Comparison of fluorescence-based semi-automated genotyping of multiple microsatellite loci with autoradiographic techniques.
The practical application of highly efficient fluorescence-based methods for the semi-automated genotyping of polymerase chain reaction-based microsatellite markers will depend on the development of robust protocols that provide accurate and reproducible data. In the present report we compare the accuracy of a fluorescence-based protocol with a benchmark radiolabeling method that depends on a known sequence ladder or amplified DNA from reference individuals for sizing by autoradiography. Three microsatellite markers, IGF1 (mfd 1), D4S174 (mfd 59), and D5S211 (mfd 154), with products overlapping in size were each labeled with a different fluorophore and run simultaneously with an internal size standard in a single electrophoretic lane. The size of each allele was compared for these markers by using both techniques for five larger CEPH families (884, 1331, 1332, 1333, and 1362). Of 462 possible alleles, four discrepancies (0.8%) were identified when the two approaches were compared. We conclude that the fluorescence-based protocol is at least as accurate as the standard radiolabeling technique since none of the sizing errors arose as a result of the fluorescence-based technique. We describe the adaptation of this fluorescence-based protocol to the simultaneous analysis of up to 24 microsatellite loci per electrophoretic lane. These highly accurate and efficient semi-automated techniques will be useful in high-resolution genomic analyses.
Department of Anesthesiology and Critical Care Medicine, Johns Hopkins Medical Institutions, Baltimore, Maryland 21287-7834.
D A
DA
Schwengel
A E
AE
Jedlicka
E J
EJ
Nanthakumar
J L
JL
Weber
R C
RC
Levitt
D000483:Alleles; D001345:Autoradiography; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004276:DNA, Satellite / Q000235:genetics*; D005069:Evaluation Studies as Topic; D005453:Fluorescence; D005819:Genetic Markers; D005821:Genetic Techniques; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012091:Repetitive Sequences, Nucleic Acid
D017931:DNA Primers; D004276:DNA, Satellite; D005819:Genetic Markers