Human PAPSS2 Polymorphisms

1
Human PAPSS2 Polymorphisms
Val291Met
I/D
Glu10Lys
Met281Leu
Arg432Lys
I/D
I/D
I/D
1
2
3
4
5
6
7
8
9
10
11
12
APS Kinase
linker
ATP Sulfurylase
Variant frequency Variant frequency 1
Variant frequency 5
2
PAPSS2 Resequencing Reactions
1
2
3
4
5
6
7
8
9
10
11
12
606
121
APS Kinase
linker
ATP Sulfurylase
3
Frequency of Polymorphisms
Number of Polymorphisms per kb
PAPSS2
Halushka, et al 1
Genes Samples Min. Allele Freq. Overall
Coding Non-Coding UTRs Introns
1 90 0.56 4.8 3.2 5.3 6.0 5.1
74 75 0.68 4.6 4.4 5.9 4.4 6.0
1 90 0.68 2.6 1.1 3.2 4.8 2.7
Including 5-Flanking Region 1Nature Genetics
22239-247, 1999
4
Human PAPSS2 Polymorphisms
Val291Met
I/D
Glu10Lys
Met281Leu
Arg432Lys
I/D
I/D
I/D
1
2
3
4
5
6
7
8
9
10
11
12
APS Kinase
linker
ATP Sulfurylase
5
PAPSS2 Sequence
Primer Name UF(-606) M13 UR(-122) M13 UF(-168)
M13 I1R133 M13 I1F(-166) M13 I2R169 M13 I2F(-96)
M13 I3R92 M13 I3F(-135) M13 I4R178 M13 I4F(-217)
M13 I5R107 M13 F610 M13 I6R186 M13 I6F(-149)
M13 I7R158 M13 I7F(-134) M13 I8R117 M13 I8F(-141)
M13 I9R174 M13 I9F(-113) M13 I10R101
M13 I10F(-122) M13 I11R131 M13 I11F(-127)
M13 R2075 M13
Primer Location 5-FR 5-FR 5-FR Intron
1 Intron 1 Intron 2 Intron 2 Intron 3 Intron
3 Intron 4 Intron 4 Intron 5 Exon 5 Intron
6 Intron 6 Intron 7 Intron 7 Intron 8 Intron
8 Intron 9 Intron 9 Intron 10 Intron 10 Intron
11 Intron 11 3-UTR
Primer Sequence
(5?3) GAGCCCAGCGGAGTGCATTGTAAG AGGTATAAAAA
GGCTCCGCAGACACG GCTGGTCAAGGGAAGTGCGACG GTGCCCCACTC
TTACTCCTCCTC CTATATTAGAATGGACAAAGGTGAGTCTCTTTCAA C
TATTTCTCACTGATGGTAGGGTTAGGACTA AAGATTTAGTTATATTTAA
AATTACTAGATTAGTTCAC GTTGGAGAGCACTTGCACTGT TTCTCTCA
AGCACTCTGCAAAGCACAAAC CTACCTTTTAATAAGCCTCTGGGCATAC
TGAATA AGCGGGATTTTGTTGACAATTTGGGGCTT CATTGGTAAAAAC
TTGAAAACTGTAAAAAGAATGCTTCAG CACCAGGTAGTGGAACTTCTGC
AAGAG GTGGCAATTCTTGGTGAGAGATTTCTTTGGA ATGGGTTAGCAT
AACAGGTGGGGAC GCTTAAACATAATAGAATGGAGCACACTGTAAATGA
T ACAACTACTTGGATTTGGGTCTTAATGCTTCT ACATTCTTCCACCTA
ATCCCAGTTTTTCTAAGT GAGAAATACTGTGCCAGAAATACTAAAGAAC
TGA TGGGCACCTGTAATCCAAGCTACTC CCAGTGGATAATGAATGCAC
AGAACAATAAAC CATGAGGAAACAGAAAGGATCCCAGAG GCAATTCTT
AGTTGACTCACATTGCTGAATTACTT GACTCTCAAGGGACTCAGTCCAC
TT GCCCACTTTTGAAGATGCAGCATTTTACAG GTATAAGAATATTGCT
TTTACCTGCTAAGATTTGGTC
PCR Cycle Conditions (35 Cycles) 94 oC 30 s
64 oC 30 s 72 oC 45 s 94 oC 30 s 66 oC
30 s 72 oC 45 s 94 oC 30 s 66 oC 30 s
72 oC 45 s 94 oC 30 s 60 oC 30 s 72 oC
45 s 94 oC 30 s 66 oC 30 s 72 oC 45 s 94
oC 30 s 66 oC 30 s 72 oC 45 s 94 oC 30
s 66 oC 30 s 72 oC 45 s 94 oC 30 s 66
oC 30 s 72 oC 45 s 94 oC 30 s 66 oC 30
s 72 oC 45 s 94 oC 30 s 66 oC 30 s 72
oC 45 s 94 oC 30 s 66 oC 30 s 72 oC 45
s 94 oC 30 s 66 oC 30 s 72 oC 45 s 94
oC 30 s 66 oC 30 s 72 oC 45 s
Table 1. PCR primers and conditions used for
human PAPSS2 resequencing. A universal M13
forward primer (5-TGTAAAACGACGGCCAGT-3) was
attached to the 5-end of all forward primers as
a universal M13 reverse primer (5-CAGGAAACAGCTATG
ACC-3) to all reverse primers. F represented
forward R, reverse U, upstream I,
intron FR, flanking region UTR,
untranslated region. Numbering for primers
located in exons and 5-FR was based on the cDNA
sequence with the A in the start codon
designated as 1. Positions 5 to that location
was assigned as negative, and those 3 to that
location as positive. Intron-based primers were
numbered on the basis of nucleotide distance from
splice junctions with 1 designated the first
nucleotide at 5-end and -1 for the 3-end of
introns.