Leiden Muscular Dystrophy data pages©

Protein Truncation Test (PTT) for DMD

(last modified on August 02, 2011)


For PTT analysis of other disease genes see;


Contents

Several sets of primers have been published to scan the entire DMD coding sequence for premature translation terminating mutations on RNA using the Protein truncation Test (PTT). The Leiden, the Roberts and the Whittock sets of primers are most frequently used. Other primers for RT-PCR amplification of DMD RNA can be found here, incl. those used in combination with direct sequencing (point mutation detection) and those used for specific amplification of transcripts in other tissues.


The LEIDEN-set:

Originally, this set was divided in five primary PCR sets (using primers A+B), which were each split in two during the nested secondary PCR, i.e. primers C+D and E+F resp. (Roest et al.). Consequently, 10 in vitro transcription/translation reactions (i.e. 1-CD to 5-EF) are required to scan the coding sequence for translation terminating mutations. Mutation scanning in 5 sets can be performed when primers C+F are combined in the secondary PCR. Currently, the Leiden group uses smaller segments for the primary PCR, i.e. primers A+H and G+B. This improves the reliability of the PCR (Roest et al.; Hogervorst et al., unpublished).

Reference: Roest et al., Hogervorst et al., unpublished.

PTT primer sequences LEIDEN-set

Set Exons PCR Name Localization Fragment PTT Sequence Reference
1   1st PCR 1h 257-280 1,345   CAAAAGAAAACATTCACAAAATGG (Hogervorst, unpl.)
      1a 1601-1578     TTAGCCAGTCATTCAACTCTTTCA (Hogervorst, unpl.)
  2-11 2nd PCR 1d 295-314 1,258   gc-[T7]-TCTAAGTTTGGGAAGCAGCA (Hogervorst, unpl.)
      1c 1511-1493     TGAGGCATTCCCATCTTGA (Hogervorst, unpl.)
    1st PCR 1b 1091-1111 1,350   CGATTCAAGAGCTATGCCTAC (Hogervorst, unpl.)
      1g 2440-2419     ACTCTGCAACACAGCTTCTGAG (Hogervorst, unpl.)
  10-18 2nd PCR 1f 1292-1312 1,174   [T7]-TTGCAAGCACAAGGAGAGATT (Hogervorst, unpl.)
      1e 2426-2405     CTTCTGAGCGAGTAATCCAGCT (Hogervorst, unpl.)
2   1st PCR 2h 2276-2297 1,327   cggatccACAAGGGAACAGATCCTGGTAA (Hogervorst, unpl.)
      2a 3594-3617     GTCTCAAGTCTCGAAGCAAACTCT (Hogervorst, unpl.)
  17-25 2nd PCR 2d 2342-2364 1,244   gc-[T7]-AGGCAGATTACTGTGGATTCTGA (Hogervorst, unpl.)
      2c 3544-3524     CCCACCTTCATTGACACTGTT (Hogervorst, unpl.)
    1st PCR 2b 3281-3299 1,538   ATTGAGGGACGCTGGAAGA (Hogervorst, unpl.)
      2g 4810-4787     cgggatcCTGCTTTTTCTGTACAATCTGACG (Hogervorst, unpl.)
  23-32 2nd PCR 2f 3320-3342 1,411   gc-[T7]-GAGCATTGTCAAAAGCTAGAGGA (Hogervorst, unpl.)
      2e 4689-4667     TCCACACTCTTTGTTTCCAATG (Hogervorst, unpl.)
3   1st PCR 3h 4365-4388 1,533   TCACATTCATTGACAAGCAGTTGG* (Hogervorst, unpl.)
      3a 5897-5876     CAATGTCATCCAAGCATTTCAG (Hogervorst, unpl.)
  31-38 2nd PCR 3d 4517-4537 1,093   gc-[T7]-GCCCAAAGAGTCCTGTCTCA (Hogervorst, unpl.)
      3c 5568-5545     TTAAACTGCTCCAATTCCTTCAA (Hogervorst, unpl.)
    1st PCR 3b 5281-5301 1,473   CACAAAGTGGATCATTCAGGC (Hogervorst, unpl.)
      3g 6745-6723     gcgaattcACTGGCATCTGTTTTTGAGGAT (Hogervorst, unpl.)
  36-45 2nd PCR 3f 5300-5318 1,450   [T7]-GCTGACACACTTTTGGATG (Hogervorst, unpl.)
      3e 6710-6691     CTTCCCCAGTTGCATTCAAT (Hogervorst, unpl.)
4   1st PCR 4h 6404-6424 1,463   GCAACGCCTGTGGAAAGGGTG (Hogervorst, unpl.)
      4a 7866-7844     CGATCCGTAATGATTGTTCTAGC (Hogervorst, unpl.)
  44-52 2nd PCR 4c 6575-6601 1,308   [T7]-GCTGAACAGTTTCTCAGAAAGACACAA (Hogervorst, unpl.)
      4c 7843-7820     CTCTTGATTGCTGGTCTTGTTTT (Hogervorst, unpl.)
    1st PCR 4b 7489-7510 1,543   AGCTCCTGGACTGACCACTATT (Hogervorst, unpl.)
      4g 9031-9008     CTCTTGAAGTTCCTGGAGTCTTTC (Hogervorst, unpl.)
  51-59 2nd PCR 4f 7643-7663 1,351   [T7]-TGGACAGAACTTACCGACTGG (Hogervorst, unpl.)
      4e 8954-8933     CCCACTCAGTATTGACCTCCTC (Hogervorst, unpl.)
5   1st PCR 5h 8580-8603 1,986   AAAAGTCTCTCAACATTAGGTCCC* (Hogervorst, unpl.)
      5a 10565-10544     ATCCATTGCTGTTTTCCATTTC (Hogervorst, unpl.)
  58-68 2nd PCR 5d 8827-8846 1,383   gc-[T7]-ACAGAGCAGCCTTTGGAAG (Hogervorst, unpl.)
      5c 10168-10146     TGGACACTCTTTGCAGATGTTAC (Hogervorst, unpl.)
    1st PCR 5b 9435-9456 ,1926   ACGAGACTCAAACAACTTGCTG (Hogervorst, unpl.)
      5g 11352-11328     gcgaattcTATTCTGCTCCTTCTTCATCTGTC (Hogervorst, unpl.)
  67-79 2nd PCR 5f 9953-9972 1,381   gc-[T7]-GGTGAAGTTGCATCCTTTGG (Hogervorst, unpl.)
      5e 11292-11272     ATCATCTGCCATGTGGAAAAG (Hogervorst, unpl.)

Legend:

*Erroneous primer sequences corrected with the help of Lara Broderick. Set: number amplified segment of the dystrophin coding sequence. Exons: exons amplified, numbering according to Roberts et al.. PCR: 1st (first) or 2nd (second) round PCR. Name: name of primer. ExX-T7 was designed to avoid amplification of exon X (and a premature stop codon). Localization: localization of the primer in relation to the dystrophin cDNA sequence (GenBank accession M18533, Koenig et al.). Length: length amplified fragment (in base pairs). PTT: length of translation product (in kiloDalton). Sequence: primer sequence, [T7] = 5'-ggatcctaatacgactcactataggaacagaccaccATG-3'. Reference: publication describing primer sequences. 


The ROBERTS-set:

This set of primers is split into 10 segments together covering the dystrophin coding region (Roberts et al.). The location of the primers has been modified to increase product yield and to decrease the number of products obtained, i.e. primers were chosen in exons showing differential splicing (Gardner et al.).

Reference: Roberts, Gardner.

PTT primer sequences ROBERTS-set

Set Exons PCR Name Localization Length PTT Sequence Reference
1   1st PCR DMD1a 13-33 1,206   CTTTCCCCCTACAGGACTCAG Roberts
      DMD1b 1218-1199     CTCTCCATCAATAGAACTGCC Roberts
  2-10 2nd PCR DMD1c 212-232 1,003   [T7]-CTTTGGTGGGAAGAAGTAGAG Gardner
      DMD1d 1175-1154     CCAAATGCTGTGAAGGAAATGG Gardner
2   1st PCR DMD2a 1091-1111 1,329   CGATTCAAGAGCTATGCCTAC Roberts
      DMD2b 2419-2399     GCGAGTAATCCAGCTGTGAAG Roberts
  9-18 2nd PCR DMD2c 1133-1153 1,303   [T7]-ACCTCTGACCCTACACGGAGC Gardner
      DMD2d 2396-2377     CAGTTATATCAACATCCAAC Gardner
3   1st PCR DMD3a 2300-2319 1,320   CATGCTCAAGAGGAACTTCC Roberts
      DMD3b 3619-3599     CTGAGTGTTAAGTTCTTTGAG Roberts
  17-25 2nd PCR DMD3c 2342-2362 1,292   [T7]-AGGCAGATTACTGTGGATTCTG Gardner
      DMD3d 3594-3574     GTCTCAAGTCTCGAAGCAAAC Gardner
4   1st PCR DMD4a 3507-3526 1,346   CAATTCAGCCCAGTCTAAAC Roberts
      DMD4b 4852-4832     CAAAGCTGTTACTCTTTCATC Roberts
  25-33 2nd PCR DMD4c 3527-3547 1,323   [T7]-AGTGTCAATGAAGGTGGGCAG Gardner
      DMD4d 4810-4790     CTGCTTTTTCTGTACAATCTG Gardner
5   1st PCR DMD5a 4740-4760 1,199   GTCTGAGTGAAGTGAAGTCTG Roberts
      DMD5b 5938-5919     CCTTTCATCTCTGGGCTCAG Roberts
  33-38 2nd PCR DMD5c 4763-4785 924   [T7]-GTGGAAATGGTGATAAAGACTGG Gardner
      DMD5d 5647-5627     ATTGAAGTCTTCCTCTTTCAG Gardner
6   1st PCR DMD6a 5823-5842 802   CTCTAGAAATTTCTCATCAG Roberts
      DMD6b 6624-6605     GCATGTTCCCAATTCTCAGG Roberts
  37-44 2nd PCR DMD6c 5528-5548 1,095   [T7]-GGAAAGGCCTCCATTCCTTTG Gardner
      DMD6d 6583-6564     CTGTTCAGCTTCTGTTAGCC Gardner
7   1st PCR DMD7a 6404-6424 1,311   GCAACGCCTGTGGAAAGGGTG Roberts
      DMD7b 7714-7694     GTCACCCACCATCACCCTCTG Roberts
  43-51 2nd PCR DMD7c 6428-6449 1,289   [T7]-CTACAGGAAGCTCTCTCCCAGC Gardner
      DMD7d 7677-7568     TCAAGCAGAGAAAGCCAGTC Gardner
8   1st PCR DMD8a 7583-7603 1,308   CTAGAAATGCCATCTTCCTTG Roberts
      DMD8b 8890-8871     CTCAGGAGGCAGCTCTCTGG Roberts
  51-58 2nd PCR DMD8c 7616-7636 1,296   [T7]-CCTGCTCTGGCAGATTTCAAC Gardner
      DMD8d 8872-8852     GGGCTCCTGGTAGAGTTTCTC Gardner
9   1st PCR DMD9a 8754-8773 1,317   GGGCCTTCAAGAGGGAATTG Roberts
      DMD9b 10070-10051     CCAGTCTCATCCAGTCTAGG Roberts
  58-68 2nd PCR DMD9c 8786-8807 1,300   [T7]-CCTGTAATCATGAGTACTCTTG Gardner
      DMD9d 10046-10027     GGGCCGCTTCGATCTCTGGC Gardner
10   1st PCR DMD10a 9953-9972 1,376   GGTGAAGTTGCATCCTTTGG Roberts
      DMD10b 11328-11308     CATGACTGATACTAAGGACTC Roberts
  67-79 2nd PCR DMD10c 9971-9993 1,332   [T7]-GGGGGCAGTAACATTGAGCCAAG Gardner
      DMD10d 11263-11243     CATTGTGTCCTCTCTCATTGG Gardner
11   1st PCR DMD11a 11185-11206 2,102   GATGGAGCAACTCAACAACTCC Gardner
      DMD11b 13286-13267     GTCAGAGGTAACAGATTTGC Gardner
  77-79 2nd PCR DMD11c 11214-11234 1,646   [T7]-GTTCAAGAGGAAGAAATACCC Gardner
      DMD11d 12859-12838     GTAACATTTGAATCAATTTGCC Gardner

Legend:

Set: number amplified segment of the dystrophin coding sequence. Exons: exons amplified, numbering according to Roberts et al.. PCR: 1st (first) or 2nd (second) round PCR. Name: name of primer. Localization: localization of the primer in relation to the dystrophin cDNA sequence (GenBank accession M18533, Koenig et al.). Length: length amplified fragment (in base pairs). PTT: length of translation product (in kiloDalton). Sequence: primer sequence, [T7] = 5'-ggatcctaatacgactcactataggaacagaccaccATG-3'. Reference: publication describing primer sequences. 


The WHITTOCK multiplex set

This set of primers is split into 5 segments together covering the entire dystrophin coding region (Whittock et al.). The primers haven been selected such that the RT-PCR and translation products can be clearly recognized as independent fragments. To enable all PCR-reactions to be performed under one set of conditions, primers were designed to have a melting temperature of at least 70°C. PCR fragments differ in length between 1.6 and 3.7 kb, translation products between 66 and 151 kDa.

Reference: Whittock et al.

PCR-conditions

PTT primer sequences WHITTOCK-set (multiplex)

Set Exons PCR Name Localization Length PTT Sequence Referenceg
1   1st PCR NW1a 7-33 3,511   CCCTCACTTTCCCCCTACAGGACTCAG Whittock
      NW1b2 3517-3491     GGGCTGAATTGTCTGAATATCACTGAC Whittock
  1-23 2nd PCR NW1c 212-238 3,272 132 [T7]-CTTTGGTGGGAAGAAGTAGAGGACTGT Whittock
  2-23   ExX-T7 242-268 3,242 132 [T7]-GAAAGAGAAGATGTTCAAAAGAAAACA Whittock
      NW1d2 3483-3457     CTGCACTGTTTCAGCTGCTTTTTTAGA Whittock
2   1st PCR NW2a 2293-2319 2,871   GGTAAAGCATGCTCAAGAGGAACTTCC Whittock
      NW2b 5163-5137     TTATCTTCCACCAACGTCTCCTTCTTG Whittock
  17-35 2nd PCR NW2c 2342-2368 2,772 112 [T7]-AGGCACATTACTGTGGATTCTGAAATT Whittock
      NW2d 5113-5086     CCTACCTCTGTGATACTCTTCAGGTGC Whittock
3   1st PCR NW3a 4734-4760 2,178   ATAAAAGTCTGAGTGAAGTGAAGTCTG Whittock
      NW3b 6911-6885     TAGCAATGTTATCTGCTTCCTCCAACC Whittock
  33-45 2nd PCR NW3c 4763-4789 2,051 83 [T7]-GTGGAAATGGTGATAAAGACTGGACGT Whittock
      NW3d 6813-6787     CTGTCTGACAGCTGTTTGCAGACCTCC Whittock
4   1st PCR NW4a 6398-6424 1,712   CAAAGTGCAACGCCTGTGGAAAGGGTG Whittock
      NW4b2 8109-8083     TGCCACTGGCGGAGGTCTTTGGCCAAC Whittock
  43-53 2nd PCR NW4c 6428-6454 1,635 66 [T7]-CTACAGGAAGCTCTCTCCCAGCTTGAT Whittock
      NW4d2 8062-8036     CTTTTGGATTGCATCTACTGTATAGGG Whittock
5   1st PCR NW5a2 7487-7513 3,842   CTAGCTCCTGGACTGACCACTATTGGA Whittock
      NW5b 11328-11302     CATGACTGATACTAAGGACTCCATCGC Whittock
  51-79 2nd PCR NW5c2 7520-7546 3,744 151 [T7]-CCTACTCAGACTGTTACTCTGGTGACA Whittock
      NW5d 11263-11237     CATTGTGTCCTCTCTCATTGGCTTTCC Whittock

Legend:

Set: number amplified segment of the dystrophin coding sequence. Exons: exons amplified, numbering according to Roberts et al.. PCR: 1st (first) or 2nd (second) round PCR. Name: name of primer. ExX-T7 was designed to avoid amplification of exon X (and a premature stop codon). Localization: localization of the primer in relation to the dystrophin cDNA sequence (GenBank accession M18533, Koenig et al.). Length: length amplified fragment (in base pairs). PTT: length of translation product (in kiloDalton). Sequence: primer sequence, [T7] = 5'-ggatcctaatacgactcactataggaacagaccaccATG-3'. Reference: publication describing primer sequences.


PCR-conditions Whittock set


(PCR-conditions have been taken from Whittock et al.)

Reverse transcription: perform five individual reverse transcription amplification (RT- PCR) reactions. Add 500-1000 ng total RNA (from muscle or peripheral blood leucocytes) to 10 ng reverse primer in a total volume of 5 ul overlaid with 50 ul mineral oil. Incubate 10 min 65oC and snap chill on ice. Add 6 ul pre-mix (containing 2 ul 5xRT-buffer [Promega], 10 nmol each dNTP, 200 units M-MLV reverse transcriptase [Promega], 40 units RNasin [Promega]). Incubate 60 min 42oC. Incubate 3 min 94oC.

First round PCR: add to RT-reaction pre-mix-2 (containing 5 ul 10xPCR-buffer [600 mM KCl, 200 mM Tris.HCl pH 8.8, 15 mM MgCl2], 20 pmol forward primer, 20 pmol reverse primer) and H2O to a total volume of 50 ul. Incubate 2 min 94oC. Add 2.5 units Taq Plus LongTM (Stratagene). Incubate 30 cycles of 10 sec 94oC / 30 sec 72oC / 4 min 72oC. Incubate 5 min 72oC.

Second round PCR: add 2 ul first round PCR-product to 48 ul pre-mix-3 (containing 5 ul 10xPCR-buffer, 10 nmol each dNTP, 20 pmol forward and 20 pmol reverse primer). Incubate 2 min 94oC. Add 2.5 units Taq Plus LongTM (Stratagene). Incubate 30 cycles of 10 sec 94oC / 30 sec 72oC / 3 min 72oC. Incubate 5 min 72oC.

Analysis: 6 ul PCR-product is analysed on agarose gel. For translation second round PCR-products were mixed in a 4:1:1:1:4 ratio (fragments 1:2:3:4:5 resp.) and translated in a total volume of 12.5 ul TNTTM T7-coupled reticulocyte lysate (Promega) with 1.5 mM MgCl2 at 90 min 30oC.



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