Screening for Mutations in Cartilage ECM Genes - Methods in Molecular Biology: Extracellular Matrix Protocols - page 133

Fig. 1. Six percent PAGE-analysis of PCR amplified COMP cDNA. Total RNA
was isolated from a skin fibroblast cell line established from a patient with pseudo-
achondroplasia and reverse transcribed into cDNA with both oligo-dt and pd(N) 6
primers as described. Lane 1, COMP C-terminal domain cDNA PCR-amplified by
primers HCt-F and HCt-R (702 bp). Lane 2, COMP Type III repeat domain cDNA
PCR-amplified by primers HT3-F and HT3-R (789 bp). M, molecular weight marker.
A white arrow indicates a putative heteroduplex. Sequence analysis of clones
confirmed that this patient was heterozygous for a COMP gene mutation.

a. 94 ° C for 1 min (template denaturation).

b. 55 ° C for 1 min (primer annealing).

c. 72 ° C for 1 min (template elongation).

2. After cycle is complete, remove 15 L of DNA loading

buffer and analyze on a 6% polyacrylamide gel in 1X TBE buffer ( see Note 3 ).
Stain the gel with ethidium bromide (10

µ L of PCR product, add 5 µ

µ g/mL) and view DNA with an UV

transilluminator ( Fig. 1 and Fig. 2 ).

3.2.2. SSCP Analysis of Genomic DNA Amplified by PCR
1.

Remove 2 µ L of PCR product to a fresh tube, add 10 µ L of denaturing DNA

loading buffer and heat samples to 95 ° C for 2 min. Place denatured DNA on ice

for 2 min.

2. Load sample onto a 6% polyacrylamide gel precooled to 4 ° C and run at 300 V for

3-4 h ( see Note 4 ).

3. After the run is complete, separate the plates gently, remove the gel, and place in

500 mL of gel-fixative solution for 20 min.

4. Wash the gel twice in dH 2 O for 5 min.