Optimization of DNA extraction from formalin-fixed tissue and its clinical application in Duchenne muscular dystrophy.
The authors report a case of Duchenne muscular dystrophy in which a deletion was determined by multiplex polymerase chain reaction using postmortem tissue from a proband who died in 1985, 2 years before the cloning of the dystrophin gene. Several extraction methods were analyzed to determine optimal conditions for recovery of DNA from fixed tissue. Variables examined were tissue lysis times and temperatures, a simple salting-out procedure for purification of DNA, polymerase chain reaction amplification of crude lysate versus purified DNA, and lysis of different tissues and tissue quantities. Extracted DNA was analyzed spectrophotometrically, electrophoretically, and by its suitability for polymerase chain reaction amplification using exon flanking primers of the dystrophin gene. Lysis at 37 degrees C for less than 24 hours led to the recovery of predominantly low-molecular-weight DNA, which was unsuitable for polymerase chain reaction in this experience. Lysis at 54 degrees C consistently yielded visible, spoolable quantities of high-molecular-weight DNA in as little as 24 hours. Direct amplification of crude, unpurified lysates was unsuccessful. However, purified samples yielded consistent amplification products. The purification step was simplified by substituting a rapid salting-out procedure for organic extractions.
Forsthoefel
K F
KF
Department of Pathology, Ohio State University, Columbus 43210.
Papp
A C
AC
Snyder
P J
PJ
Prior
T W
TW
D000293:Adolescent; D001483:Base Sequence; D002872:Chromosome Deletion; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification*; D005557:Formaldehyde; D006801:Humans; D008969:Molecular Sequence Data; D009136:Muscular Dystrophies / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013696:Temperature; D013997:Time Factors; D016707:Tissue Fixation
D005557:Formaldehyde; D004247:DNA