1
Supplemental Digital Content 1
Methods
PCR-LDR protocol of four target SNPs:
The PCR reactions were performed with 1L DNA sample, 1 × GC-I buffer
(TAKARA), 3.0 mM Mg2+, 0.3 mM dNTP, 1 U HotStarTaq polymerase (Qiagen Inc),
2L multiple PCR primers and ddH2O in a total volume of 20L. The PCR cycling
program were: 95ºC for 2min, followed by 11 cycles of 94ºC for 20s, 65ºC (decreased
0.5ºC per cycle) for 40s, 72ºC for 90s plus 24 cycles of 94ºC for 20s, 59ºC for 30s,
72ºC for 90s, with a final extension at 72ºC for 2min. Then 1 U shrimp alkaline
phosphatase and 1 U Exonuclease I were added in the PCR product, incubated at 37ºC
for 1h and inactivated at 75ºC for 15min for purification. The LDR reactions were
performed in a final volume of 10L containing 1L 10×ligase reaction buffer,
0.25L Taq DNA ligase, 0.4L 5’ ligase primer mixture (1µM), 0.4L 3’ ligase
primer mixture (2µM), 2L purified PCR product and 6L ddH2O. The LDR
reactions were cycled as follows: 38 cycles of 94°C for 1min and 56°C for 4min, and
kept at 4°C. 0.5L LDR product were mixed with 0.5μL Liz500 SIZE STANDARD
(Applied Biosystems, Foster City, CA, USA) and 9μL Hi-Di (Applied Biosystems,
Foster City, CA, USA), inactivated at 95°C for 5min and then sequenced by
ABI3130XL sequencer (Applied Biosystems, Foster City, CA, USA). Finally the raw
data was analyzed by GeneMapper 4.1 (Applied Biosystems, Foster City, CA, USA)
and the nucleotide at each SNP site can be read and recorded. The primers for four
target SNPs were shown in (Supplemental table 1).
2
PCR-RFLP protocol of APOE SNPs:
ApoE genotypes (rs429358 and rs7412) were determined by the restriction fragment
length polymorphism (RFLP) method. The PCR reactions were performed with 1L
DNA sample, 1L PCR primers (2µM), 1×GC buffer I (TAKARA), 2.0 mM Mg2+,
0.2 mM dNTP, 1 U HotStarTaq polymerase (Qiagen Inc) in a total volume of 10L.
The PCR cycling program were: 95ºC for 15min, followed by 11 cycles of 94ºC for
20s, 65ºC (decreased 0.5ºC per cycle) for 40s, 72 ºC for 100s plus 24 cycles of 94ºC
for 20s, 59ºC for 30s, 72ºC for 90s, with a final extension at 72ºC for 2 min. The
digestion of endonuclease was performed at 37ºC overnight, with 10L PCR product,
1 U of restriction endonuclease (AflIII and HaeII, New England Biolabs), 1×buffer
and ddH2O in a final volume of 20L. Then the products were diluted 10 times and
analyzed by capillary electrophoresis. The primers for two SNPs were shown in
(Supplemental table 1).
Supplemental table 1
Product size and primers of SNPs within SORT1 and APOE
genes
SNP
rs12740374
Product size
(bp)
202
PCR primer sequence
Ligase reaction primer sequence
rs12740374F:
CAGCCCAGGTGTT
TGCTCAGTT
rs12740374R:
GGACACCAGAAC
CCAGACTTGAA
rs12740374FG:
TCTCTCGGGTCAATTCGTCCTTTGGCTCGGCTG
CCCTGATGG
rs12740374FT:
TGTTCGTGGGCCGGATTAGTTGGCTCGGCTGC
CCTGACGT
rs12740374FP:
TGCTCAATCAAGCACAGGTTTCATTTTTTTTTT
TTT
rs646776FC:
rs646776F:
3
rs646776
rs599839
rs464218
183
386
122
rs429358
317
rs7412
185
aF
GGTCGGGAGACT
CAGACACCAC
rs646776R:
GCCTCTCCCACCG
TAGAAGTCC
rs599839F:
CCCAGATCGYGCC
ATTAAAC
rs599839R:
ACTGTTGTGGTCA
GCCCCAGAG
rs464218F:
GAGCCCAGGGAT
GGTACCAAGT
rs464218R:
TGGAATTCGAAGG
GACCTTTTCA
TTCCGCGTTCGGACTGATATGCTGATAAGCCTG
TCCCTCTGACC
rs646776FT:
TACGGTTATTCGGGCTCCTGTGCTGATAAGCCT
GTCCCTCTGACT
rs646776FP:
ATGTCCATGACACTGCTCCCATTTTTTTTTTTTT
TTTT
rs599839RG:
TTCCGCGTTCGGACTGATATGAATGTATTTTTAT
ATKCTCTGTATATCTGGAACTC
rs599839RA:
TACGGTTATTCGGGCTCCTGTGAATGTATTTTTA
TATKCTCTGTATATCTGGAACTT
rs599839RP:
GATCCTGCTCCTATTTCTTTCTYTTTTTTTTTTT
TTTTTTT
rs464218RG:
TCTCTCGGGTCAATTCGTCCTTCCCACTTCTGT
GTGTTCTGCATTGC
rs464218RA:
TGTTCGTGGGCCGGATTAGTCCCACTTCTGTGT
GTTCTGCATCGT
rs464218RP:
GCCTGCCCCAGGSCATCTGCTTTTTTTTTTTTT
T
rs429358F:
AGGGCGCTGATG
GACGAGAC
rs429358R:
GCCCCGGCCTGGT
ACACT
rs7412F:
GGCGCGGACATG
GAGGAC
rs7412R:
GCCCCGGCCTGGT
ACACT
indicates forward primer and R indicates reverse primer.