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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
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
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
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
CTGGATTACTCGCTCAGAAG [clamp]-GCAGCACAAAATGAGTACAG DMD-18bF/GC18bR
19 GCGCGGATTCACAGTCCTTGTATTG [clamp]-GTGTTTATCAAATCCCTAAG DMD-19F/GC19R
20 [clamp]-TCAGTCTGTGGGTTCAGGGG CAGTAGTTGTCATCTGCTCC DMD-GC20aF/20aR
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
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
[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
[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
GAAGAAAGAGGAGCTGAATG [clamp]-AGTTGCAAACACATACGTGG DMD-41bF/GC41bR
42 [clamp]-TAATGGAGGAGGTTTCACTG GCGCCACCTTGTAAAATACGAATG DMD-GC42F/42R
43 [clamp]-CTATAGACAGCTAATTCATT GCTGCTGTCTTCTTGCTATG DMD-GC43aF/43aR
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
GATTTCAACCGGGCTTGGAC [clamp]-AAAGAAAAACTTCTGCCAAC DMD-51bF/GC51bR
52 GAAGAACCCTGATACTAAGG [clamp]-CATCTTGCTTTGTGTGTCCC DMD-52F/GC52R
53 [clamp]-TCCTCCAGACTAGCATTTAC AGACCTGCTCAGCTTCTTCC DMD-GC53aF/53aR
TTCAGAACCGGAGGCAACAG [clamp]-CAAATGTAACCAGTATTTTA DMD-53bF/GC53bR
54 [clamp]-AAAAACTGACATTCATTCTC TAATCCCGGAGAAGTTTCAG DMD-GC54aF/54aR
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
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
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
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
50oC 45 s,
72oC 1 min
10 - 60 4 h
Dp427m - II -588 - -450 138 tagcaggaaaacaTgtccc* /
[GC]-cccttgggattcaatc
48oC 45 s,
70oC 1 min
30 - 80 4 h
Dp427m - III -588 - -198 390 [GC]-tagcaggaaaacatgtccc /
cagtggacatagtacttgc
54oC 45 s,
72oC 1 min
15 - 60 4 h
Dp427m - IV -239 - 50 288 [GC]- ttgtgtttagaaggc /
AGTGAGTGATCCCAACAC
54oC 45 s,
72oC 1 min
30 - 80 4 h
Dp427m - V 1 - 324 324 TCCTGGCATCAGTTACTGTG /
[GC]-ctaaacgttatgccacag
48oC 45 s,
72oC 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;

Denaturation was at 94oC.



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