Screening for Mutations in Cartilage ECM Genes - Methods in Molecular Biology: Extracellular Matrix Protocols - page 127

encoding cartilage oligomeric matrix protein (COMP) have been shown to
result in PSACH and some forms of MED (5-6,14-16) . All of the COMP
mutations reported to date have been found in exons, which encode the
type III repeats, and C-terminal domains. MED can also result from muta-
tions in the gene encoding the 2 chain of type IX collagen ( COL9A2 ) (4 , 17)

and there is evidence that mutations in the genes encoding the

α

α 1(IX)

( COL9A1 ) and 3(IX) ( COL9A3 ) chains of type IX collagen (19) may also

result in MED phenotypes.

α

1.3. Ectopic (Illegitimate) Transcription
To complement mutation screening in genomic DNA by SSCP-analysis, we
have used cultured patient skin fibroblast and transformed lymphoblastoid cell
lines as a source of mRNA. Under normal culture conditions these cell lines
can maintain a low basal transcription (“ectopic” or “illegitimate” transcrip-
tion) of cartilage specific genes (20-22) . Using such cell lines it is possible to
amplify by the polymerase chain reaction (PCR), cDNA from the COMP,
COL9A1 , COL9A2 , and COL9A3 genes for direct mutation screening using
DNA sequencing.

2. Materials
2.1. RNA and DNA Isolation
1. Trizol (Gibco-BRL, Gaithersburg, MD).

2. 99.7-100% v/v ethanol, chloroform (AnalaR-BDH, London, UK).

3. Isopropanol, diethyl pyrocarbonate (DEPC) (Sigma, St. Louis, MO).

4. QIAamp Blood Midi/Maxi Kit and QIAamp Tissue Kit (Qiagen, Chatsworth, CA).

2.2. Reverse Transcription of mRNA and Amplification of cDNA
and Genomic DNA by the Polymerase Chain Reaction (PCR)

1. Superscript™ Preamplification system for first strand cDNA synthesis (Gibco-BRL).

2. Taq DNA polymerase.

3. 100 m M solutions of dCTP, dATP, dGTP, and dTTP (Boehringer-Mannheim,

Mannheim, Germany).

4. Mineral oil (Sigma).

5. Oligonucleotide (PCR) primers.

2.3. Polyacrylamide and SSCP Gel Analysis
1.

Protean II xi cell (20 cm × 20 cm) gel electrophoresis equipment (Bio-Rad).

2. DNA loading buffer: 30% glycerol (AnalaR-BDH), 0.25% bromophenol blue

(Sigma), 0.25% xylene cyanol (Sigma) in 10 m M Tris-HCl, pH 7.5 (Gibco-BRL).

3. Denaturing DNA loading buffer: 95% Formamide (Sigma), 10 m M NaOH

(AnalaR-BDH), 0.25% bromophenol blue (Sigma), 0.25% xylene cyanol (Sigma)
in 10 m M Tris-HCl, pH 7.5 (Gibco-BRL).