General Technique
Micro-satellite genotyping is performed using two laser DNA Analyzer 4200 sequencing machines from LI-COR Inc. In most cases, Polymerase Chain Reaction (PCR) primers and PCR conditions are available in public repositories. Otherwise, PCR assays are developed and optimized. Primers with infrared dyes attached are used to label the PCR products. Since the length of the micro-satellite allele is reflected in the size of the PCR product, alleles can be distinguished by size separating the PCR products with gel electrophoresis. Alleles are then identified by SAGA, an allele calling software with sizing accuracy of 0.2 base pairs.
Five to 10 ng of DNA are dispensed into 96-well plates using a Hydra robot, vacuum dried and stored at 4 degrees C until use. The PCR mix is added and the samples are cycled (35 times at 94 degrees C for 15s, 50 degrees C -58 degrees C for 15s and 72 degrees C for 15s) using a Peltier Thermal Cycler (MJ Research). Markers are optimized for annealing temperature and magnesium concentration. Each reaction contains 10 ng of DNA, infrared-labeled primers (0.2μM each), 50mM KCl, 10mM Tris, 1-2.5 mM MgCl2, 0.5 units of Taq. Up to five markers can be pooled after PCR using a liquid handling robot and filtered through G-75 Sephadex in a 96 well filter plate. Samples are loaded onto LI-COR 4200 sequencing machines for electrophoresis.
After electrophoresis, the gel image is evaluated and sent to a UNIX workstation for allele calling. Our in-house genotyping software (SAGA) is used to call alleles in a batch mode to allow gels to go from the raw state to the ´called´ state without any user interaction. Upon completion of allele calling, results can be checked manually. This is facilitated by simultaneous display of an autoradiograph-like image and the extracted chromatogram view. Calls are then exported to the SBEAMS database genotyping module where data is stored, curated and used to construct input files for analysis.
Purpose/use/application of the technique:
Short tandem repeat (STR) or micro-satellite markers have been extensively used in linkage mapping of human diseases. Until recently, they have been the DNA marker of choice for conducting Genome Scans due to their high heterogeneity and ease to genotype using a conjunction of PCR and gel electrophoresis. STRs are scattered throughout the genome in abundance. One of the first efforts of the human genome project was to identify and map at least one STR marker per centimorgan (cM). Currently, more than ten thousand STRs have been mapped at high density to produce genetic maps.
Example(s) of projects at ISB that use this technique:
- Linkage mapping of hereditary prostate cancer susceptibility
Representative publication(s):
Janer M , Fredrichsen D, Stanford J-L, Badzioch M, Kolb S, Deutsch K, Peters M , Goode E L, Welti R, DeFrance H B, Iwasaki L, Li S, Hood L, Ostrander E, and Jarvik G. Genomic Scan of 254 hereditary prostate cancer families. Prostate, 57(4): 309-19, 2003..
Friedrichsen DM, Stanford JL, Isaacs SD, Janer M, Chang BL, Deutsch K, Gillanders E, Kolb S, Wiley KE, Badzioch MD, Zheng SL, Walsh PC, Jarvik GP, Hood L, Trent JM, Isaacs WB, Ostrander EA, Xu J. Identification of a prostate cancer susceptibility locus on chromosome 7q11-21 in Jewish families.
Proc Natl Acad Sci U S A. 2004 Feb 17;101(7):1939-44
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