Linkage analysis with multiplexed short tandem repeat polymorphisms using infrared fluorescence and M13 tailed primers
- Univ. of Minnesota, Minneapolis (United States); and others
The use of short tandem repeat polymorphisms (STRPs) as marker loci for linkage analysis is becoming increasingly important due to their large numbers in the human genome and their high degree of polymorphism. Fluorescence-based detection of the STRP pattern with an automated DNA sequencer has improved the efficiency of this technique by eliminating the need for radioactivity and producing a digitized autoradiogram-like image that can be used for computer analysis. In an effort to simplify the procedure and to reduce the cost of fluorescence STRP analysis, we have developed a technique known as multiplexing STRPs with tailed primers (MSTP) using primers that have a 19-bp extension, identical to the sequence of an M13 sequencing primer, on the 5{prime} end of the forward primer in conjunction with multiplexing several primer pairs in a single polymerase chain reaction (PCR) amplification. The banding pattern is detected with the addition of the M13 primer-dye conjugate as the sole primer conjugated to the fluorescent dye, eliminating the need for direct conjugation of the infrared fluorescent dye to the STRP primers. The use of MSTP for linkage analysis greatly reduces the number of PCR reactions. Up to five primer pairs can be multiplexed together in the same reaction. At present, a set of 148 STRP markers spaced at an average genetic distance of 28 cM throughout the autosomal genome can be analyzed in 37 sets of multiplexed amplification reactions. We have automated the analysis of these patterns for linkage using software that both detects the STRP banding pattern and determines their sizes. This information can then be exported in a user-defined format from a database manager for linkage analysis. 15 refs., 2 figs., 4 tabs.
- OSTI ID:
- 437162
- Journal Information:
- Genomics, Journal Name: Genomics Journal Issue: 3 Vol. 30; ISSN 0888-7543; ISSN GNMCEP
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
INCLUDING NUCLEAR AND PARTICLE DETECTORS
55 BIOLOGY AND MEDICINE
BASIC STUDIES
AUTOMATION
BANDING TECHNIQUES
COST
DATA BASE MANAGEMENT
DETECTION
DNA SEQUENCING
EFFICIENCY
ELECTROPHORESIS
FLUORESCENCE
GENE MUTATIONS
GENES
GENETIC MAPPING
HUMAN CHROMOSOMES
MULTIPLEXERS
OLIGONUCLEOTIDES
POLYMERASE CHAIN REACTION
RELIABILITY
RFLPS
STATISTICS