PTT for Emery-Dreifuss Muscular Dystrophy

(last modified September 24, 2004)

Contents

• Introduction
• A result (with scanned image)
• PTT protocol
  • RNA-isolation
  • reverse transcription and PCR
  • table of primer sequences
  • general Leiden-protocol for non-radioactive PTT

Introduction

The Protein Truncation Test for point mutation detection in the EMD-gene (Emery-Dreifuss Muscular Dystrophy) has been published as;

• de Koning Gans PA, Ginjaar I, Bakker E, Yates JR, den Dunnen JT. (1999). A protein truncation test for Emery-Dreifuss muscular dystrophy (EMD): detection of N-terminal truncating mutations. Neuromuscular Disorders 9(4): 247-250.

EMD entry in OMIM  /  EMD entry LocusLink

PTT protocol

RNA isolation

RNA was purified from peripheral blood lymphocytes or from cultured Epstein-Barr virus transformed lymphoblastoid cell lines following standard procedures{ref}.

Reverse transcription and PCR

One µg total RNA was reverse transcribed in a 60 µl reaction, with 400 U Superscript II RT in 5*First Strand Buffer (Gibco BRL), using 1 µg random hexamer primers (Promega), 0.01M DTT, 1 mM dNTP's and 40 U RNasin at 42ºC for 1 h. 5 µl of the cDNA reaction was used for a first round PCR amplification. PCR conditions: 0.5 µM of each dNTP's, 15 pmol of each primer (Em2F-T7 and Em2R), 0.75mM MgCl2, 10*buffer 3 (Expand long template kit, Boehringer Mannheim), 0.75 µl enzyme mix (Expand long template kit) in 25 µl final volume. PCR was for 2 min. at 94ºC, followed by 35 cycles of 30 sec. at 94ºC, 30 sec. at 64ºC, 1 min. at 72ºC and a final elongation of 7 min. at 72ºC. 0.1 µl of this PCR reaction was used in a second round PCR amplification. PCR conditions: 0.2 µM of each dNTP's, 3.5 pmol of each primer (Em1F-T7 and Em1R-M13), 2 mM MgCl2, 10*buffer (Perkin Elmer), 2 U Taq polymerase (Perkin Elmer) in 50 µl final volume. PCR was for 2 min. at 94 ºC, followed by 28 cycles of 30 sec. at 94ºC, 30 sec. at 64ºC, 1 min. at 72ºC and a final elongation of 7 min. at 72 ºC.

Table of primer sequences

Legend:
Name: laboratory primer name. Location: Location of primer on the emerin cDNA-sequence (accession nr M...., GenBank). Sequence: primer sequence, T7- = ..., M13- = ....

In vitro transcription translation (non radioactive)

General protocol from Leiden including SDS-PAGE analysis of translation products, electro blotting, detection of proteins, interpretation, protein marker, estimation of the part of the PTT fragment to be sequenced and troubleshooting.

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