Fig. 1
From: Mutation Detection in Machado-Joseph Disease Using Repeat Expansion Detection
Autoradiograph of RED products from part of a MJD/SCA3 disease family using a (CTG) 10 oligonucleotide
In RED, a thermostable ligase is used to ligate repeat oligonucleotides following annealing at adjacent bases to repeat sequences in genomic DNA. Products are size separated on a denaturing Polyacrylamide gel, electroblotted, and hybridized to a complementary 32P-labeled probe. The longest reaction product corresponds to the longest repeat size present in the genome. Reactions were run as previously (6), with cycling conditions being 94°C for 5 min followed by 396 cycles of either 80°C for 30 sec and 94°C for 10 sec. The lowest band in each lane represents 90 bp and each additional band represents an additional 30 bp. Note RED products with largest band size at 210 bp in affected individuals in generation 1 and 2 whereas the affected offspring in the third generation (fourth lane from left) has a 240-bp product correlating with a larger repeat size. Unaffecteds only show products of 120 bp.