High throughput visualization and analysis of human short tandem repeat polymorphisms (STRP`s) using an infrared based automated DNA sequencer
- LI-COR, Inc., Lincoln, NE (United States); and others
Short tandem repeat polymorphisms (STRPs) including di-, tri-, and tetranucleotide repeats are very useful markers for gene mapping, genetic diagnosis, and forensics because they are highly polymorphic, abundant throughout the mammalian genome, and amplifiable by the polymerase chain reaction (PCR). In order to meet the demand for large scale gene mapping and genetic diagnosis, the overall speed of genotyping STRPs should be increased. Automated detection systems using laser irradiation along with infrared fluorescently labeled PCR primers or dATP`s have been used for improved detection of PCR amplified STRPs when compared to conventional radioactive detection methods. Here, we report protocols that provide high throughput visualization and analysis of PCR amplified STRPs using the LI-COR Model 4000S DNA Sequencer. Short (15 cm separation distance) gels were used which produced rapid migration of the DNA fragments to the scanning detector (less than 1 hour from sample loading to detection of up to 350 base long DNA fragments). Seven percent denaturing acrylamide gels with a constant 2000V were employed for fast and adequate resolution. A 64 well format was used for loading (60 samples with 4 lanes for standard markers). Multiple loading of samples (using the same gel up to 3 times) has been achieved. In addition, we have multiplexed more than one locus per lane. By applying these conditions (60 samples x 3 loci x loads/gel x 2 gels/day) it is possible to generate images for over 1000 person loci in less than a day. Images were analyzed using Scanalytics RFLPscan software. The output data gave each allele size in number of base pairs.
- OSTI ID:
- 134035
- Report Number(s):
- CONF-941009-; ISSN 0002-9297; TRN: 95:005313-0771
- 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
DNA SEQUENCERS
AUTOMATION
EFFICIENCY
RESOLUTION
NUCLEOTIDES
STRUCTURAL CHEMICAL ANALYSIS
GENOTYPE
GENES
GENETIC MAPPING
DETECTION
SIZE
INFRARED SPECTROMETERS
BIOLOGICAL MARKERS
POLYMERASE CHAIN REACTION
FLUORESCENCE
MULTIPLEXERS