the suitable procedures of
DNA isolation and quantification, primer design and

Key words APOA1; HRM; optimization

High resolution melting (HRM) is a post-PCR technique for variant screening and genotyping based on
different melting points of DNA fragments.1 The mainprinciple of HRM is the integration of intercalating dye
to double stranded DNA (dsDNA) and disintegrationof the fluorescence dye when the dsDNA are separated
to single stranded DNA (ssDNA). By using a real timemeasurement of the fluorescence level, a melting curveof fluorescence level against time is formed. The melting profile is mainly influenced by the length of DNA
fragment, the GC content and heterozygosity.1–3 Thus,
the advantages of this technique are that it is fast, simple, efficient and high throughput result, particularly forscreening of a large number of samples.1, 3, 4
APOA1 is located in chromosome 11q23 and encodes apolipoprotein A1 (apoA1). APOA1 spans about
2.2 kb and contains 4 exons.6 ApoA1 composes up to
70% of HDL-C.7 In the structure of HDL, apoA1 is agroup of proteins arranged in an anti-parallel double-belt
structure.8 It is a cofactor for lecithin cholesterolacyltransferase (LCAT), substrate for scavenger receptor
class B member 1 (SR-B1).9 Deletions in the APOA1
result in a decrease of HDL-C level to less than 40 mg/
dL.10 The monogenic disorder caused by APOA1 mutation comprises about less than 5% of low HDL-C levelcases,11 but it accelerates the atherosclerosis process incontrast to other mutations in genes which encode themain protein of reverse cholesterol transport (RCT).12
This study aimed to obtain an optimal quantitative
polymerase chain reaction (qPCR)-HRM condition forscreening of APOA1 variance. So far, it is the first study
which screens variance in APOA1 using HRM technique; therefore optimization of the technique is very

Corresponding author: Udin Bahrudin, MD, PhD
bahrudin00@gmail.com
Received 2016 November 21
Accepted 2016 December 28
Abbreviations: APOA1, apolipoprotein class A member 1 gene;
apoA1, apolipoprotein class A member 1 protein; dsDNA, double
stranded DNA; HDL-C, high density lipoprotein cholesterol; HRM,
high resolution melting; LCAT, lecithin cholesterolacyl transferase;
qPCR, quantitative polymerase chain reaction; RCT, reverse cholesterol transport; SR-B1, scavenger receptor class B member1; ssDNA,
single stranded DNA

24

HRM technique for APOA1 variance screening

important in order to get a reliable result.13 In addition toa suitable procedure for DNA isolation and quantification, primer design and an estimated PCR product size,
this study found that appropriate annealing temperatureand PCR cycles were important factors in optimizationof HRM technique for variant screening in APOA1.

primers were designed to cover all exons from 2 to 4 andtheir flanking regions.
DNA isolation and quantification

The genomic DNA was isolated using the salting out
method. Then, the DNA concentration was quantified
using the Nanovue Plus (GE Healthcare Bio Sciences
AB, Buckinghamshire, UK). The DNA samples werediluted using distilled water to a concentration of 10 ng/
μL. The genomic DNA from normal healthy donors wasused in the optimization of qPCR-HRM. The criteria of
normal healthy donors were that their HDL-C level > 40
mg/dL, cholesterol total level < 200 mg/dL, LDL-C level< 160 mg/dL, triglyceride level < 150 mg/dL, and their
DNA sequence were similar to the reference sequence in
the NCBI database. Informed consent for participation inthis study was obtained. The investigation was approvedby the Institutional Review Board of the Diponegoro
University Faculty of Medicine and Dr. Kariadi Hospital(number 190/EC/FK/RSDK/2013) and conformed to theprinciples outlined in the Declaration of Helsinki.15

MATERIALS AND METHODS
Primer design

The first step in this study was designing primers for
APOA1. The referent sequences were taken from thedatabase of UCSC Genome Bioinformatics Human Genome Browser Gateway (http://genome.ucsc.edu/cgi-bin/hgGateway?redirect=manual&source=genome.ucsc.edu)
and Ensembl (www.ensembl.org) with the accession
numbers were GRCh38/hg3814 and ENSG00000118137,6
respectively. The primers were designed with PCR
product sizes 100–400 bps using the Lightcycler Primer
Design Software V.1.0.R.84 (Idaho Tech, Salt Lake City,
UT). The concentration of primers both forward and reverse primers was 10 pmol/μL.
Based on the gene sequence database, the primers
were designed from the start codon at exon 2. Exon 1 isan untranslated region, and thus it was not included in
the analysis. The primers for amplicon 2 and amplicon
3 have a PCR product size of less than 250 bps as an
ideal. They were located in intron with the purpose ofcovering the entire exon area. Exon 4 was divided into 3
amplicons, i.e. 4A, 4B and 4C in order to get optimum
HRM results. The details are listed in Table 1. The

HRM technique and its data analysis

APOA1 was amplified using qPCR RotorGeneQ 5Plex
HRM (Qiagen, Valencia, CA) with total volume 10 µL
in each tube. Each tube consisted of 5 μL Type-It HRM
Master Mix (Qiagen, Cat. No. 206542), 1 μL of forward
and reverse primers each with a concentration of 10
pmol/μL, 2 μL of DNA with a concentration of 10 ng/
μL and 2 μL of H2O. The curve analysis was done with

Table 1. List of primers, nucleotide positions, PCR products size and their GC content
Exon

Amplicon Primer Sequences

Nucleotide Positions

F:
GRCh38/hg38: chr11:
GCCAGGCCCTTCTTCTC
116836271-116836287
R:
GRCh38/hg38: chr11:
GGTTGGCTCCCTAGGTT
116836370-116836386
F:
GRCh38/hg38:
chr11:
33
GGCCTGATCTGGGTCTC
116836512-116836528
R:
GRCh38/hg38: chr11:
TCATCCCACAGGCCTCT
116836741-116836757
F:
GRCh38/hg38:
chr11:44A
CCAGCCCTCAACCCTTC
116837258-116837274
R:
GRCh38/hg38: chr11:
CCCTCTTGGAGCTCTGC
116837531-116837547
F:
GRCh38/hg38:
chr11:
44B
GCAGGAGGAGATGGAGCTCTA
116837485-116837505
R:
GRCh38/hg38: chr11:
CCTTGGCGGAGGTCCTC
116837795-116837811
F:
GRCh38/hg38: chr11:
44C
TCAGCGAGAAGGCCAAG
116837769-116837785
R:
GRCh38/hg38: chr11:
AGGAAGTCCCTGCTCCA
116838025-116838041
bp, base pair; F, forward primer; R, reverse primer; GC, guanine and cytosine.
2

2

25

Primer GC Productcontent (%) size (bp)Amplicon GC
content (%)

64.7

116

59.0

246

58.6

290

60.3

327

69.7

273

63.4

58.8
64.7
58.8
64.7
58.8
57.1
64.7
58.8
58.8

H. Wahyuningsih et al.

Rotor Gene–Pure Detection version 2.1.0 (build 9) Software (Qiagen). The PCR product was visualized withthe amplification curve and confirmed with1.5% agarose
gel for 30 minutes. The melting profile was analyzed
through melting curve.

the idea that appropriate annealing temperature and
PCR cycles were also important factors in optimizationof HRM technique for variant screening in APOA1.
The primers covering exons 2, 3 and 4 were designed using a well-established software, the Lightcycler
Primer Design Software V.1.0.R.84 (Idaho Tech). The
maximum PCR product size was 327 bps in exon 4B
which was qualified for maximum PCR product sizeto achieve 100% sensitivity and 100% specificity of the
HRM technique.1, 13 The primers of amplicons 2 and 3
were ideal primers with PCR product sizes of less than
250 bps,13 while the size of amplicons 4A and 4C were
290 bps and 273 bps, respectively. These primer designs
and the estimated PCR product size were clearly important in the optimization of PCR-HRM technique forvariant screening in APOA1. In addition to target length,
primer sequences should be carefully checked for known
polymorphisms. Failed identification of any SNP in theprimer sequence will reduce the success rate of HRM
analysis since the HRM melting profile is influenced by
DNA heterozygosity.1–3
Initially, the experiment used annealing temperature
60 °C and 40 PCR cycles and it showed that amplicons
2, 3, 4B and 4C have crossing points of less than 30 (Fig.
1A). It means that the sample was adequate and the amplification was efficient.13 On the other hand, amplicon
4A had a crossing point more than 30 cycles, meaningthat the sample may not be adequate and the amplification may not efficient.13 Amplicons 4A and 4C had
double bands on agarose gel. These could be caused byunspecific PCR products, and thus required an increasein annealing temperature to obtain optimal qPCR-HRM
conditions. Other possible causes for results of amplicon
4A which had a crossing point more than 30, double
peak melting curve and double band on agarose gel wereless efficient DNA amplification, a low annealing temperature leading to unspecific PCR products, and a CG
rich content (60.3%) in the DNA sequence.13
As shown in Fig. 2, an annealing temperature of 62°C and 40 PCR cycles also failed to show an optimal
condition for amplicon 4C. Thus, further increases ofannealing temperature and PCR cycle, i.e. 63 °C and
50 cycles were performed to get a single peak of HRM
curve and single band on agarose gel (Fig. 4).
Although a single peak of HRM curve could beobtained for amplicon 4A by increasing the annealing
temperature from 60 to 62 °C, the amplification curve
did not reach a plateau pattern (Fig. 2) suggesting thatthe optimum temperature was obtained but an additional
PCR cycle was needed. Although a PCR with a temperature of 62 °C and 45 cycles produced better results,a thin smear remained at the upper the estimated PCR

RESULTS
Optimization of APOA1 was done in exon 2 to 4 whichwere divided into 5 amplicons. They were amplified
by using 5 pairs of primers. Because of its length, exon
4 was divided into 3 amplicons, i.e. 4A, 4B and 4C.
Exon 1 were excluded. At 60 °C annealing temperatureand 40 PCR cycles, the HRM result of amplicon 2, 3,
4B and 4C showed a crossing point less than cycle 30thand single peak of melting curve (Fig. 1A). Clear bands
matched with the estimated PCR product sizes wereseen in the agarose gel. However, double peaks of melting curve (Fig. 1B) and double bands on the agarose gel(Fig. 1C) were seen for amplicon 4A and 4C.
In order to get better HRM curves which represent
correct product for amplicon 4A and 4C, we modified
the annealing temperature and number of PCR cycle.
For amplicon 4A, we tried to increase the temperature
to 62 °C and 40 cycles. Although the PCR result showed
one correct band in gel electrophoresis, the relative thinband and the amplification curve pattern (Fig. 2) indicated that PCR could be further optimized. PCR withtemperature 62 °C and 45 cycles produced better results,
i.e. the amplification curve increased after the 30th cycle(Fig. 3A), a single peak of melting curve (Fig. 3B), and a
thick band matched with the estimated PCR product size
was seen in the agarose gel (Fig. 3C). However, a smear
was seen at the upper end of the estimated PCR productsize. Further increasing the temperature to 63 °C and 50
cycles resulted in no amplification in the raw material ofqPCR-HRM (data not shown).
Optimization of amplicon 4C was done with higher
annealing temperature and PCR cycle than that in Fig. 2,
i.e. 63 °C and 50 times, respectively. This condition resulted in an increase of amplification curve after the 30th
cycle (Fig. 4A), a single peak of melting curve (Fig. 4B),
and a clear single band matched with the estimated PCR
product size in the agarose gel (Fig. 4C).
The summary of optimum qPCR-HRM condition of
amplicons 2, 3, 4A, 4B and 4C are listed in Table 2.
DISCUSSION

It has long been known that the important factors in
the optimization of HRM technique for genetic variant
screening are a suitable procedure of the DNA isolationand quantification, primer design and an estimated PCR
product size,13 and data from this study clearly supports
26

HRM technique for APOA1 variance screening

A
	
  

C

70

12
10

4B

65

55

500 bp300 bp200 bp100 bp

2

50

8

3

45

Fluorescence

Fluorescence

60

40

6

4C

35
30

4

M

25
20

2

15

4A

10
5

00

	
  B

5

10

5

15

10

20

15

25

20
Cycle

30

25

Cycle

3.0

35

30

40

35

40

3.00
2.75

4B

3

2.50

2.0

2.00

dF/dT

dT/dF

2.25

1.75

4C

2

1.50

4A

1.25

1.0

1.00
0.75
0.50
0.25

0

A
	
  

0.00

65

66

67

68

69

70

70

71

72

73

74

75

76

77

78

79

80
ºC

81

82

83

84

85

86

87

75
80
85
o
Temperature ( C)

88

89

90

91

92

90

93

94

95

95

H

2

3

4A 4B 4C

Fig. 1. The result of optimization of APOA1 using
annealing temperature 60 °C and 40 times of PCR
cycles.
(A) qPCR amplification curve of amplicons 2, 3,
4B and 4C showed crossing point less than 30,
while amplicon 4A showed that of more than 30.
(B) HRM curve of amplicons 2, 3 and 4B had single peak, while amplicon 4A and 4C had double
peak of melting. (C) Amplicons 2, 3 and 4B had
clear single band on the agarose gel matched withthe estimated PCR product size, but amplicons 4A
and 4C had nonspecific band; the temperature ofqPCR was set for starting at 65 °C. bp, base pair;dT/dF, delta temperature per delta fluorescence; H,
H2O; HRM, high resolution melting; M, molecularweight marker; qPCR, quantitative polymerase
chain reaction; 2, 3, 4A, 4B, and 4C are names ofamplicons.

C

8

5.0

6

4.0

4C

3.5

4

Fluorescence

Fluorescence

4.5

3.0

H2O

2.5

2

500 bp300 bp200 bp100 bp

4A

2.0

M

1.5

0

	
  B

5

0

10

5

15

10

20

15

25

20
Cycle

30

25

Cycle

35

30

35

4C

40

1.8

4C

1.6

1.4

2.0

1.2

dF/dT

dT/dF

4A

40

2.0

3.0

1.0

0.8

H2O

1.0

0.6

4A

0.4

0

H

1.0

0.2

0.0

65

66

67

68

69

70
70

71

72

73

74

75
80
85
o
Temperature ( C)
75

76

77

78

79

80
ºC

81

82

83

84

85

86

87

88

89

90

90
91

27

92

93

94

95
95

Fig. 2. The result of optimization of amplicon 4A
and 4C APOA1 using annealing temperature 62 °C
and 40 times of PCR cycles.
(A) qPCR amplification curve of amplicons 4A and4C showed crossing point more than 30. (B) HRM
curve of amplicons 4A and 4C had single peak ofmelting. (C) Amplicon 4A had clear single band on
the agarose gel matched with the estimated PCR
product size, but amplicon 4C had a nonspecificband. bp, base pair; dT/dF, delta temperature perdelta fluorescence; H, H2O; HRM, high resolution
melting; M, molecular weight marker; qPCR,
quantitative polymerase chain reaction; 4A and 4C
are names of amplicons.

H. Wahyuningsih et al.

Fig. 3. The result of optimization of amplicon 4A
APOA1 using annealing temperature 62 °C and 45
times of PCR cycles.
(A) qPCR amplification curve of the amplicons
showed that amplification was started at the 30th
cycle. (B) Its HRM curve had single peak of melting. (C) The amplicon had a clear single band on
the agarose gel matched with the estimated PCR
product size, with very thin smear at the upper ofthe correct band. bp, base pair; dT/dF, delta temperature per delta fluorescence; H, H2O; HRM, highresolution melting; M, molecular weight marker;
qPCR, quantitative polymerase chain reaction; 4A isa name of an amplicon.

Fig. 4. The result of optimization of amplicon 4C
APOA1 using annealing temperature 63 °C and 50
times of PCR cycles.
(A) qPCR amplification curve of the amplicon 4C
showed crossing point more than 30. (B) Its HRM
curve had single peak of melting. (C) The amplicon
had a clear single band on the agarose gel matchedwith the estimated PCR product size. bp, base pair;dT/dF, delta temperature per delta fluorescence; H,
H2O; HRM, high resolution melting; M, molecularweight marker; qPCR, quantitative polymerase
chain reaction; 4A is a name of an amplicon.

28

HRM technique for APOA1 variance screening
Table 2. Profile of qPCR-HRM of APOA1
Amplicon

Program2, 3 and 4B

Initial denaturation
Denaturation
Annealing
Elongation
HRM

Temperature (°C)

95
95
60
72
65–95 oC, 0.1 oC temperature increment
Wait for 2 s for each steps afterwards
HRM final step
40
4A
Initial denaturation
95
Denaturation
95
Annealing
62
Elongation
72
HRM
65–95 oC, 0.1 oC temperature increment
Wait for 2 s for each steps afterwards
HRM final step
40
4C
Initial denaturation
95
Denaturation
95
Annealing
63
Elongation
72
HRM
65–95 oC, 0.1 oC temperature increment
Wait for 2 s for each steps afterwards
HRM final step
40
HRM, high resolution melting; qPCR, quantitative polymerase chain reaction.

Time

Cycle

5 min
10 s
10 s
10 s

1
40
1

1 min
5 min
10 s
20 s
30 s

1
1
45
1

1 min
5 min
10 s
10 s
10 s

1
1
50
1

1 min

1

REFERENCES

product size (Fig. 3), and further increasing of temperature to 63 °C and 50 cycles resulted in no amplification
detected in the raw material of qPCR-HRM. Thus, atthis point, considering that the amplification curve after
the 30th cycle and a single peak of the melting curve
could be achieved by using an annealing temperature of
62 °C and 45 cycles, this method could be accepted.
Our results indicated that there are primers whichhave similar PCR conditions for HRM, and thus facilitate multiple primers for a qPCR-HRM running. In
contrast, some primers may need different conditions toachieve optimum conditions as in APOA1. To evaluate
whether methods of APOA1 variant screening showedin this study are appropriate for detection of mutant
APOA1, further qPCR-HRM analysis using either genomic DNA or plasmid DNA harboring APOA1 mutation is warranted.
In conclusion, in addition to a suitable procedureof DNA isolation and quantification, primer design andan estimated PCR product size, the data in this study
showed that appropriate annealing temperature and PCR
cycle were important factors in optimization of HRM
technique for variant screening in APOA1.

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