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;

• PTT for Emery-Dreifuss Muscular Dystrophy (EMD)
• Protein Truncation Test (PTT)
• Leiden-protocol for non-radioactive PTT

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.

• Leiden
• Roberts
• Whittock (multiplex PTT, incl PCR conditions)

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the primary PCR, i.e. primers A+H and G+B. This improves the reliability of the PCR (Roest et al.; Hogervorst et al., unpublished).

Reference: Roest et al., Hogervorst et al., unpublished.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

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

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)

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 65^oC 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 42^oC. Incubate 3 min 94^oC.

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 94^oC. Add 2.5 units Taq Plus Long^TM (Stratagene). Incubate 30 cycles of 10 sec 94^oC / 30 sec 72^oC / 4 min 72^oC. Incubate 5 min 72^oC.

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 94^oC. Add 2.5 units Taq Plus Long^TM (Stratagene). Incubate 30 cycles of 10 sec 94^oC / 30 sec 72^oC / 3 min 72^oC. Incubate 5 min 72^oC.

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 TNT^TM T7-coupled reticulocyte lysate (Promega) with 1.5 mM MgCl₂ at 90 min 30^oC.

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