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SGCE primers for mutation screening

(last modified December 9, 2005)

Contents

DNA

SEQ

ExonLength
(in bp)		Forward primer /
reverse primer		T ann.
(in oC)		Name
1301TGCTGAACTGGCCAAGCTGG /
AGAGAGGCTGGTGCCCAAAG		641F / 1R
2434GGCGTATCTCATTATTTGTC /
AGGTAGATCACTTGTCAGAG		552F / 2R
3311CATGTGTGAAAATAACTGTC /
GGTAACTTTAGTTTCAACAC		533F / 3R
4290ATGAAAATGGAAAGAATGAC /
AGTTATATTAGGTATGTGGC		554F / 4R
5393CCAGGATTATGACAGAACTC /
GCAATAGGCCATCTTCCATC		555F / 5R
6343AGGGATGAGTCTAGTTAATC /
CAAACGTTAACTCCAGCCAC		576F / 6R
7348GAATGCTTTAGTGTATCCAG /
GTTGTTATCTTAGCAGGATC		537F / 7R
8171GCATATAGTCTTAATGTTCC /
CACATGTATGGAGCATGATG		538F / 8R
9341AATTGATGACCCATCAGGCT /
CACAACAACAGAAAGCTCTG		559F / 9R
10574CAGTTGCATTTGGCAGACC /
TTCTGCATAGCCATTCCATC		5210F / 10R
11220TCATTCTAGTATGTCTGCTC /
TTTGGTGAAGATAAAGCTTC		5311F / 11R
11b304GGCATTGTGGTAGGGAAAC /
GCTTACAAAGTAGCACCAAC		5811bF / 11bR
12158GTATCCATGCCCTGACTAAC /
AGCTCATGCATTATTGGAAG		5512F / 12R

Legend:
Primers for PCR and sequence analysis of the SGCE gene (Tezenas et al., 2005).
NOTE: primer sequences not checked.

SSCA

Amplified Length Forward primer /
reverse primer		 Name PCR Reference
Annealing T
(oC)		 no. of
cycles
exon 1 304 ctgatgctgaactggccaag /
agagaggctggtgcccaaag		 1F/R 60 35 Klein  2002
exon 2 295 ctgaattatcaagggcgtatg /
ccatttgaaataatgttaatg		 2F/R 56  35 Klein  2002
exon 3 355 agacagaatgttttgattgaaac /
accaccatcaggtaa C tttag a		 3F/R 60 35 Klein  2002
exon 4 339 ttctcattgcccagagaagg /
tcagttatattaggtatgtggc		 4F/R 58 35 Klein  2002
exon 5 306 cttcattaaagatatgcatgc /
ataagtttgataagatcaccg		 5F/R 58 35 Klein  2002
exon 6 335 taaatcctgcttttaaggtgg /
ttattcctaaaagcagtt C ag b		 6F/R 58 35 Klein  2002
exon 7 348 aagaatgctttagtgtatccag /
ttgttatcttagcaggatctc		 7F/R 58 35 Klein  2002
exon 8 309 gacaatgtcagcatttccac /
gttttagtttctacccctcct		 8F/R 58 35  Klein  2002
exon 9 295 catgcatattaataattatgg C ctc c /
caaattgatgacccatcaggc		 9F/R 60 35 Klein  2002
exon 10 448 taatgtagcctagtgccac /
agccaacttcatgacttctag		 10F/R 55 40 Klein  2002
exon 11 249 ctggggtcatagtttacccg /
atttggtgaagataaagcttc		 11F/R 60 35 Klein  2002
exon 12 230 gatggaaactttctccttgcc /
caacatgcataacatatgccag		 12F/R 60 35 Klein  2002

Legend:
Primers for PCR and SSCA of the SGCE gene (Klein et al., 2002).  a C in genomic sequence (AC069292), G in Klein et al.; b C in genomic sequence (AC069292), G in Klein et al.; c extra C in genomic sequence (AC069292), - in Klein et al.

DHPLC

ExonLengthPrimer Forward /
Primer Reverse		DHPLC oven temperature (°C) Start%
B-buffer 		Reference
1327CTGGGAGGGAAGAAGAAAGG /
AGGGTGTACTGAAGGAGGGGTA		68, 6453, 58Valente 2005
2TGAATTATCAAGGGCGTATCTCA /
TTAGACCATTTGAAATAATGTTAATGA		5653Valente 2005
3CCAAAGCAACATGTGTGAAAA /
GTATAGTTTTGCTCTTTCTAGGTG		55, 58.555, 49Valente 2005
4TCATTGCCCAGAGAAGGAAT /
CATCAGTTATATTAGGTATGTGGCATT		5356Valente 2005
5ATGCCCTTTTTCACCAAAATTAG /
GCAATAGGCCATCTTCCATCTAT		55, 58, 6058, 55, 53Valente 2005
6TCCTGCTTTTAAGGTGGATTGTT /
CAAACGTTAACTCCAGCCACAT		54.5, 5657, 55Valente 2005
7TTTGCAACGATTAATTTGTTGTGT /
TGAAACTTTGCTTTTAATGGAATC		54.2, 5858, 54Valente 2005
8GACAATGTCAGCATTTCCACATC /
AGTTTTAGTTTCTACCCCTCCTAAA		54.4, 56.458, 55Valente 2005
9AAATTGATGACCCATCAGGCTAA /
CAACAGAAAGCTCTGTTCTTTACA		58, 5954, 53Valente 2005
10CATGACTGGGGTCATAGTTTACC /
CACAAGTGTTTTGCCTTATTTGG		52.6, 56.8, 56, 52Valente 2005
11GAAGATGGAAACTTTCTCCTTGC /
CAACATGCATAACATATGCCAGA a 		56.1, 57.155, 53Valente 2005

Legend:
Primers for PCR and DHPLC of the SGCE gene (Valente et al., 2005).
a sequence not unique in genome.

Other DNA-primers

Amplified

Length

Reference

forward primer

reverse primer

Name
promoter 375 Grabowski
 (for bisulphite treated DNA)
gtgttatgttttataaatagataag		 aactcatatacctctacaattc BSTx_F/
BSTx_R
ex7-in.. McNally GCAAGTGTCCACCTATCAGG cttagcaggatctctaattatc
human SGCE pseudogene
McNally CATAGAATTCATTGAGATGCAGTC AAGTGAGACACAGGAAGTGTTGATG
murine Sgce gene
ex6/ex7 McNally GTTTATGTCATGGTTGGTGCCGATG TACTTCCTGATAGGTGGACACTTGC 2F/1R
ex6/in6 McNally GTTTATGTCATGGTTGGTGCCGATG ttccaacagcaattcgggttcc 2F/3R

Legend:
Exonic sequences are in upper case, intronic and gene flanking sequences in lower case and added primer tails in italics. Amplified: region amplified. Numbering of exons is according to McNally et al (1998), except for exons 9b-11 (designated 10-12 by McNally et al [1998]). Length: length of PCR-product in basepairs. Reference: publication describing the primer(s). Forward primer: sequence of forward primer. Reverse primer: sequence of reverse primer. Name: name of the primers.

RNA-primers

Amplified Reference Length
(in bp)		 Forward / reverseprimer Name
1-12 Tezenas 2005  (1437) GAATCGAGGACGGACGGC / 
AGCTCATGCATTATTGGAAG		 PF / 12R
8-9 McNally   GACGGGAAGGCGTGGAAAAGAGAA /
AGCACTGTGATGGACCAGTTGG		  

Legend:
Exonic sequences are in upper case, intronic and gene flanking sequences in lower case and added primer tails in italics. Amplified: region amplified. Numbering of exons is according to McNally et al. 1998, except for exons 9b-11 (designated 10-12 by McNally et al. 1998). Length (in bp): length of PCR-product in base pairs. Reference: publication describing the primer(s). Forward primer: sequence of forward primer. Reverse primer: sequence of reverse primer. Name: name of the primers.

intron 3 CA-repeat

Allele size (bp) Allele frequency
162 0.51
164 0.31
166 -
168 0.16
170 0.01
172 0.01

Legend:
Polymorphic CA-repeat localized in intron 3 of the SGCE-gene. Allele size (bp): size of the amplified fragment in base pairs. Allele frequency: frequency of the alleles identified (McNally et al. 1998), using 45 unrelated, normal individuals of mixed ethnicity. Amplification was performed using primers ESG-E3-3C (5'-gttatctactgtgtttccatgcac-3') and ESG-E3-F2 (5'-CAACAATCATTGAGgtaatttacacc-3'). PCR-conditions; 50 ng genomic DNA in a 10 ul volume (Messina et al.1997) with addition of 8% DMSO, using as cycling parameters 10 min 94oC and 35 cycles of 1 min 55oC, 30 sec 72oC and 20 sec 94oC.

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