Reuse of Denaturing Polyacrylamide Gels for Short Tandem Repeat Analysis

Abstract

Denaturing polyacrylamide gel electrophoretic analysis of amplified polymorphic short tandem repeat (STR) loci using fluorescent markers is a mainstay of forensic and paternity testing. To reduce the drawback of preparing gels or using expensive precast gels, we have developed a simple and rapid method to reuse gels between 2 and 8 times over a period of several days. Following the initial electrophoresis and scan, the original samples are removed from the gel by a 1-1.5-h reverse-electrophoresis step. This step heats the gel for the next set of samples and can be performed several days after the initial electrophoresis. Sample bands remain sharp on subsequent runs, but edge effects (frowning of the outside lanes) become progressively worse and ultimately limit gel reuse. Well distortions and separation of the gel from the plates become problems if the gel is used more than twice. However, degassing the gel solution and bonding the gel to both plates eliminate these problems. Precast gels also can be used multiple times. Using this technique, we have successfully analyzed samples amplified with a nine-locus multiplex system and characterized the separated products using a fluorescent scanner and software.

Development of Ligase-Assisted Spacer Addition for the Measurement of Microsatellites

“…One commonly used method for detecting length variability consists of resolving PCR-amplified alleles on 6% denaturing polyacrylamide sequencing gels and visualization by autoradiography, either by PCR primers end-labeled with radionucleotides or radioactively labeled deoxynucleotides incorporated during amplification. Several modifications have aimed to simplify this procedure, including alternative staining methods (7,17,22,24), modified gel matrices to increase the level of resolution (9), and the recycling of polyacrylamide gel electrophoresis (PAGE) gels to reduce the cost of reagents and time (23).…”