Fluorescent detection of Southern blots and PCR-based genetic typing tests
- Molecular Dynamics, Inc., Sunnyvale, CA (United States)
- Laboratory of Parasitic Diseases, Bethesda, MD (United States); and others
The Southern blot is used to study gene organization, to identify disease-causing genomic rearrangements, or for typing RFLP markers in forensic, paternity, or prenatal diagnostic testing. Fluorescence offers a much greater dynamic range and a more linear response than film used in radioactive or chemiluminescent detection of RFLPs. We therefore investigated using the Fluorimager{trademark} 575 (Molecular Dynamics, Inc.) for analyzing Southern blots. Using a single-locus probe to D2S44 (YNH24) (Promega Corp.), we detect as little as 100 ng (0.05 attomole) genomic DNA. The alkaline phosphatase-labeled probe is detected using AttoPhos (JBL Scientific), and the developed membrane is scanned with the Fluorimager. Biotinylated hybridization probes can also be developed using a streptavidin-alkaline phosphatase conjugate and AttoPhos. The instrument scan parameters can be adjusted to prevent overexposure and accompanying loss of resolution in images of blots, gels, or 96-well microplates. We have used these other sample formats in PCR-based genetic typing assays. We use FluorKit DQS (Molecular Dynamics) to accurately quantify PCR template DNA (1-500 ng) in 96-well microplates scanned using the same instrument. Mutation detection assays run include heteroduplex gels (5% polyacrylamide, 2.7 M urea), short tandem repeat (STR) markers, amplified fragment length polymorphisms (AmpFLP), competitive priming PCR, and allele-specific oligotyping. These assays are run using either 1- or 2-color labeling. We detect unlabeled PCR products, such as the AmpFLP marker D1S80 (Perkin-Elmer) by post-staining gels for 10 minutes with SYBR Green 1 (Molecular Probes) and scanning the wet gel. The Fluorimager scans a 20 x 25 cm sample within three minutes, allowing rapid optimization of fluorescent protocols and high sample throughput.
- OSTI ID:
- 134069
- Report Number(s):
- CONF-941009-; ISSN 0002-9297; TRN: 95:005313-0805
- Journal Information:
- American Journal of Human Genetics, Vol. 55, Issue Suppl.3; Conference: 44. annual meeting of the American Society of Human Genetics, Montreal (Canada), 18-22 Oct 1994; Other Information: PBD: Sep 1994
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
BASIC STUDIES
44 INSTRUMENTATION
INCLUDING NUCLEAR AND PARTICLE DETECTORS
GENES
STRUCTURE-ACTIVITY RELATIONSHIPS
GENE MUTATIONS
DETECTION
GENOME MUTATIONS
DNA
PROBES
DNA HYBRIDIZATION
EVALUATION
STRUCTURAL CHEMICAL ANALYSIS
FLUORESCENCE
RESOLUTION
EFFICIENCY
POLYMERASE CHAIN REACTION
RFLPS
DIAGNOSTIC TECHNIQUES
BIOLOGICAL MARKERS
BIOASSAY