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SGCB primers for mutation screening

(last modified August 18, 2005)

DNA analysis
- SSCA
  - Sylvius 2003
  - Bönnemann 1996
- SEQ
  - Duclos 1998
- DHPLC
RNA analysis

DNA

SSCA - Sylvius

Exon	Forward primer / reverse primer	Size (bp)	Amplification
1	tggggaggggagggtgtgagcag / cctcccccgctcCtccag ^*	208	72^oC to 50^oC (-1^oC/cycle, 1 min); +12 cycles, 50^oC, 1 min
2	tagataaatgcaccAaaacgag ^** / ttccccatggcaattaaaatgag	313	72^oC to 50^oC (-1^oC/cycle, 1 min); +12 cycles, 50^oC, 1 min
3	tggtgataatattttctacttgttttcc / gcccctctcctgtttgcatttctttc	312	35 cycles, 50^oC, 1 min
4	attgttcaggaattttgtttgcagtcttc / attctctcccattagtaaaacaaagcc	298	72^oC to 50^oC (-1^oC/cycle, 1 min); +12 cycles, 50^oC, 1 min
5	gcttctatttctctatctctgataac / ccaagaacctaataattctcttaagctc	256	35 cycles, 55^oC, 1 min
6	agttttgtttactgactttgttctg / agtcaagatataaacatgttggtgacc	315	35 cycles, 55^oC, 1 min

Legend:
Primers for PCR and SSCA of the SGCB gene (Sylvius et al., 2003). The primer sequences published (^*cctcccccgctcA tccag and ^**tagataaatgcaccCaaacgag) differ from the human genome sequence as shown.

SEQ - Duclos

Exon	Size (bp)	Forward primer / reverse primer	Name
2	287	agttccaatggtcctgtaaaga / aataaagtctatgatttcactata	BF9 / BR1
3	318	ggcctaccaacagcggac / aatatctctttccaataatcaactag	BF3 / BR3
4	307	ttcaggaatttTgtttgcagtct^* / actctagagaataattctctc	BF4 / BR4
5	254	tctgataacaattctcacattaat / tatggatttatgtacccaagaa	BF5 / BR5
6	291	cctcatggaggtaaataaatc / aagtcaagtcaagatataaa	BF6 / BR6

Legend:
Primers for sequence analysis of the exons of the SGCB gene (Duclos et al., 1998). The primer sequence published (^*ttcaggaattt_gtttgcagtct) differs from the human genome sequence as shown.

SSCA - Bönnemann

Amplified	Reference	Length	Forward primer / reverse primer	Name
promoter/ exon 1	Bönnemann	476	tggtccgagaaacaaagagagc / cctcccccgctcatccag	pro-5/e1-R4
promoter/ exon 1	Bönnemann 1996	208	tggggaggggagggtgtgagcag / cctcccccgctcatccag	pro-1/e1-R4
exon 2	Bönnemann 1996	313	tagataaatgcacccaaacgag / ttccccatggcaattaaaatgag	e2-Fb/R
exon 3	Bönnemann 1996	312	tggtgataatattttctacttgttttcc / gcccctctcctgtttgcatttctttc	e3-Fb/Rb
exon 4	Bönnemann 1996	298	attgttcaggaattttgtttgcagtcttc / attctctcccattagtaaaacaaagcc	e4-F/R
exon 5	Bönnemann 1996	256	gcttctatttctctatctctgataac / ccaagaacctaataattctcttaagctc	e5-Fb/Rb
exon 6	Bönnemann 1996	314	agttttgtttactgactttgttctg / AGTCAAGATATAAACATGTTGGTGACC	e6-F/R

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 Bönnemann et al. (1996). 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

RT-PCR

It should be noted that the entire coding region goes from 1-957.

Amplified	Reference	Length	Forward primer / Reverse primer	Name
-42-456	Duclos 1998	499	gCacagtcgggcgggga^* / CTTTGTTGTCCCTTGCTGA	BF0 / BR0
-40-267	Duggan	308	ACAGTCGGGCGGGGAGCT / CACGGCCCAAATAACAAGTG	-
1-1123	Lim	276	ATGGCGGCAGCGGCGGCG / AATCTTCTCAGAGAGCCTAA	-
37-1123	Duclos 1998	1087	AGTTCCAATGGTCCTGTAAAGA / ttaggctctctgagaagatt	BF9 / BF8
120-398	Bönnemann	279	TAAAGCTGGATACATTCCG / ATTTTCATTTCGCCTTCCTC	A3b1F/R
349-672	Bönnemann	324	GGAGTGATCCACCCTCTTTA / AGCACGCCCATCAACTTTTA	A3b2F/R
612-887	Bönnemann	276	TACTGAAAGGATTACCAGCAA / AAGAGCGTCCCATCAGCAC	A3b3F/R
773-982	Duggan	210	ATGGATCTGTGATGGTCAGC / CATGTTGGTGACCTCTGGG	-

Legend:
The primer sequence published (^*gGacagtcgggcgggga) differs from the human genome sequence as shown (NOTE: primer located at extreme 5' end SGCB-transcript). Amplified: region amplified. Numbering of nucleotides is according to the coding DNA Reference Sequence, with the first base of the Met-codon counted as position 1. 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.

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