III.

RESEARCH METHOD

III.1. Research aspects

III.1.1 Research field

This research project is in the field of human genetics particularly molecular genetics and

ophthalmogenetics.

III.1.2 Research location

Patient selection was based on medical records and information from families member

and these patients mostly from java island. DNA extraction was performed at Molecular

and Cytogenetic Laboratory of Center for Biomedical Research, Faculty of Medicine

Diponegoro University Semarang Indonesia and genetic detection was done at blindness

group Antropogenetica Department St. Radboud University Nijmegen, Netherlands.

III.1.3. Research period

The research was conducted within 14 months, from October 2008 to December 2009

III.1.4. Research design

Design of this study was cross sectional design.

III.2 Material

III.2.1 Population

Target population: RP individuals with Indonesian citizenship.

Reachable population: RP individuals from Dr. Kariadi, William Booth, and Panti

Wilasa Hospital and referal from ophthalmologist.

III.2.2 Specimens

Specimens were taken from all non syndromic RP patients and their family members by

using consecutive random sampling from Dr. Kariadi Hospital, William Booth Hospital
 and Panti Wilasa Citarum Hospital and also referral from other island. It was included the

arRP, adRP and X-linked RP. Informed consent was obtained from all samples in

accordance with the tenets of the Declaration of Helsinki (Edinburgh, 2000;

www.wma.net).

III.2.2.1 Inclusion criteria.

All of non-syndromic RP patients in every ages or sex, diagnosed clinically by

ophthalmology examination and also their families whose agree to join the study.

III.2.2.2 Exclusion criteria

Unilateral diseases, nystagmus/wandering eye, exudative retinal detachment, retinal

vasculitis, non cooperative pupils, and non RP disease were excluded.

III.2.3 Data collection.

Clinical examination

The non-syndromic RP patients have been examined to check the visual acuity

with Snellen chart and funduscopy by ophthalmologist and subsequently performed

history taking and made the pedigree.

After clinical diagnosis of RP were established, 5 mL EDTA blood (for DNA

analysis) were drawn from each subjects and then by salting out procedure for extracting

the DNA, the DNA were put on the freezer.

III.3. Operation definitions:

Results /
No Variable Definition Scale
measurements
1 RP is a group of inherited retinal Yes / No Nominal
(dependent degeneration disorder
variable)
characterized by night blindness,
progressive loss of peripheral
vision and characteristic
pigmentary retinopathy (bone
spicules), attenuated arterioles,
waxy pallor of optic disc which
diagnosed based on funduscopy
2 Mutated
gene Yes / No Nominal
is a change in the DNA of a gene
(independent
variables)
I N H E R I T E D
3 Homozygote is identical pairs of genes or
alleles for a specific trait Yes / No Nominal
4 Heterozygote The genes for a specific trait are Yes / No Nominal
different (different alleles at a
particular gene locus on
homologous chromosome)
5 X-linked Single gene disorders that reflect Yes / No Nominal
diseases the presence of defective genes
on the X chromosome.

6 Dominant One mutated copy of the gene in Yes/ No Nominal

inheritance each cell is sufficient for a person
to be affected
7 Recessive Two mutated copies of the gene Yes / No Nominal
inheritance are present in an affected person
is a method for mapping the
human genome, used to detect
genes that cause disease only
Homozygosity Yes/No Nominal
8 when both copies in an individual
mapping are mutated (i.e. the genes
are homozygous, or the same)

III.4. Research Scheme

 History taking, Eye examination and
Confirmed as RP, Pedigree Analysis
of RP Patients

Informed Consent

Blood sampling
/DNA Sampling

Measuring DNA concentration

Autosomal Recessive Autosomal Dominant

/ isolated

Consanguineous family Non consanguineous family

Affymetrix 5.0 (SNP Array) Illumina 6K SNP (SNP Array)

((SNP array
Homozygous region

Linkage analysis (CA Markers)

Candidate gene
Confirmation Test
DNA sequencing (Using Restriction
Enzym on conrol
Mutation
samples)

III.5. Data Collection

 1. Primary Data:

Personal identity of each RP patients including: date of birth, age of onset, clinical data

and pedigree.

The data from sixteen families with thirty six affected individuals and twenty nine

unaffected persons were joined with this study. All DNA from Affected persons was

screened using SNP array to find the homozygous region.

2. Secondary Data:

Medical records from hospital, information from the other member of the families

III.6. Methods

III.6.1 Procedure

Patients and families members who met the selection criteria were asked to participate

the research project by signing written informed consent forms and also their family

members.

Clinical features and pedigree of patients were reviewed and confirmed by

ophthalmologist and geneticist. Blood samples were collected from the affected and other

available family members by vein puncture. The DNA was extracted from the blood by

the salting out method.

III.6.2 DNA concentration and Gel Electrophoresis

Before measuring the concentration make sure that the DNA quality is quite good

by running the DNA by agarose gel electrophoresis. The concentration of agarose 0.8 %
 and put 5 µl loading buffer and 2 µl DNA (70ng/µl) and set the electrophoresis on 50V

for at least 2 hours After Checking the quality of the DNA then all the DNA was

measured the concentration using the nano-drop and made a working solution for sending

the DNA to SNP arrays (70 ng/mikroliter) and also working solution for PCR (20

ng/mikroliter). Labeling and give the number for all DNA samples and put on the -200

fridge.

III.6.3 Primers and Condition for PCR

Good primer design is essential for successful reactions. The important design

considerations described below are a key to specific amplification with high yield.

1. Length: The optimal length of PCR primers is 18-22 bp, enough for adequate specificity,

and shorts enough for primers to bind easily to the template at the annealing period.

The Primers that has been used:

a. PDE6A gene primers

This primers has been used to screen the PDE6A gene on chromosome chr5:149,215,712-

149,306,548 with 22 exons.

Table 3 : Primers for PDE6A gene

Primer name Primer number Sequence Size

PDE6A ex1 fw 27435 CATTGGAAGGCCTCCGTC 599
PDE6A ex1 rev 27436 ACCCCACCTGTACCCCAG
PDE6A ex2 fw 27437 GGAAGCACCTTTATTCCCTTG
343
PDE6A ex2 rev 27438 ACTGAGGCCAGGTCAAACAG
PDE6A ex3 fw 27439 GCCAGAGGATGGATTTCTTC
202
PDE6A ex3 rev 27440 CAATGCTTCAGGGAAAGGAC
PDE6A ex4 fw 27441 TTATTCTCCAGCTAAGTGAGCTACTAC
277
PDE6A ex4 rev 27442 CTCTAGCCGTCAGTCAGTGTC
 PDE6A ex5 fw 27443 GCTGACTCATGGAGGTGGG
224
PDE6A ex5 rev 27444 CAGGGTTTACTGTGCCTTGC
PDE6A ex6 fw 27445 CTCCTGTTGAACTTCTATTTGACTC
597
PDE6A ex6 rev 27446 TGATTAACATGTTCTTTGATCTTTG
PDE6A ex7 fw 27447 TTTAGTCCAGCAATCCCTGC
182
PDE6A ex7 rev 27448 CCTTCCACTCTTTCTTCCACG
PDE6A ex8 fw 27449 CACTGGGCACCCTCTCAG
170
PDE6A ex8 rev 27450 TCTAATGCTCTTTGGGGAGG
PDE6A ex9 fw 15001 ATCTATCATCGTTGCCTCTG
264
PDE6A ex9 rv 15002 GCAAGCTGACTCTGAATCC
PDE6A ex10 fw 27451 GAGGCCTAGAGACTCTTGCTATG
318
PDE6A ex10 rev 27452 ATGGAGTTGCAAGTTTTGCC
PDE6A ex11-12 fw 27453 TTGAGCTGGTGGTTCTGTTG
673
PDE6A ex11-12 rev 27454 TGCAACATGACTAATTCTTTAAATCC
PDE6A ex13 fw 27455 CAACCCTCATGAGACCATTTC
259
PDE6A ex13 rev 27456 AAGAACAACGCTGTTGCTACC
PDE6A ex14 fw 27457 TCCTTACACCCGCCTTTTC
250
PDE6A ex14 rev 27458 GGAGCCACACAGAGCTGG
PDE6A ex15 fw 27459 TGCTCTGGGGCTAGAGATTG 583
PDE6A ex15 rev 27460 CACACTCAGGCTCCTTGTG
PDE6A ex16 fw 27461 TTTCACAAGGAGCCTGAGTG
607
PDE6A ex16 rev 27462 AGCAGTGAGCTGTGATGGTG
PDE6A ex17 fw 27463 TAGCAGCTCAGGTGTTGCC
247
PDE6A ex17 rev 27464 GCAAGAGCTGTCAGTGCATC
PDE6A ex18-19 fw 27465 GGTGAGAGAGATGGAATTAACCAG
570
PDE6A ex18-19 rev 27466 TCTAGGTCAGGATGGAGCAC
PDE6A ex20 fw 27467 TGCTTCATAGATAGGGTAGGTTTC
218
PDE6A ex20 rev 27468 TGCTGTTGTCTGGAAATCCC
PDE6A ex21 fw 27469 TTCCAGTGTTGGACGTTTCC
342
PDE6A ex21 rev 27470 GCCTGAATGAGACTCCGTG
PDE6A ex22 fw 27471 TCCAAATTAGGTCAAAGGGG
214
PDE6A ex22 rev 27472 GGCTTGAGTCATCTCTTCCC

b. NR2E3 gene primers.

The primers below were the primers to amplify the NR2E3 with 9 exons on chr15:69,887,948-

69,899,648.

No Exon Primer no Sequence sizes

1 NR2E3 ex1 fw 30132 GTTCATGGACTGAGGCAAAG
265
2 NR2E3 ex1 rev 30133 GAACCTCTGGCCCTTACCC
3 NR2E3 ex2-3 fw 30134 AAGGGACCCGAGGGAAG
547
4 NR2E3 ex2-3 rev 30135 CAGGAAGGGTCAGGACGAC
5 NR2E3 ex4 fw 30136 AGGTGACAAGAAATGGGCAG
368
6 NR2E3 ex4 rev 30137 GAGGGAGGATCAAGGCAAG
 7 NR2E3 ex5 fw 30138 CTCCAAGTACTCCCTGCCAC Table 4.
312
8 NR2E3 ex5 rev 30139 GTCTGGTTGACTCGAGTGGG Primers for
9 NR2E3 ex6-7 fw 30140 ACTTCCATTCCTTGGGTGC
408 NR2E3 gene
10 NR2E3 ex6-7 rev 30141 AGTGAGAGGCAGATGGAAGTC
11 NR2E3 ex8 fw 30142 CATGTCTGCAGCCAGAACC
266
12 NR2E3 ex8 rev 30143 TGGGAGAGGCAGAGAAAGG c. RHO
13 NR2E3 ex9 fw 30144 CTCTGGGGACCTCATCTCTC gene
335
14 NR2E3 ex9 rev 30145 ATTGCTGGGGCAAAGAGTAG
primers

RHO was the causative gene for adRP with five exons on chr3:130,728,172-130,738,876.

Table 5. Primers for RHO gene

No exon primer no sequence size

1 RHO ex1 fw 30230 GAGCCCTGAGTGGCTGAG 495
2 RHO ex1 rev 30231 AGACCCCTCCATGCTCCC
3 RHO ex2 fw 30232 GGGAGTGCACCCTCCTTAG 302
4 RHO ex2 rev 30233 TGGGTTTGAGTCCTGACTGG
5 RHO ex3 fw 30234 ACCTTGGCTGTTCCCAAGTC 295
6 RHO ex3 rev 30235 AGCAGGAGCCGCAGATG
7 RHO ex4 fw 30236 ATCTGCGGCTCCTGCTC 377
8 RHO ex4 rev 30237 CCCTGGGAAGTAGCTTGTCC
9 RHO ex5 fw 30238 GTGCCAGTTCCAAGCACAC 250
10 RHO ex5 rev 30239 TGGTGGGATGGCTGTGG

d. CRB1 gene primers

These genes have twelve exons and located on chr1:195,502,031-195,716,207.

Table 6: Primers for CRB1 gene.

No exon primer no Sequence primer no sequence
1 exon 1 2820 CAGCAACACACCAGAGGATG 3002 ATAATACGCCAGAACTAAACCAG
2 exon 2 fr 1A 2943 CGTTGAGGCAGCACAAAGGTC 4834 ATGTTGTCACAGTCTTTGTCC
3 exon 2 fr 1B 4833 GTTCTTGTTCAGACACAGCC 5004* CAGGAGTTCTTGCCACAGG
4 exon 2 fr 2 2735 GTACAGTGGGACAATCTGTG 2734 CCAAGTCGCAGTGTCTGCC
5 exon 2 fr 3 2712 GATGGAATTGATGGTTACTCC 2799 TCACCTCTGCCTCTGCCAC
6 exon 3 fr 1 2800 GCTCTGGTAAACAAAGCATTG 2732 GAATCCAGGGGCACAGTCG
7 exon 3 fr 2 2762 GACGAATGTTGGTCCCAGC 2801 CAGAGTGGTAAAATAGTTCATG
8 exon 4 2818 GAAACAGTATAAAGATATCTGATC 2803 GCTATAAGCGATATGTGTATTC
9 exon 5 3113* CAACGTGATAGATCGATGCC 2806* CACAGCTCTTCCTGCTAATAC
10 exon 6 fr 1 2807 ACAAGTAAATTACGTGAAACTTC 2710 AGTGAGGGATGCATGTTCC
11 exon 6 fr 2 2709 ATTCTCCTGGGCTGTACC 2711 GCTATGTTACAAACTGAGCC
12 exon 6 fr 3 2662 GCGATGGCTTCCTGTGGG 2664 TGCCACTCTCCATCGCTGG
13 exon 6 fr 4 2663 CAGGTCAATAATCAGTCAAAGG 2666 CAAACGAAGGTGTGGATGGC
14 exon 6 fr 5 2665 ACCAGTGGGAATGACCAGC 2668 CTGTGGCAGTCACACTGG
15 exon 6 fr 6 7194* CAACCTTGTCAAAGCAGAGGA 2670 CTCTGAGGCATGGCACTCC
16 exon 7 fr 1 2671 TTCTCCTCCTCCTCTATTTTG 2669 ACACGGATATATTGATAAGTGC
17 exon 7 fr 2 2654 CTCCATGTTTGTCCGAACGC 2641 TCTTGCTTGTCAGGTAGGC
18 exon 7 fr 3 2655 TCAGTCTTCACAAAACCTAGG 2648 ATAAAGTAAAAGTTTAGCATACAG
19 exon 8 2554 CAACATTTTTCTATTTAGTTGCC 2544 CTCAAATGTCGCAACTTAACTG
20 exon 9 fr 1 AATGATCATTACTATTAATAACGG GTGCCATCATTCACTGACTG
21 exon 9 fr 2 2517 GTGGCAACAGCTTTTATATGC 2518 CATGAACATTTTCAAAGTAAGAG
22 exon 9 fr 3 2549 ATATAAAGGGCCTGCAAGGG 2526 GCTGCAACTCTGTCAGAGC
23 exon 9 fr 4 2527 GAACTCAACATCGATGAATGC 2546 CAGTGATGCAGAGTATAGCTTC
24 exon 10 2545 CTTGAATGAGATGAACAAGATG 2547 GAGGAGAAGATGAACTTTGAG
25 exon 11 fr 1 2215 ACCAATGTATTCAACAGGGACC 2455 TTACGTCCACCTCGCAGC
26 exon 11 fr 2 2458 TCTGTGCCAGGACTTACTC 2530 CAGAGATCTAAAATGAATCAAG
27 exon 12 4354 CCTGAGTAGTTCCATTGTCC 4355 ATTCACAGTGTGTGGATCCC

e. EYS gene primers

This gene was located on chr6:66,101,527-66,346,418 with forty four exons and span for
at least 2 MB.
 Table 7: Primers for EYS gene

fw
Exon rv primer length Condition
primer
1 24521 24522 286 55°C, 3mM MgCl2 (1.5µl)+ W1
2 24523 24524 315 58°C, 2mM MgCl2
3 24525 24526 472 55°C, 3mM MgCl2 + W1
4A 23111 23112 498 TD54, 3mM MgCl2
4B 24357 24358 376 59°C, 3mM MgCl2
4C 24359 24360 486 GUCY2D, 3mM MgCl2
5 26746 26747 485 59°C, 3mM MgCl2
6 23117 23118 403 TD54, 3mM MgCl2
7 23119 23120 311 TD54, 3mM MgCl2
8 23121 23122 285 TD54, 3mM MgCl2
9 23123 23124 559 TD54, 3mM MgCl2
10 23125 23126 427 TD54, 3mM MgCl2
11 23127 23128 368 TD54, 3mM MgCl2
12 25002 25003 570 59°C, 3mM MgCl2
13 24649 24650 293 59°C, 3mM MgCl2
14 25348 25349 655 59°C, 3mM MgCl2
15 25350 25351 273 59°C, 3mM MgCl2
16 24527 24528 447 58°C, 2mM MgCl2
17-18 24774 24775 551 GUCY2D PCR prog, 3mM MgCl2 + W1
19 24531 24532 425 58°C, 2mM MgCl2
20 24663 24664 395 59°C, 3mM MgCl2
21 24665 24666 322 59°C, 3mM MgCl2
22 25222 25223 531 59°C, 3mM MgCl2
23 25224 25225 486 59°C, 3mM MgCl2
24 25226 25227 572 59°C, 3mM MgCl2
25 24681 24682 378 59°C, 3mM MgCl2
26A 24683 24684 525 59°C, 3mM MgCl2
26B 24685 24686 434 59°C, 3mM MgCl2
26C 24687 26750 549 59°C, 3mM MgCl2
26D 24689 24690 439 59°C, 3mM MgCl2
26E 24691 24692 410 59°C, 3mM MgCl2
27 24695 24696 443 59°C, 3mM MgCl2
28 24697 24698 273 59°C, 3mM MgCl2
29 27922 27923 521 59°C, 3mM MgCl2
30 25230 25231 335 59°C, 3mM MgCl2
31 24709 24710 475 59°C, 3mM MgCl2
32 25437 25438 552 59°C, 3mM MgCl2
33 25571 25572 465 GUCY2D, 3mM MgCl2
34 24719 24720 405 59°C, 3mM MgCl2
35 24721 24722 447 59°C, 3mM MgCl2
36 24731 24732 458 59°C, 3mM MgCl2
37 24735 24736 410 59°C, 3mM MgCl2
38 24737 24738 438 59°C, 3mM MgCl2
39 24739 24740 410 59°C, 3mM MgCl2
40 24741 24742 438 59°C, 3mM MgCl2
41 24743 24744 432 59°C, 3mM MgCl2
42 24745 24746 323 59°C, 3mM MgCl2
43 24747 24748 444 59°C, 3mM MgCl2
44A/B 24749 24752 836 59°C, 3mM MgCl2
44C/D 24753 24756 810 59°C, 3mM MgCl2
 f. MERTK gene primers

This gene has nineteen exons and located on chr2:112,370,662-112,505,415.

Table 8: Primers for MERTK gene

No Primer name Primer No Sequence Size

1 MERTK_exon1_for 15400 gccactcggcACTCACTG 258
2 MERTK_exon1_rev 15401 agaggcccctgcttcctc
3 MERTK_exon2_for 15402 cctaagaagttgggaacctac 586
4 MERTK_exon2_rev 15403 ctgggctacagaatgatactc
5 MERTK_exon3_for 15404 AGTTGAAGAAGTTTCCATCC 241
6 MERTK_exon3_rev 15405 ATTTAACACAGGCCCTAAAAC
7 MERTK_exon4_for 15406 GGCTCTGTCTCTGTTTTCAG 294
8 MERTK_exon4_rev 15407 GCCAAACTTTTGATCCTGTC
9 MERTK_exon5_for 15408 AGAATTGTAGTGAACAGCAGC 217
10 MERTK_exon5_rev 15409 GCAAGCTCATGCTGTACC
11 MERTK_exon6_for 15410 GGTAGCTGTAGCCTGTCATC 247
12 MERTK_exon6_rev 15411 AATCCTTAAACCCACAGAGAG
13 MERTK_exon7_for 15412 CCTGACATTCCCACCAC 302
14 MERTK_exon7_rev 15413 GGAAGGGTTTGTTGAATCAC
15 MERTK_exon8_for 15414 AAAACCAAACACTTGAAAACC 292
16 MERTK_exon8_rev 15415 ACCAGCAAGTTGAAAGGAG
17 MERTK_exon9_for 15416 CAGTTTGCCCAGACCTC 282
18 MERTK_exon9_rev 15417 CAGGTTACTTTCTGGCAATC
19 MERTK_exon10_for 15418 TGTTACAAGCCAGTGTTTCTC 294
20 MERTK_exon10_rev 15419 AACAGGAAAGGCATAATCAC
21 MERTK exon 11 for 14842 GAAGCTCTGTAGCATCCTTG 235
22 MERTK exon 11 rev 14843 TGATCCTTCTTTGTTCTCAAC
23 MERTK_exon12_for 15420 TTATCAAGTGAAAGAAAACACG 371
24 MERTK_exon12_rev 15421 TGTATGTGCTAGGCATTGAAG
25 MERTK_exon13_for 15422 CACTGTAGCATTTCTGTGGTC 209
26 MERTK_exon13_rev 15423 ACCCAATACTGAAGCAACTG
27 MERTK_exon14_for 15424 ACCCACTCCCCTTAATTG 211
28 MERTK_exon14_rev 15425 CACAGAGCAGATCAGCAG
29 MERTK_exon15_for 15426 CGAGGGCTTTTCTGGTC 259
30 MERTK_exon15_rev 15427 TTAGATGATTTGGTTGTCCTG
31 MERTK_exon16_for 15428 AACTGCTTGCAAGTTTTCC 247
32 MERTK_exon16_rev 15429 AGGTCCTCTCACTAACCCTG
33 MERTK_exon17_for 15430 GACCAGTAATTTAAGGCATTG 285
34 MERTK_exon17_rev 15431 GTCATTTCTTTCAATATGCCC
35 MERTK_exon18_for 15432 AAAGTCCATTCAGGCTTTG 279
36 MERTK_exon18_rev 15433 CTGTGTTTCCGAGGTCAG
37 MERTK_exon19_for 15434 aatgaatgctgattaaaatgtg 688

38 MERTK_exon19_rev 15435 ACAATTGGATTCTCCTACAGC

g. ABCA4 gene primers

This gene was contained 50 exons and located on chr1:94,228,981-94,361,292.

Table 9 : Primers for ABCA4 gene

Primer name Primer number Size Sequence

ABCA4 exon2 fw 21050 gggctgtgtctgctctgg

ABCA4 exon2 rv 21051 ggcccagaccaaagtctc
2. Melting ABCA4 exon3 fw 1635 249 CCTGCTTGGTCTCCATGAC
ABCA4 exon3 rv 1636 ACGTGAAGGGGTGTGCAAC
ABCA4 exon4 fw 2100 259 GCTATTTCCTTATTAATGAGGC
ABCA4 exon4 rv 2101 CCAACTCTCCCTGTTCTTTC
ABCA4 exon5 fw 1681 230 GACCCATTTCCCCTTCAAC
ABCA4 exon5 rv 1649 AGGCTGGGTGCTTCCCTC
ABCA4 exon8 fw 1752 356 GAGCATTGGCCTCACAGCAG
ABCA4 exon8 rv 1684 TTAACCAACATGAGAGGCC
ABCA4 exon10 fw 2531 222 GACACACCAAAAGTTCTCTCT
ABCA4 exon10 rv 2532 TCCCCTCCCCTCCCCATC
ABCA4 exon12 fw 1744 286 GGTCCTCCTCACACTCTCT
ABCA4 exon12 rv 1688 ATTTCCCACTGACTTTGGAG
ABCA4 exon13 fw 1745 282 GAGGTGTGAGTGAGCTATCC
ABCA4 exon13 rv 1746 CCCATTAGCGTGTCATGG
ABCA4 exon14 fw 1747 330 CCTCTACCAGGTACAGAGC
ABCA4 exon14 rv 1689 GGGAAAGGAACCAAAGTATTC
ABCA4 exon15 fw 1562 407 AGGCTGGTGGGAGAGAGC
ABCA4 exon15 rv 1563 AGTGGACCCCCTCAGAGG
ABCA4 exon16 fw 1564 330 CTGTTGCATTGGATAAAAGGC
ABCA4 exon16 rv 1565 GATGAATGGAGAGGGCTGG
ABCA4 exon19 fw 1570 322 TGGGGCCATGTAATTAGGC
ABCA4 exon19 rv 1571 TGGGAAAGAGTAGACAGCCG
ABCA4 exon20 fw 1572 325 ACTGAACCTGGTGTGGGG
ABCA4 exon20 rv 1573 TATCTCTGCCTGTGCCCAG
ABCA4 exon21 fw 1574 301 GTAAGATCAGCTGCTGGAAG
ABCA4 exon21 rv 1575 GAAGCTCTCCTGCTCCAAGC
ABCA4 exon22 fw 1965 291 CACCCTCCACAGCCCCTTAAC
ABCA4 exon22 rv 1577 TCGTTGTGGTTCCTGTACTCAG
ABCA4 exon24 fw 1580 212 GCATCAGGGAGAGGCTGTC
ABCA4 exon24 rv 1581 CCCAGCAATATTGGGAGATG
ABCA4 exon27 fw 1584 493 GCTACCAGCCTGGTATTTCATTG
ABCA4 exon27 rv 1585 GTTATAACCCATGCCTGAAG
ABCA4 exon31 fw 1789 193 TAAGTCCTCAAGTTCCAAGG
ABCA4 exon31 rv 1790 TCTTCTACAGGGCAGCCAG
ABCA4 exon32 fw 21054 tgggaaagaaagttaacggc
ABCA4 exon32 rv 21055 catggctgtgaggtgtgc
ABCA4 exon33 fw 1586 265 TTCATGTTTCCCTACAAAACCC
ABCA4 exon33 rv 1587 CATGAGAGTTTCTCATTCATGG
ABCA4 exon34 fw 1588 286 GCTTAACTACCATGAATGAG
ABCA4 exon34 rv 1589 ATTCCTTGCTAGATTTCAGC
ABCA4 exon35 fw 1590 255 GCAGCGTCTCCAATGTCCTC
ABCA4 exon35 rv 1591 AAGAGTGGAGAAGGTGACAAG
ABCA4 exon37 fw 1793 226 TTGCAGAGCTGGCAGCAG
ABCA4 exon37 rv 1794 CCACCAGGCTTCTCTTCAG
ABCA4 exon38 fw 1795 253 GGAATGGAATGTGGAACTCC
ABCA4 exon38 rv 1796 CACATACTCTACTATCCTAC
ABCA4 exon39 fw 2314 268 TGCTGTCCTGTGAGAGCATC
ABCA4 exon39 rv 1798 TCCCAGCTTTGGACCCAG
ABCA4 exon41 fw 1801 319 GGACACTGTACAGCCAGC
ABCA4 exon41 rv 1802 GACGAGTTATAACACAGGG
ABCA4 exon42 fw 2102 214 CTCCTAAACCATCCTTTGCTC
ABCA4 exon42 rv 2103 AGGCAGGCACAAGAGCTG
ABCA4 exon43 fw 1594 277 CTTACCCTGGGGCCTGAC
ABCA4 exon43 rv 1595 CTCAGAGCCACCCTACTATAG
ABCA4 exon46 fw 1600 256 GAAGCAGTAATCAGAAGGGC
ABCA4 exon46 rv 1601 GCCTCACATTCTTCCATGCTG
 Temperature(Tm): by definition is the temperature at which half of the DNA duplex will

dissociate becomes single stranded and indicates the duplex stability. Primers with

melting temperatures in the range of 52-58 oC generally produce the best results

3. Annealing temperature: The reaction temperature is lowered to 50–65 °C for 20–40

seconds allowing annealing of the primers to the single-stranded DNA template.

Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the

primers used.

4. GC Content: The GC content (the number of G's and C's in the primer as a percentage of

the total bases) of primer should be 40-60%.

5. GC Clamp: The presence of G or C bases within the last five bases from the 3' end of

primers (GC clamp) helps promote specific binding at the 3' end due to the stronger

bonding of G and C bases. More than 3 G's or C's should be avoided in the last 5 bases at

the 3' end of the primer.

Actually there was no single set of conditions can be applied to all PCR

amplifications, so individual component concentrations (time and temperature

parameters) should be adjusted within proposed ranges for specific product. Control

DNA was used to adjust the condition for PCR. If it worked properly then DNA from the

patients can be used.

The protocol that could be used / set per reaction for PCR as follow: H20 16,25

µl, PCR buffer 10x 2,5 µl, 50 mM MgCL2 1,5 µl, dNTPs 10 mM 0,5 µl, 10 pmol Primer

forward 0,5 µl, 10 pmol Primer reverse 0,5 µl, TAQ 5U/ul 0,25 µl, DNA 3 µl so the total

amount was 25 µl
 The PCR machine setting for the most of the genes was 94 Celsius degree for 3

minutes (one cycle), 94 degree for 30 second , 58 degree for 30 second and 72 degree for

30 sec all these three with 35 times of cycle and ended with 72 degree for 5 minutes.

To check whether the changes on nucleotide are the real mutation, the next step

was checking the DNA with the normal DNA by using RFLP. RFLP (Restriction

Fragment Length Polymorphism) protocol that have been used: First purified PCR

product and add 10 µl DNA and 10 µl restriction mix per sample, and the restriction

mixture per reaction can be made as follow: buffer 2 µl, restriction enzyme 0,2 µl and

H20 7,8 µl with total volume 10 µl and then incubate PCR machine at 37 degree for 4

hours

III.7. Data analysis

Data will be analyzed with descriptive method and will be presented in tables and

graphics.

III.8. Research Ethics

Informed consents will be provided from the initial consent given by parent or caregiver

at the time of diagnosis that allows the use of material left anonymously. Ethical

clearance was provided by Health Research Ethical Committee of Faculty of Medicine,

Diponegoro University and Dr. Kariadi General Hospital Semarang.