SEMIAUTOMATED RESOLUTION OF OVERLAPPING STUTTER PATTERNS IN GENOMIC MICROSATELLITE ANALYSIS

Microsatellites are polymorphic, short nucleotide repeating units scat tered more or less randomly throughout the genome, They are readily de tectable by polymerase chain reaction (PCR) and often used as genomic markers. One problem in the analysis of microsatellite data is the app earance of secondary bands during PCR that result in extended banding patterns. These ''stutter'' patterns may overlap in heterozygous allel es and obscure the overall pattern, severely interfering with analysis . This paper develops a model that successfully predicts the general s hape of stutter patterns, It then presents techniques for measuring th e intensity of the individual contributing alleles, The model is based on the assumption that there is a certain probability of losing or ga ining a microsatellite repeat unit during each PCR cycle, The effect i s cumulative, with the chance of losing a repeat unit being much great er than that of gaining one, which leads to a gradual reduction in the mean length of the pattern with increased PCR cycles. This can be mod eled quantitatively to predict the shape of the stutter pattern, a pre diction borne out by experiment. Next, a least-squares technique is pr esented that is used to analyze the overlapping stutter patterns and d etermine the relative concentration of each microsatellite in heterozy gous alleles. The technique is based on the observation that, at least for microsatellites of approximately the same length, the relative in tensity of each band in the stutter pattern is approximately the same for each allele. The stutter shape is most easily determined from homo zygous alleles. It can also be approximated from heterozygous samples if the difference between the lengths of the primary microsatellite ba nds can be determined. (C) 1997 Academic Press.