*
SNP 1360020 T/G
LEFT
PRIMER GGTAGCCTACTGTGGCCTTG
RIGHT
PRIMER TGGGCTTTCCATGCAACTAT
PRODUCT
SIZE: 498,
GGTAGCCTACTGTGGCCTTGAACCACCATATATGAGAAAGGGCTGCCAAATTCAGAGGAAGT
CAAAAGAATTGGCGTGGGAAGATGCCAGGAGAGCATGCCTGTGGCTACAGACCACAGACA
AAGCCAGCCACATCCATTTTTCCAAGATGTTGCAAATTATGAGAGAGAACACCAACAATC
TCTTTTTTTGTAACAGACTGGGGTAAGTGCACTGATGGCAAGAGCTTCCCTAATCCCATC
TGGCACAGAAAAGGCAGGGAGGGATGAGAAGTCAGGAAGCAGTCGTCAGTGAGTGGGACA
GGCTGACTACAGGAGAAGGTTTGCATTGATTGCTGTCCTGCAGCATCAATCTCTGCTGGC
TAGGGGAAAGATTAGAATCTCTCTCCCTCTCTTGGTGGAATACTAGACATCGGAGAAGTG
AAGGAGAATGAGGGATGAGAAATTACCTAATGGGTACAATGTGCACCATCTGTGTGATAG
TTGCATGGAAAGCCCA
* SNP 1410659 T/C
LEFT PRIMER
TGCTGTGAGGGGAAATCAAT
RIGHT PRIMER
ATGCAATTAACAGGCCCACT
PRODUCT
SIZE: 506,
TGCTGTGAGGGGAAATCAATTTATGTTACTGAGCTATATGAACCTCCAGTAAAACCTAATTCTTTTG
CTCTTCTGCATATGATCATCCTTAGCTTTTCATATGGCCCACAATTAAATGCAAAAGATGATTCTGA
TCCCTTCTTATAAAGCAGAGGAATGATTCATTAAAAAAAACTTCTCAAAAGCATTTATCTCGGGTTT
GTTTGAAATAGCCATTACCTTTCCATCTTGAATAAAATTCAAGTTTACAAAATAAGTAAATTAATAA
AATATAGTTCAAGTTTACAAAAATTAGCCACAATTCTTTCAGAACACATCGATTACATATATACAT
ATCTCCTTACATATTGTGAGGAGCACTCAGCCACCTACTTATTTGATCTATTCAATAGTTATGGTAC
TCACACTGTTACGCATTGGGCTACATCTATAAAGTAGAACAGCATATCTTAAGATCCTCACAGGCAG
TGAGAACACTGTAACTATAGTGGGCCTGTTAATTGCAT
*SNP 1854352 G/A
LEFT PRIMER CATCTCAGCAGTGTGACCAAG
RIGHT PRIMER
GCCTGTAATCCCAGCACTTT
PRODUCT
SIZE: 471, CATCTCAGCAGTGTGACCAAGATCTGAGCACATGCTGGCAGCCAGCATGACAATATAGTTTCCTT
ATTAGGAGGTGAGCAGACATAACAATCTGAGGGATGAAGCTCAGGGGAATATGTAGTCAACACAC
TCCTCAATTAATTTCCCTTCATAAAATATCTACTACATTCCAAGCGAAATAAAAATTGTATCCAA
CAAACAAAACATTCTTCTTTCTTTCTTTCTCTGAGGCAGAGTCTCGTTCTGTTGCCCAGGCTGGA
GTGCAGTGGTGTGATCTCAGCTCACTGCAATCTCCTGGGTTCAAGTGATTCTCATGCCTCAGCCT
CTTGAGTAGCTGGGATTACAGGCATGCACCACCAGCTAATTTTTGTATTTTTAGTAGAGACAGG
GTTTCACCATGTTGGCCGGGTTGGTCTCGAACTCCTGACCTCAGGTGATCTGCCTGCTT
CCCAAAGTGCTGGGATTACAGGC
*
SNP 2149436 G/A
LEFT PRIMER
CCTTGCTTCCCTGGGATAAG
RIGHT PRIMER
GGAAAACCTGGAAGAAATGGA
PRODUCT
SIZE: 497,
CCTTGCTTCCCTGGGATAAGTCTCACTTGAAAATGGTAGATGATCTTTTTAACGTATTGTTGAGT
TCAGTTTGCTGATGCGTTGAGAATTTTTACATCAATGTCCATCAGAAAAACTGAGCTGTAGGGTT
TTTTTGTGTGTGTTTTGTTTGTTTTTGTTTTTTGATGTGTTGTTCTGATTTTGGTACGAGGG
TAATACAGGTCTCATAGAACAAGTTTTGAAATATTTCCTTCCCTATTTTTTTCAACAGTTTG
AGTAGAATTGGCATTAGTTCTTCTTTAAATGCTTGGCAAAATTCAGAATTCAGTAGAGAAGCT
CTCAGGTACGGTTTTGTTTTTTTTTTTTTTTTCCCCCTTTCTTTTTGTTGCAGGAGACTTTTT
ATTACCGCTTCAATTTTGTCACTTCTTATTGATCTATTCAGGTTTTGGATTTCTTCGTGGTTC
AATCTTAGTAGGTGGTATGTGTCTAGGAATTTATCCATTTCTTCCAGGTTTTCC
*
SNP 1410655 T/C
LEFT PRIMER
CATGCTGAGTGCAACCAAAT
RIGHT PRIMER
TGAGATCAGAAAAGAGAGGTGCT
PRODUCT
SIZE: 500, CATGCTGAGTGCAACCAAATTTTAATATTTGCTGTTTATCTTCATTTCCCTTAATTTCTGAT
GTGTTTAATGCAAAGACTACATGGGGCTATGTTGTTAAACTTCTGTGCAAGGCATATAGCTC
TATAATTTGTGATTCCCTGGGACTTTCTTGAAAATAAGCCAGAAATTGCTGCAGGTGCAGAA
TTCAGCAGCTTGAAAACTGCATTCAAGGAGAAGACCAATTTCATTACATGAAGACAGGGTAT
GTAGAGTTTTCTATAGGCTTAGGCATAAGAACCCAACCTACTTGTATTTTGCATCGAATCA
AGAATTTGGCTGGGCAGTTATAGCCCTGCCCCTCTGATTTTAACAAAAGCTTTAGTCAAAAT
GGGAGTTCCATCTCTAGAAATGTCTCCTCAGAGATGTACAAGTTTTGGAAGGGTAATTTTAA
GAGTTAAAGATGTGTTAAATAACCAACTGCCTGCATAAATAATGAGCACCTCTCTTTTCTGATCTCA
*SNP
1410656 G/A
LEFT PRIMER
TGCTGGGCCTTAGAAGGTAA
RIGHT
PRIMER CCAAGAGCAGCACCCATATT
PRODUCT
SIZE: 527, TGCTGGGCCTTAGAAGGTAATTATAATTTGAGGAAGTGAAAAGAAGGTAAGTGCAATTAATT
TCTAGTTAATTAAATAAACTGAAATAGAAACTTATTGGGAATAATGAATGCTTCTCTCTTGT
TCTAAAGAGGAGAAGCTAGGGATGTTGATTAGTTTCACAGAGGACATTGAAGGTTCGGGTCT
GGAGTCTGGATGGTTCCCTCAAAGACAACAGGAAATCAACAAATGGGAGAGTGGCTGAAAGA
AGTGCCTGGGGAAAATAAAACCAGAGTTTGTGTGCAGCAGGTCTAGAAAAACATTAGCCAA
GTGATATTGCAATAGTCCAAGTAAGCAATGGCATAACTGTAACAATGCTTCTAAGCTCCCTA
GGGTTAAAAAAAAATTCAGATAACTTTTCCACATTTGTCATCCGGCAGGTGGGAAAGGTGGT
AAGAGAATATAAGAAAAGATAGTAAGATTTCCTTTTAAGAACATCTTTTACCCACTCTGTCC
TACCTGCTCATCAATATGGGTGCTGCTCTTGG
*
SNP 2503314 G/A
LEFT PRIMER
AAGAAGGGCAGTCAACAGGA
RIGHT PRIMER
ACACGCTGGGCATTATGTCT
PRODUCT
SIZE: 422, AAGAAGGGCAGTCAACAGGAGACTAGAGTGAGGTATGAGGAATCCAGTGACAGAGGTTACAA
AAGGATGTTCCTGAGGGAGTTGAGTGAGAGACTTCATGTGGAAGAGCACATTCCACCCAGTG
GAAAGGAAGCCAAGCCTCACACATTTCAGAAGCCCTTAGAGGATCCATTTTATAGCACTGTG
CAATGGCTACAATAGCCAGTCTTCTTGAGGCATTGTAGCACAGGGGAAACAGGCTGGCCCCC
TGGAGTCAGAAACATGTAAATGCAAATCCCAGCTCTGCACATTAGTAGCTTTGTGACCACGG
GCAGATAACTCACATACAGTTTCCTCATCTGTACGTTATGGACATTTTAGCAAAGCTGCCT
GACAGTTTTAAGATGAGAGGTGATGTACCCAAGACATAATGCCCAGCGTGT
*
SNP 754325 A/G
LEFT
PRIMER
GAACAAACACAGAGGCCACA
RIGHT PRIMER
AAGACTGGAAGGCAGGAGAA
PRODUCT
SIZE: 298,
GAACAAACACAGAGGCCACACAAAGATATTAACTGTGGGAAGCAGCTGCC
CCCATAGGGCTGGGAGAATAAAGGGGAAACTCTGGAGTGATTAGAACTAGAAACTCAGAG
AGGAACTCAGGAGCTAGGATCCTGACCTCAGGGAAGTGGTGTGGATACGGCTGGTTCTGG AAGTGTGGAAAGAAACTCACAACCAGAACTACCTACCACTGCTCAGTTTAGAGGCCAGTC
CTGGGGCACTGTGGCAAGGACACGAGACAAGCAGGAAAGAGTAAGTCCTTCTCCTGCCTT CCAGTCTT
* SNP 1018579 A/G & 1018578
T/G
LEFT PRIMER
AAGGCCCCACCTCTTAACAC
RIGHT PRIMER
CCCTCCAGGTTCATGAGCTA
PRODUCT
SIZE: 519, AAGGCCCCACCTCTTAACACCAACACATTAGAGATTAAATTTCCACATATGAATTTTGGAG
AGACACAAATACCCAGACCATAGTAACCTTTCATGAATTCTAAAGCCACTTGTGTGCCTAC
GATTTGCAAGTATTAGAGCTAGGATATGACTCAGCTCCTGCCTTTCAGAAACTTACTTATA
TTCAAAATTGCAATGCAATGTTAGGTGCCACAATAGAGGTGTGGCTGTACAAGGGCTAAA
GGTGCACTGAAGAGTGCTTAGCTTTATCTAAGCAGTTCAAAAAGTTTCCTTGGGGAATGTG
CATTTGAGCTGGGAACTGAAAAACATGTAAGTTTGTAAGGTAAATTGTGCATGGTGAGAA
GTGTGTTGTGGGATAGGCAAAAGGAACAGCATGTGCAAAATCATGATGGTATAAGAGCATG
GGGTGTTTAGATTATAATTAGGAGGGAAGTAGCTTGATACAGCTTCAGCATAGGATGGAAT
TATGAGGGAGCTGTAGCTCATGAACCTGGAGGG
SNP 1051064 A/G
LEFT PRIMER
GGGTTTTCATGGCAAGTCAG
RIGHT PRIMER
CCCAGAATATCTGCCATGATT
PRODUCT
SIZE: 516,
GGGTTTTCATGGCAAGTCAGAATTTTTAAGCAAGTTGCAAGTAACTTCTATATTTTTAAGT
AAGTTGCAAATATCTATTCATATTTAATTTAGCAAAATATAACTTGTTTTTAAATTTCTTT
TGTTTCTGAAGTCTGTGTCAGCATTTGCTCCAATTTGCAACCCTGTACTCTGTCCCTGGGG
CAAAAAAGCCTTTAGTGGATATTTGGGAACAGATCAAAGTAAATGGAAGGTAGGTACTGA
ATGGCTGTCTTAATACTCATTCTTAAGTTTCAAGTTTGTTTAAAAGGTTGGACATATATTA
TATTCCTACCCTGCTCATGTTAGGGCTCTAGAGCTGAGACAAATTAGCAAAAGATATATAT
ATATCGATATATATATCAATATATGAGATATATATTATTATATATATTATTTTATATATAT
TATTATATATATTATTTTATATATATATATATATAAATCAACAAAAAACCCTACTTTCTAA
TTGTATTGAATCATGGCAGATATTCTGGG
*
SNP 2230948 A/C
LEFT PRIMER
GGAAGATGCTGAGGTCAAGG
RIGHT PRIMER
ACATAGGCTCAGGGTGGCTA
PRODUCT
SIZE: 505, GGAAGATGCTGAGGTCAAGGTCTCCCAGGCTACCATGGAAGGAGCCAATGGGCAGCTAGA
GATTTAGGTCTGGAACTGAGAGAAATAATCAGCTATGAAGGTACAGACTTGGCCACAAAC
ATATTGAAGGCATGAGAGTGGTTGAATTTGTCCAGGGAGAATGTGAAGAACAGAAGGTGA
TAGAACTGTAGCAAAACCCAACTATTGAGGAAAATATTTTCATGCCTGCAAAAAGAGAGA
GATCGAACTGCCTCCTTTAGAAACCTACTTGGGAAAGGGCTTTCTTTACAAGTAAGGCAA
GTTGTCATTACTGGTGGGGGAAAAAAATTGGTACTGCGAAACCAACTTATTTCCTACCAC
ATGTGAGGTTTTGAATAATATCTGGGTGGCATATTTCTGCTGAAGAAAATAAGCCAGTT
CAATGGTGTATCTATTAAATAAAAATAGTGCTAATAGTTTATCAGAGTTATCACCACAGA
CGTGCCTAGCCACCCTGAGCCTATGT
*
SNP 6312 A/G
LEFT PRIMER TACTCCATTCCCGTGGAAAC
RIGHT PRIMER
GACAACTTTCCTCCCTGGAA
PRODUCT
SIZE: 486,
TACTCCATTCCCGTGGAAACCAGGAGTCCCTTGGTGCAGACAGCTCTTCCTACTTTCCCA
TGCAGTTCTTTTGTGCGACTTTGAGGGGCTCGTGAATGATTTCTAAATGTGTGCCTGCTG
AGGCGAGCCGCACAGGGAGGGAGGAACCCAGCCGAGCCGTGCCAGAGGAAGCCAACAGGA
TCCTAGCAGTGCGGGAGCTGGCTCAGCTCTTGCATGCAGTTTTTGAAGTCAGCAAAACAG
AAACCAAATTACTATCATATTATGCTGGTGGAAGATCAAGAAGAGGGGACTCTACACCAG
TTTAATTACTGTGAGAGATGCAGCGAGTCACAGAATAACAAATGTATCTCATGTGTGGA
CCCTGAAGACAAATGTAAGTTCTCATGCCGCTATATTTTATTGCTGTGTAATTTTCTTTC
CGGTTTGAAATCATGCTTGGCCAACATGTAATCATTTCAATGAGAATTTCCAGGGAGGAAAGTTGTC
*
SNP 2298086 T/C
LEFT PRIMER
AGCCTCAGCAATTTCTGCTC
RIGHT PRIMER
CTTGTGCCATTTGGGAAGTT
PRODUCT
SIZE: 479, AGCCTCAGCAATTTCTGCTCTTGGAGAATCAAGCGGATTAAAATTTTTTTTCAAGTCTGA
AATTTCACACTTACTTCAAAATCTTAAATTATTTCCCTTTTTCCTCAGAATTGAACATAT
TGCATAGTCATTTTACTTTCACTGCAATAGAGCAAGTTAAAGTTTATATAATTTATTCAA
TATTATTTCTCAAGAAAGGTTGAGACAAAAAAGCATGTACAAAGCTTTGCATCGATTCAA
TGGTTCAAACTCATATATTAAATAAAATTTTTACTAGAACACAGTTATATGAAGTAAGTT
TGATGTTACCTCTGGTACTCCTAATTTTCGGCATGCTTCCAAAAAGTTTTCCACATTTC
TTCTGCATTTGGCCATGCTAAGTTTGGGCTATAAATGCAAATATACGGAAATAATATATT
ATTTAAAAGAGAAACCATTTTAATAACATGTTAAATTTTAAACTTCCCAAATGGCACAAG
* SNPs
912125 C/A & 912124 G/A
LEFT PRIMER
ACACACCCAGCCATACATACA
RIGHT PRIMER
AGCTGACAGTATCGTCAGTGGA
PRODUCT
SIZE: 269,
ACACACCCAGCCATACATACATATAACATATACATATTATGTGCACATATACATAAATAT
ACATGATTGCTACAATCTGTTTCAGCAATGACACCCTATGATGTGTGTGTGTGTGTGTG
TGTGTGTGTGTGTGACCGGGGTCAACCTTTTTGATGGGTAAGTCAATCACAAAGTGGTTT
GTACAAATCCAAGATTTTAATGGCTGTTAAATAATAAAAGGAAGGATATTTGCACTATA
TACATTCTGTCCACTGACGATACTGTCAGCT
*
SNPs 2298085 T/A & 2298084 T/C
LEFT PRIMER
ATTCTTCCAGACACTGTGTTCA
RIGHT PRIMER
TGTTGATGGGGAAGTTGATG
PRODUCT
SIZE: 274,
ATTCTTCCAGACACTGTGTTCATATATGCCTACATAGATATACATATATGCCTATGTATA
TATATATAGATGTTTGTATATCATGTATATACACTGATGTATATGTATGCAAAATTTTC
TGCATACATATGTATATATGTATACACACGTATGTTTATATTGTTTATATATATGCACA
CACACACGTATATATAGTTTTTACGGGATCATACTGTTGTGTAACCTGTTTTAGTTTGCC
TAAAGTACACCATAAACATCAACTTCCCCATCAACA
*
SNP 1886221 T/C
LEFT PRIMER
CTTCAAAAACGGCAAATCCT
RIGHT PRIMER
AATGGAGCTGGTATGTTACCG
PRODUCT
SIZE: 263, TTCAAAAACGGCAAATCCTGCAAACTTCAGGAGCTGTAAATTACAGGAAGCCTTTAAATT
CCAGGAACTGTGTCTGTCACAGTGAAGTTTCAAAGCTGTTCCTCTCCCCACTTAATGTC
TTTATCATATACCTGCCCTCAAACAACAGAGGCTCTACCTTGTCCAACCGTAGCAAAAAG
GTTTTCTGAATACCTTGAGAGGCTATGATCAGTCAACCAACAAAAAAGAGCATCTTAAAA
ATCGGTAACATACCAGCTCCATT
Internal Primers
*1854352-FS1
AGTAGCTGGGATTACAGGC
*1854352-FS2
AAAATTAGCTGGTGGTGCA
*1410659-FS1
AATTCTTTCAGAACACATCGA
*1410659-FS2
GGAGATATGTATATATGTA
*1360020-FS1
GACTACAGGAGAAGGTTT
*1360020-FS2
TGCAGGACAGCAATCAATG
*1018579-FS1
ATGCAATGTTAGGTGCCACA
*1018579-FS2
GCCCTTGTACAGCCACACCTCTA
*1018578-FS1
TGGGAACTGAAAAACATG
*1018578-FS2
GCACAATTTACCTTACAAACTT
PCR Conditions for HTR2A SNPs
10xPCR buffer 1.0ul
dNTP(5mM) 0.4ul
MgCl2(25mM) 0.6
(for FP, use 1.2ul)
F-primer(20uM)
0.125ul (for FP, use 0.025ul)
R-primer(20uM)
0.125ul (for FP, use 0.025ul)
Taq Polymerase(5U/ul)
0.05ul
DNA(20ng/ul) 3.0ul
Sterile water ---
Total 10.0ul
PCR
reaction conditions
Initial
denaturation
95° C for 4min(if
it is AmpliTaq Gold do it for 10min)
35 cycles of
94° C for 45sec
60° C for 45
sec(see table below for
change in temp)
72° C for 1min
followed by
final extension at
72° C for 7min.
FP
method :
SAP
treatment :
10X SAP buffer
1.0ul
Alkaline
Phosphatase 2.0ul
Exonuclease I 0.1ul
Sterile water --
Total 10.0ul
37° C for 1hr
30min, followed by 95° C for 15min.
FP
labeling :
10uM Detection
primer 1.0ul
Thermoseq buffer
1.0ul
25uM ddNTP 0.05ul
Thermoseq (8u/ul)
0.1ul
Sterile water
Total 10ul
Reaction
conditions
94° C for 1min
followed by
94° C for 10sec
55° C for 30sec
(for SNP18 at 60° C)
for 35 cycles
4° C forever
|
Marker
|
A
temp
|
|
|
1410656
|
60
|
|
|
1360020
|
60
|
|
|
2149436
|
55
|
|
|
1854352
|
60
|
|
|
1410659
|
60
|
|
|
1018578
|
60
|
|
|
1018579
|
60
|
|
|
2298086
|
60
|
Increase
mg cl & primer
concent.
|
|
6312
|
60
|
|
|
2503314
|
60
|
|
|
1051064
|
50
|
Increase
Mg cl concent.
|
|
754352
|
60
|
|
|
1018578
|
60
|
|
|
2503314
|
60
|
|
|
2230948
|
60
|
|
|
1410655
|
55
|
|
|
1886221
|
60
|
|
