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Fig. 2. Six percent PAGE analysis of PCR amplified COL9A1 and COL9A2 cDNA.
Total RNA was isolated from a transformed lymhoblastoid cell line established from a
patient with MED and reverse transcribed into cDNA with both oligo-dt and pd(N) 6
primers as described. Lane 1 , COL9A2 cDNA PCR-amplified by primers 9a2ex3-F
(5'-aca atg ggc ccc ctg gaa aag c-3') and 9a2ex10-R (5'-cct tcc aga ccc tgg atg gtt-3')
(348 bp). Lane 2, COL9A1 cDNA PCR-amplifed by primers 9a2ex8-F (5'-cct gga gtt
cca ggc atc gat-3') and 9a1ex16-R (5'gga tct cca tca tga aag cca a-3') (380 bp). M ,
molecular weight marker (2 Kb). These PCR-amplifed cDNA fragments encode
equivalent regions of the COL3 domain of the
α
1(IX) and
α
2(IX) chains (19) .
Fig. 3. Silver-stained 6% polyacrylamide gel showing SSCP analysis of PCR
amplified genomic DNA from exon 13 of the COMP gene. Lanes 1-4 , Four different
patients with pseudoachondroplasia, the black arrows indicate the position of abnor-
mally migrating single stranded DNA in patients 1 and 4. (For this particular DNA
fragment, the single strands migrate at about 300-bp.) Sequence analysis of clones
confirmed patients 1 and 4 were heterozygous for COMP gene mutations.
5. Stain gel for 15 min in 300 mL of gel-staining solution.
6. Wash the gel twice in dH 2 O for 5 min.
7. Develop for 10-20 min in 150 mL developing solution.
8.
Terminate developing by soaking gel in 150 mL of stop solution for 5 min.
9.
Dry gel onto 3M Whatman paper or alternatively use a gel drying kit ( Fig. 3 ).
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