Leiden Muscular Dystrophy pages^©

Primers for DGGE analysis of the dystrophin gene

(modified last April 20, 2001)

NOTE

Until our DGGE-work has been published, the information displayed below has is confidential (see disclaimer)

For PCR-conditions and other details see "DGGE analysis of the dystrophin gene". For additional information contact Dr. Johan T. den Dunnen (ddunnen @ lumc.nl) or Robert W.M. Hofstra (r.w.m.hofstra @ med.rug.nl).

Primers

The primer sequences shown below are for PCR on genomic DNA and subsequent analysis using Denaturing Gradient Gel-Electrophoresis (DGGE). To improve the detection of sequence changes in the amplified products, a GC-clamp has been added to the 5'-end of either the forward or reverse primer, indicated by [clamp]. The sequence of the clamp is listed in the Table Legend. To facilitate DGGE-detection of sequence changes, 16 exons had to be split into two partly overlapping segments.

Table

Exon	Sequence forward primer	Sequence revers primer	Primer names
1m	AGCCTACTGGAGCAATAAAG	[clamp]-AGCTTGTCACAAACTAAACG	DMD-01F/GC01R
2	[clamp]-ATGGAAAGTTACTTTGGTTG	CAGGTACATAGTCCATTTTG	DMD-GC02F/02R
3	[clamp]-TAAATTGAGTGTATTTTTTT	TTTTGCCCTGTCAGGCCTTC	DMD-GC3aF/3aR
3	CTTCAGTGACCTACAGGATG	[clamp]-TTAACTTTCTTAAAAATAAG	DMD-03bF/GC03bR
4	[clamp]-CTCTCTGCTGGTCAGTGAAC	GTGTGTCACAGCATCCAGAC	DMD-GC04F/04R
5	[clamp]-GCATTTGGTCTCTTACCTTC	TTTGTTTCACACGTCAAGGG	DMD-GC05F/05R
6	[clamp]-GGTTCTTGCTCAAGGAATGC	GCTCAGGAGAATCTTTTCAC	DMD-GC06aF/06aR
6	AAATATCATGGCTGGATTGC	[clamp]-ATCAGAGTCTAAATCACCAC	DMD-06bF/GC06bR
7	GCGCGTTTGTCTTTGTGTATGTGTG	[clamp]-TACTAAAAGCAGTGGTAGTC	DMD-07F/GC07R
8	GCGCGCCAAAAATTGATGTGTAGTG	[clamp]-ATCTTGAATAGTAGCTGTCC	DMD-08F/GC08R
9	[T7]-CCCTCCTCTCTATCCACTCC	[clamp]-GGAAGCAGTTCTCTGGTTTG	DMD-T709F/GC09R
10	GCGCGGACATTAATTGTGTAACACC	[clamp]-GGATGACTTGCCATTATAAC	DMD-10F/GC10R
11	CACCGATTTACCTAGAGTTC	[clamp]-TCCAAAACTTGTTAGTCTTC	DMD-11F/GC11R
12	[clamp]-AAGTTGCTTTCAAAGAGGTC	GTACTATACACAGAGTTTGC	DMD-GC12F12R
13	[clamp]-CATTTCAACACACATGTAAG	AGCAGCAGTTGCGTGATCTC	DMD-GC13aF/13aR
13	CTCTCACTCACATGGTGGTG	[clamp]-CTTTTAAAGGACATATTTAG	DMD-13bF/GC13bR
14	GCGCGGTTTGCTGATGCTTGATTG	[clamp]-CCCCGTGTCTTTTACAGCTA	DMD-14F/GC14R
15	TATGTTTATTTATTCCTTGG	[clamp]-TACTGGGTTTTTATAAGACC	DMD-15F/GC15R
16	[clamp]-TGCAACCCAGGCTTATTCTG	..GCGCGTCTCTGAGATAGTCTGTAGC	DMD-GC16F/16R
17	AGCAATGTCTGACCTCTGTT	[clamp]-GCTGTAAATGAGTTTTCTCC	DMD-17Fnw/GC17R
18	[clamp]-AGAAGAAAGAGATAATCAAG	AGTTGCCTTCCTTCCGAAAG	DMD-GC18aF/18aR
18	CTGGATTACTCGCTCAGAAG	[clamp]-GCAGCACAAAATGAGTACAG	DMD-18bF/GC18bR
19	GCGCGGATTCACAGTCCTTGTATTG	[clamp]-GTGTTTATCAAATCCCTAAG	DMD-19F/GC19R
20	[clamp]-TCAGTCTGTGGGTTCAGGGG	CAGTAGTTGTCATCTGCTCC	DMD-GC20aF/20aR
20	GGCTGGAGTATCAGAACAAC	[clamp]-GGAATGCCAAGAAATACCTA	DMD-20bF/GC20bR
21	ATGTATGCAAAGTAAACGTG	[clamp]-TGGAAAATGTCAAGTTAGCC	DMD-21F/GC21R
22	[clamp]-CAATTAAGTGATTCTCATTC	GCGTGCTTTATTGTTTTGAC	DMD-GC22F/22R
23	[clamp]-TTCATCAATTAGGGTAAATG	AGGAGAGCTTCTTCCAGCGT	DMD-GC23aF/23aR
23	CGCCCTCTGAAATTAGCCGG	[clamp]-GTAAATAAAAATGAGGGTAG	DMD-23bF/GC23bR
24	AATCAGCACACCAGTAATGC	[clamp]-TTATTCATATTAAAGGCATC	DMD-24F/GC24R
25	[clamp]-TTTTCAGCTGGCTTAAATTG	TTAAGTACGTTGAGGCAAGC	DMD-GC25F/25R
26	[clamp]-CATCACTGTCAATAATCGTG	[T7]-TCAAGATACTCTTCTTCAGC	DMD-GC26aF/T726aR
26	[T7]-CTATCAGAGATGCACGAATG	[clamp]-TTTACCTTCATCTCTTCAAC	DMD-T726bF/GC26bRnew2
27	[clamp]-TTTCATGCTATTAAGAGAGC	AACTATGACCATGTATTGAC	DMD-GC27F/27R
28	[clamp]-TTCACATTTACTTTTCTACC	CTGCATATAAATCATCATCA	DMD-GC28F/28R
29	[clamp]-CATTTGCTGATAATCCAATG	[T7]-ATTAGCTCATCCATGACTCC	DMD-GC29aF/T729aR
29	[T7]-ATTCGCATATTGGCACAGAC	[clamp]-ATGCAAATTAGATTAAAGAG	DMD-T729bF/GC29bR
30	[clamp]-TACAGAAAAGCTATCAAGAG	CAAACAAAAAGAATGGAAGC	DMD-GC30F/30R
31	[clamp]-GTTAGTTGTTCTTTGTAGAG	GCCCAACGAAAACACGTTCC	DMD-GC31F/31R
32	[clamp]-GGCAAAATTAAATCAGTGCC	TAATGAGGAAAGTCAAGGGG	DMD-GC32F/32R
33	[clamp]-TAACTCTACTGATTATCATG	GTGGAAAGAAGTGTTTGTGG	DMD-GC33F/33R
34	[clamp]-ATTTGAATTAAAGAGTAAAC	ATGTGTTTTCACGTATGTTC	DMD-GC34F/34R
35	[clamp]-CAAGACATTACTTGAAGGTC	CGCGCACAGAGAAGGGTGTAAAAGC	DMD-GC35F/35R
36	GTCTAACCAATAATGCCATG	[clamp]-GTAACTGGTGTACAATTTGG	DMD-36F/GC36R
37	[clamp]-CTAACTTCAAGTCCTATCTC	CGCAAGAGACCATTTAGCAC	DMD-GC37F/37R
38	[clamp]-CTTTTGGTTTATGTTTCTTA	TTTATTTCCACTCCTAGTTC	DMD-GC38F/38R
39	CTCTGTTAACAATGTACAGC	[clamp]-AAACCACAGGCAAGGTATAT	DMD-39F/GC39R
40	..GCGCGATTTTAATAATGTCTGCACC	[clamp]-AAAATCTGGTATTGACATTC	DMD-40F/GC40R
41	[clamp]-ATGTGGTTAGCTAACTGCCC	GCTGGATCTGAGTTGGCTCC	DMD-GC41aF/41aR
41	GAAGAAAGAGGAGCTGAATG	[clamp]-AGTTGCAAACACATACGTGG	DMD-41bF/GC41bR
42	[clamp]-TAATGGAGGAGGTTTCACTG	GCGCCACCTTGTAAAATACGAATG	DMD-GC42F/42R
43	[clamp]-CTATAGACAGCTAATTCATT	GCTGCTGTCTTCTTGCTATG	DMD-GC43aF/43aR
43	AGTCTACAACAAAGCTCAGG	[clamp]-TCATTTCTGCAAGTATCAAG	DMD-43bF/GC43bR
44	[clamp]-CGCTATATCTCTATAATCTG	TAAAGAGTCCAGATGTGCTG	DMD-GC44F/44R
45	[clamp]-GACATGGGGCTTCATTTTTG	GTTTTCATTCCTATTAGATC	DMD-GC45F/45R
46	[clamp]-GTTTGAGAACTATGTTGGAA	ACTTCTTTATGCAAGCAGGC	DMD-GC46F/46R
47	AGGTAGTTGGAATTGTGCTG	[clamp]-GCACCCAGGAAACAAAATAC	DMD-47F/GC47R
48	[clamp]-GGCTTATGCCTTGAGAATTA	CAGTGATATTGCCATTTTTC	DMD-GC48F/48R
49	[clamp]-ACTGTGAAGTTAATCTGCAC	CAACAGGCGAAGCATAACCC	DMD-GC49F/49R
50	[clamp]-CGAATAAGTAATGTGTATGC	TTGAACAAATAGCTAGAGCC	DMD-GC50F/50R
51	[clamp]-TCTTCTTTTTTCCTTTTTGC	GATCAAGCAGAGAAAGCCAG	DMD-GC51aF/51aR
51	GATTTCAACCGGGCTTGGAC	[clamp]-AAAGAAAAACTTCTGCCAAC	DMD-51bF/GC51bR
52	GAAGAACCCTGATACTAAGG	[clamp]-CATCTTGCTTTGTGTGTCCC	DMD-52F/GC52R
53	[clamp]-TCCTCCAGACTAGCATTTAC	AGACCTGCTCAGCTTCTTCC	DMD-GC53aF/53aR
53	TTCAGAACCGGAGGCAACAG	[clamp]-CAAATGTAACCAGTATTTTA	DMD-53bF/GC53bR
54	[clamp]-AAAAACTGACATTCATTCTC	TAATCCCGGAGAAGTTTCAG	DMD-GC54aF/54aR
54	CCAGTGGCAGACAAATGTAG	[clamp]-CCCATTATTACAGCCAACAG	DMD-54bF/GC54bR
55	[clamp]-TTTATGAGTTCACTAGGTGC	ACAAATGCTGAGAATTGTTC	DMD-GC55F/55R
56	TCTGCACATATTCTTCTTCC	[clamp]-ATTTGTGGCCTTTTTGCTCC	DMD-56Fnw/GC56Rnw
57	GATATTCTGACATGGATCGC	[clamp]-GTCACTGGATTACTATGTGC	DMD-57F/GC57R
58	TTGAGAAGAATGCCACAAGC	[clamp]-TCACCACTGATCCTTCTATC	DMD-58Fnw/GC58R
59	[clamp]-AAAGAATGTGGCCTAAAACC	GCGGCCGCCAAAGGGCCCTGAAGCAAAG	DMD-GC59F/59R
60	ATTCTCATCTTCCAATTTGC	[clamp]-TTACTGTAACAAAGGACAAC	DMD-60F/GC60R
61	[clamp]-GAATGAGAGAACATAATTTC	TGAGATGCTGGACCAAAGTC	DMD-GC61aF/61aR
61	GTCGAGGACCGAGTCAGGCA	[clamp]-AGATGCAATAAAGTTAAGTG	DMD-61bF/GC61bR
62	ATTTGACCTCCTTGCCTTTC	[clamp]-GTTAGTCACAATAAATGCTC	DMD-62F/GC62R
63	[clamp]-GTTTTCTTGACTACTCATGG	CCTGTCATTTAACTTGGAGG	DMD-GC63F/63R
64	GGCAAATCACTGGGCGTCGG	[clamp]-AGCAAAGACATAGTATCAAG	DMD-64F/GC64R
65	[clamp]-GGAAGGTTTTACTCTTTGAG	CTAAGCCTCCTGTGACAGAG	DMD-GC65F/65R
66	GGTCTAGTAATTGTTTTCTG	[clamp]-CTAGGGTAATTAGCCAACAT	DMD-66Fnw/GC66R
67	TTGGATGTCAGGTTCTGCTG	[clamp]-GAAAGAATAAATATGTTACC	DMD-67F/GC67R
68	[clamp]-CTTTCCTTTCATCCTTTGGC	TTGGACACTCTTTGCAGATG	DMD-GC68aF/68aR
68	AGAAACTGCCAAGCATCAGG	[clamp]-AGGAGATAAAAGATCAAGTC	DMD-68bF/GC68bR
69	[clamp]-CGAAGAAATACATACGTGTT	TGAACTAACTCTCACGTCAG	DMD-GC69F/69R
70	[clamp]-AGTTTTGAAATCATCCTGTC	CATCAAACAAGAGTGTGTTCTG	DMD-GC70F/70R
71	AGAAAGCGTGTGTCTCCTTC	[clamp]-AGCGAATGTGTTGGTGGTAG	DMD-71F/GC71R
72	GATGGTATCTGTGACTAATC	[clamp]-TCAATCAATATTTGCCTGGC	DMD-72F/GC72R
73	[clamp]-TCACATAAGTTTTAATGAGC	ACCTCTAAATCCCTCAAAGC	DMD-GC73F/73R
74	[clamp]-TGAGTCCCTAACCCCCAAAG	GCACTCTGCATACCAATGAC	DMD-GC74F/74R
75	[clamp]-TTTACTTTTTTGATGCC	CCTTTGTGTTGACGCAGTAG	DMD-GC75aF/75aR
75	CGCCGCCGGCCAATAGGAATCTGCAAGC	[clamp]-GTTTGTAAAAATCCCATCTC	DMD-75bF/GC75bR
76	[clamp]-TGAAAAGTAATTCTGTTTTC	TCTACCTTTCTTCAGACAAC	DMD-GC76F/76R
77	[clamp]-ATGTAATTTTCCATTATTTG	GATCCCAGCAAATCTGAGTC	DMD-GC77F/77R
78	[clamp]-CCCTTTCTGATATCTCTGCC	AAGCAGGATGAGACAGACAG	DMD-GC78F/78R
79	[clamp]-TGATGCTATCTATCTGCACC	TTCTGCTCCTTCTTCATCTG	DMD-GC79F/79R

Legend:
Exon: number of exon in the human DMD-gene. Sequence forward primer: sequence of the forward primer. Sequence reverse primer: sequence of the forward primer. Primer names: name of the primer. [clamp] = is GC-clamp tail sequence added to the 5'-end of the primer (5'-CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCGCGGCCGC-3'), [T7] = T7 RNA polymerase promoter tail sequence added to the 5'-end of the primer (5'-TAATACGACTCACTATAGGG-3').

Primers replaced by others

Exon	Forward / reverse primer sequence	Primer names
17	CCAAGCAGTCTTTACTGAAG / AGTTGCCTTCCTTCCGAAAG	DMD-17F/GC17R, detects additional polymorphism
56	[clamp]-TCTGCACATATTCTTCTTCC / ATTTGTGGCCTTTTTGCTCC	DMD-GC56F/56R
58	CCACAAGCCTTTCTTAGCAC	DMD-58F
66	CCTCTAGGAAAGGGTCTAGT	DMD-66F

Primers Tubiello

Exon	Region	Length (in bp)	Forward / reverse primer sequence	PCR condition	Denaturant	Gel run
Dp427c - A		262	[GC]-CGTTGTAGCAGCTGCATA / aatcctacCTTCCATGCCA		30 - 80	4 h
Dp427c - B		427	gtacagaagagcgagtaga / [GC]-aatcctacCTTCCATGCCA		30 - 80	5 h
Dp427m - I	-850 - -550	299	[GC]-aagctttctattggcaaag / gtgtgtgtataggtctca	50^oC 45 s, 72^oC 1 min	10 - 60	4 h
Dp427m - II	-588 - -450	138	tagcaggaaaacaTgtccc^* / [GC]-cccttgggattcaatc	48^oC 45 s, 70^oC 1 min	30 - 80	4 h
Dp427m - III	-588 - -198	390	[GC]-tagcaggaaaacatgtccc / cagtggacatagtacttgc	54^oC 45 s, 72^oC 1 min	15 - 60	4 h
Dp427m - IV	-239 - 50	288	[GC]- ttgtgtttagaaggc / AGTGAGTGATCCCAACAC	54^oC 45 s, 72^oC 1 min	30 - 80	4 h
Dp427m - V	1 - 324	324	TCCTGGCATCAGTTACTGTG / [GC]-ctaaacgttatgccacag	48^oC 45 s, 72^oC 1 min	20 - 70	5 h

Legend
Data from Tubiello et al. (1995). [GC]- = 45 nucleotide GC-rich clamp. ^*T in sequence was 'r' in paper. PCR conditions for are;

Dp427m sequences; 300 ng genomic DNA in 50 ul with 30 pmol primers, 0.5 U Taq-polymerase
- fragments I, III and IV - 200uM of each dNTP, 50 mM KCl, 10 mM Tris.HCl (pH8.0), 1.5 mM MgCl₂, 0.01% gelatine
- fragments II and V - 1.5 mM each dNTP, 16.6 mM (NH2)SO4, 67 mM Tris.HCl (pH8.8), 6.7 mM MgCl₂, 10 mM 2-mercaptoethanol, 0.17 mg/ml BSA
Dp427c sequences; 250 ng genomic DNA in 50 ul with 30 pmol primers, 0.5 U Taq-polymerase, 200uM of each dNTP, 50 mM KCl, 10 mM Tris.HCl (pH8.0), 1.5 mM MgCl₂, 0.01% gelatine.

Denaturation was at 94^oC.