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SGCD primers for mutation screening

(last modified March 7, 2004)

DNA analysis
- PCR
  - Tsubata
  - Nigro
- SSCA
- DHPLC
- DGGE
RNA analysis

DNA

DNA-primers

Amplified	Length	Reference	Primer forward / primer reverse	Name
exon 2	227	Tsubata	cctgccttctggaagtaatc / aaaatgaccatgagcagggc	D-Sarc2F/R
exon 3	246	Tsubata	tgcttctctcttgcctcgtt / gctaaacaaacctagatggt	D-Sarc3F/R
		Nigro	cagcggtttaatgtgagtgcttct / ccggaagaaagctaaacaaacctaga	2F/2R
	187	Duggan	TGCCTCAGGAGCAGTACACT / AATTGTGAAGTTCATGACTTTG	deltaE2F/R
exon 4	208	Tsubata	ttacagcctgaggtgttttg / gcaacaataatgcctccttc	D-Sarc4F/R
exon 4		Nigro	tcatatcttccctgttatctctgtg / caataatgcctccttctctcagcag	3F/3R
exon 5	222	Tsubata	ccccttggagagttgtaatg / tattctgagtgcctcgcatg	D-Sarc5F/R
exon 5		Nigro	tcatgtctttctcttattttcttattg / ggttcatatggtgtttcctttgacc	4F/4R
exon 6	245	Tsubata	gatgagactaatggtgtttt / aaaatgtacacaGTagcatc*	D-Sarc6F/R
exon 6		Nigro	gcgatgagactaatggtgttttctc / tgggaggacatatcctgttcataaca	5F/5R
exon 7	188	Tsubata	caggtgactccagtatctcc / tggccagttgcacagagcaa	D-Sarc7F/R
exon 7		Nigro	caggtgactccagtatctccaatc / gcacagagcaaggcaatagccatt	6F/6R
exon 8	199	Tsubata	aaaagggatctttattgacg / tgtagctctttgaaCttctgt*	D-Sarc8F/R
exon 8		Nigro	ccttgagcatgaacttccttttgt / tggcctgttgaagctgtagctct	7F/7R
exon 8b	174	Tsubata	aagctggcaatatggaagcc / ggctccttttgttgatacac	D-Sarc9pF/R
exon 9	243	Tsubata	ctgaccaatgctttccttcc / atgctgccaacaatgtccac	D-Sarc9mF/R
		Nigro	tctgaatgtcaagagaagagacgacag / cagccagtgtgtaaagccaaaaa	8F/8R

Legend: (primer sequences obtained from Nigro, with the assistence of Eloisa de Sa Moreira)
*primer sequences listed have genomic targets but differ from those published (Tsubata, D-Sarc6R has GT in stead of TG, D-Sarc8F/R has a C inserted).
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 Tsubata et al. 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.
PCR-conditions (Nigro primers); initial denaturation 30 sec at 99^oC, 30 cycles with denaturation 15 sec at 96^oC and 3 min annealing-extension at 61^oC.

SSCA

Exon	Forward primer / reverse primer	Size (bp)	Amplification
2	cctgccttctggaagtaatc / aaaatgaccatgagcagggc	229	65^oC to 55^oC (-2^oC/2 cycles, 1 min); +30 cycles, 55^oC, 1 min
3	tgcttctctcttgcctcgtt / gctaaacaaacctagatggt	243	70^oC to 60^oC (-1^oC/2 cycles, 1 min); + 30 cycles, 60^oC, 1 min
4	ttacagcctgaggtgttttg / gcaacaataatgcctccttc	208	70^oC to 60^oC (-1^oC/2 cycles, 1 min); + 30 cycles, 60^oC, 1 min
5	ccccttggagagttgtaatg / tattctgagtgcctcgcatg	218	65^oC to 55^oC (-1^oC/2 cycles, 1 min); + 30 cycles, 55^oC, 1 min
6	gatgagactaatggtgtttt / aaaatgtacacaGTagcatc ^*	244	65^oC to 55^oC (-2^oC/2 cycles, 1 min); + 30 cycles, 55^oC, 1 min
7	caggtgactccagtatctcc / tggccagttgcacagagcaa	188	65^oC to 55^oC (-2^oC/2 cycles, 1 min); + 30 cycles, 55^oC, 1 min
8	aaaagggatctttattgacg / tgtagctctttgaaCttctgt ^**	196	60^oC to 50^oC (-2^oC/2 cycles, 1 min); + 30 cycles, 50^oC, 1 min
9a	ctgaccaatgctttccttcc / ATGCTGCCAACAATGTCCAC	239	60^oC to 50^oC (-2^oC/2 cycles, 1 min); + 30 cycles, 50^oC, 1 min
9b	AAGCTGGCAATATGGAAGCC / GGCTCCTTTTGTTGATACAC	173	60^oC to 50^oC (-2^oC/2 cycles, 1 min); + 30 cycles, 50^oC, 1 min

Legend:
Primers for PCR and SSCA of the SGCD gene (Sylvius et al., 2003). The primer sequences published (^*aaaatgtacacaTGagcatc and ^**tgtagctctttgaa_ttctgt) differ from the human genome sequence as shown here.

RNA-primers

RT-PCR

Amplified	Reference	Length	Forward primer	Reverse primer	Name
-26-278	Duggan	304	GTGTTGAGTGAAGGGACCAG	TCTTTGGCGTAGAGAGGTTG	delta1F/R
192-486	Duggan	294	TGATGGAATGGGAAACCTGA	TTCAGCTCCTACTACCACTTCA	delta2F/R
436-748	Duggan	312	GGAAAATTGCTCTTCTCTGCA	ATCCATGAGGCAGTCTAGGT	delta3F/R
643-938	Duggan	295	GGCAATATGGAAGCCACCTG	GCAGGCAGTGTGTAAAGCCA	delta4F/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 Tsubata et al. 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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