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Primers for CAPN3 analysis

(last modified December 29, 2005)

DNA-analysis
- SSCA (Richard et al.)
- DHPLC (Piluso et al. 2005)
- DHPLC (Hermanova et al. 2006)
RNA-analysis
- Richard & Beckman (2001)
- Chrobakova et al. (2004)

DNA-primers

Amplified	Length	Reference	forward primer	reverse primer	Name
	185	Richard	ATaAGATGACTGCCTGCCAACGTACAAC	ttcctgtgagtgaggtctcg	550delA-F
	182	Richard	TAGATGACTGCCTGCCAACGTACAC		550delA-R
645-765	121	Richard	CTACGAAGCTCTGAAAGGTG	GGCTTTCTTCATGATCTTGT	G222R-F
645-765	121	Richard	CTACGAAGCTCTGAAAGGTA	GGCTTTCTTCATGATCTTGT	G222R-R
i6 - i7	325	Richard	ctctggttactgctctacaa	agcacgaaaagcaaagataaa	IVS6-1G>A-F
i6 - i7	325	Richard	ctctggttactgctctacag	agcacgaaaagcaaagataaa	IVS6-1G>A-R
1447-i11	197	Richard	GCCCTGATGCAGAAGAACC	ccaggagctctgtgggtca	R489W-F
		Richard	GCCCTGATGCAGAAGAACT	ccaggagctctgtgggtca	R489W-R
		Richard	GCCCTGATGCAGAAGAACCG	ccaggagctctgtgggtca	R489Q-F
		Richard	GCCCTGATGCAGAAGAACCA	ccaggagctctgtgggtca	R489Q-F
1697-i13	114	Richard	AGGGGGAATTCATCCTCCG	ttcaacctctggGagtgggcc	R572W-F
		Richard	AGGGGGAATTCATCCTCTG	ttcaacctctggGagtgggcc	R572W-R
		Richard	AGGGGGAATTCATCCTCCG	ttcaacctctggGagtgggcc	R572Q-F
		Richard	AGGGGGAATTCATCCTCCA	ttcaacctctggGagtgggcc	R572Q-R
		Richard	AAGAATGGGGTTGATTTGGAG	GCATTTCGCATCTCGTAGCT	S744G-F
		Richard	AAGAATGGGGTTGATTTGGAG	GCATTTCGCATCTCGTAGCC	S744G-R
		Richard	AAGAATGGGGTTGATTTGGAGA	TGCGTCGTTGACTGCATTTC	R748Q-F
		Richard	AAGAATGGGGTTGATTTGGAGA	TGCGTCGTTGACTGCATTTT	R748Q-R
		Richard	AAGAATGGGGTTGATTTGGAGA	ATGTGTTTGTCTGCGTACC	R769Q-F
		Richard	AAGAATGGGGTTGATTTGGAGA	ATGTGTTTGTCTGCGTACT	R769Q-R
		Richard	CACCAGAGCAAACCGTCCAC	CAGGGCTTGTTTTGCCTTTG	1872C>T-F
		Richard	CACCAGAGCAAACCGTCCAC	CAGGGCTTGTTTTGCCTTTA	1872C>T-R
		Richard	CAAGGACCTGAAGACACACG	ACCCTGTATGTTGCCTTGG	2069delCA-F
		Richard	CACAAGGACCTGAAGACACG	ACCCTGTATGTTGCCTTGG	2069delCA-R
		Richard	CATCTGCTGCTTCGTTAG	GGAGATTATCAGGTGAGATGCC	2362change-F
		Richard	TGCTGCTTCGTTTCATCT	GGAGATTATCAGGTGAGATGCC	2362change-R

Legend:
....

DHPLC

Exon	Length (in bp)	Forward primer	Reverse primer	DHLPC (in ^oC)
Prom	296	ttcagtacctcccgttcacc	gatgcttgagccaggaaaac	58.5
1A	210	CTTTCCTTGAAGGTAGCTGTAT	CGGCTGATGATGGCTGAATAGATGC	64.5
1B	279	GAGGCTGGGGGTGGAAACCCAAGTG	gaggtgctgagtgagaggac	59
2	239	actccgtctcaaaaaaatacct	attgtccctttacctcctgg	58.5
3	185	ctcacgatctgtgccctgtgtc	cgctggccctcttaactacactctc	61.4
4	227	ggacacatttcctaacagtaatttgagt	ctagccccacctcacattctaaag	61.7
5	262	gtgaccccaaattggtcttcat	ggtaaaaggcttcctgacacttg	60.7
6	220	ccctgtgttgttccctacattct	ggagggatggtcagttctgata	59.5
7	169	aatgggttctctggttactgc	cttcatgaggttaacccaggag	63
8	239	acagaagattccctttccagaga	atcttcaatactctcatggctcagt	63.5
9	159	cctgcttccttaattcctccatttt	ctcttccccacccttacccttct	60.3
10	252	cctcctcacctgctcccata	tttttcggcttagaccctcc	64
11	242	gtgaatgcgtgcttcctttctg	aaagtatgtttcctgggctggag	62.7
12	93	attcacatctgaagcatcttcctt	gtagtgctgcacacaactgctg	56.3
13	337	tgtggcaggacaggaTgttc^*	ttcaacctctggagtgggcc	63
14	191	gggttctctagaggctggttct	gacttttcgcacagaagatctaaac	62
15	94	tcacttattctgcatttactgtttcc	agtacacacacttcatgctctctacc	52.4
16	205	cagactgtaatcctcccttccttcctg	tgtagcgtgctggccaccccac	62.1
17	146	acctctgacccctgtgaacc	gcgtcctgggcttggaggtac	60
18	154	tcctgaaccatgaccctcctct	ctgctttgagacaacgggactc	60
19	159	tcctcactcttctccatccc	accctgtatgttgccttgg	62.5
20	134	gtgtgctgtgtagccctgacctc	cttgactcccacgcttcattttct	60.3
21	170	gaatggggttgatttggagattcag	cacatccctgccaccccttc	61.4
22	179	ctccacgtccacctctaacatgg	agagggcagcccccctctccca	61.4
23	137	tctggcctgtgcattctttcac	ccctccaccttgagttttaatg	58.5
24	131	gggtcactcttttctgatctaca	AGGGTGAAACTGAAATCCTGAG	60

Legend:
Primers for DHPLC analysis of the CAPN3 gene (modified from Richard et al., as published by Piluso et al. [2005]). PCR at 60^oC. Exon: exon amplified. Length (in bp): length of amplified fragment in base pairs. Forward primer: sequence of forward PCR-primer. Reverse primer: sequence of reverse PCR-primer. DHLPC (in ^oC): DHLPC melting temperature.
^*primer published is gtggcaggacaggaCgttc; present in only 1/3 GenBank sequences (potential polymorphism).

DHPLC

Exon	Length (in bp)	Forward / reverse primer	T anneal (in ^oC)	DHPLC (in ^oC)
1	780	ctaccatgcagtctctcttg / AGGTGCTGAGTGAGAGGA	55	56.0 and 61.0
2	301	agccaagattgcaccagt / TTACTGGAAGCAAGCACAGA	60	57
3	366	GGGTTGACACCTCTGTAAGG / GAAGTTTCCTCCAGCTTCTG	60	61.2
4	279	GAAGGACACATTTCCTAACA / CTTCCTGTGAGTGAGGTCT	52	59.4
5	379	GAACATCACAGCATCCAAGA / CAGTTCCTGCTTCCTCAAAC	58	58.8
6	390	CCTTTGTCTGTCCCATCTCT / TATTTCAACCCAGACCCTTG	60	60.6
7	290	cacactttcagggagcaga / CCCAACAGCTAAGTCCTGA	60	56.0 and 60.2
8	275	acaccctcgcgtaagagat / ATGGCTCAGTTCCCAACAC	60	61
9	335	TGCTATTGGGTTTCACACTG / CAAGGGCTGGAATCTGATAA	60	58
10	402	AAAGGAAAGACAGGCTCCTC / GGACGTCTGGAACAGAGAGA	60	61
11	289	CTCTGTCTCATCCCCTTC / CCTCTCTGGGAAAGTATGTT	55	60.8
12	353	ATACCCAGTGACCCACAGAG / CCCTCTCCTATCACAGTCCT	60	62
13	451	TATTTAAGCCTTGGGAGTCG / GGAGGAGACGTGTCTAGCTG	60	57.0 and 62.0
14	317	GGGTTCTCTAGAGGCTGGTT / GCCAAAGTAGGATTTGCAGT	60	60
15	287	CTTTTTGGCTCCATGTGATT / AGCAGAAGCTCAGGAAGGA	60	58
16	397	GCTTGTCAGTGGCAGAGATA / TTCAAACCTCCAGATGTTCA	60	61
17	314	CTCACCAGGTCTGCATTT / AGTCCAAAGTGACCTCTACC	55	61.2
18+19	318	taatagcaccgacagggatt / TCAGGGTCTCCTTAAGTTTCC	58	56.0 and 60.0
20+21	586	aatctctgactggtggtgga / ATGTTAGAGGTGGACGTGGA	60	61
22	368	GGGAGGGGACTAGGCTACTA / GATGGAGCAGACTGGAGATT	60	60
23	293	ATTCTCTGCCTGCACATCTT / CACCACTGTGTGGAAATGAG	60	59.4
24	697	CTCTTGCCCCGTAAGATTC / ATTGGAAGCCATTCAGTCAT	60	62

Legend:
Primers for DHPLC analysis of the CAPN3 gene (Hermanova et al. [2006]). Exon: exon amplified. Length (in bp): length of amplified fragment in base pairs. Forward / reverse primer: sequence of forward / reverse PCR-primer. T anneal (in ^oC): annealing temperature PCR. DHLPC (in ^oC): DHLPC melting temperature.

Richard & Beckman

Set	Amplified	Length	Nucleotides	Forward primer / Reverse primer	Name
A	1-4	696	-189_-170 507_526	CAGCTCGGTTTTTAAGATGG / CGTCCACCCACTCTCCATAG	A1a/A1m
A	1-3	552	-64_-43 488_468	CTTTCCTTGAAGGTAGCTGTAT / AAGATCCCTGCGTAGTTTTCG	A2a/A2m
B	1-10	1006	274_293 / 1279_1260	AAGTTCCCCATCCAGTTCGT / GAAGCTTGTCAGACTGCAGA	B1a/B1m
B	2-10	914	341_360 / 1254_1235	ATGGAGCCAACAGAACTGAC / GGCCGTGAGGTTGCAGATCT	B2a/B2m
C	7-16	906	998_1017 / 1903_1884	GTCACGCCTACTCTGTCACG / GCTTTTGCTTATCAGGGCTT	C1a/C1m
C	8-16	759	1085_1104 / 1843_1824	AGGTGGAGTGGAACGGTTCT / GCTCCTTGTTGCTGTTTGCT	C2a/C2m
D	14-24	869	1751_1770 / 2619_2600	TTGAAAATACCATCTCCGTG / GGGGTAAAATGGAGGAGGAA	D1a/D1m
D	16-24	721	1802_1821 / 2522_2503	CCATCATCTTCGTTTCGGAC / GGGTGAAACTGAAATCCTGA	D2a/D2m

Legend:
Primers for nested RT-PCR. Nucleotides: nucletoide numbering according to the CAPN3 coding DNA Reference Sequence. Reference: primers according to Richard I & Beckmann J (2001) Molecular diagnosis of calpainopathies. Muscular Dystrophy; Methods and Protocols (Ed. Bushby KMD & Naderson LVB) Humana Press, pp 273-287.

Chrobakova

RT-PCR set	Location	Forward / reverse primer
A	-256 - 340	CCAAGTCAACATTGCTTCAG
		CAATGATAAATCGGGGATTC
B	222 - 826	TTATGTGGACCCTGAGTTCC
		TTCCATAGGTCATGTTCGTG
C	643 - 1243	TCCTACGAAGCTCTGAAAGG
		TGCAGATCTCCAACTTTGTG
D	991 - 1588	GTCAGAGGTCACGCCTACTC
		AGGCGTTGTACAGGAAGAAG
E	1402 - 2001	GACGATGACCCTGATGACTC
		CTCCATGTCATCTCCTGCTA
F	1937 - 2536	AGGAAAGTGAGGAACAGCAA
		GCTTTGGAAATAGAGGGTGA

Legend:
RT-PCR set: RT-PCR sets described by Chrobakova et al. (2004). Location: position of primer sequences in relation to the CAPN3 coding DNA Reference Sequence (NOTE: location has not been verified yet). Length (in bp): length of amplified segment in base pairs. Forward / reverse primer: sequence of forward and reverse primer.

Leiden Muscular Dystrophy pages©

Primers for CAPN3 analysis

DNA-primers

DHPLC

DHPLC

RNA-primers

Richard & Beckman

Chrobakova

Leiden Muscular Dystrophy pages^©