LINKAGE ANALYSIS WITH MULTIPLEXED SHORT TANDEM REPEAT POLYMORPHISMS USING INFRARED FLUORESCENCE AND M13 TAILED PRIMERS

The use of short tandem repeat polymorphisms (STRPs) as marker loci fo r linkage analysis is becoming increasingly important due to their lar ge 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 eliminatin g the need for radioactivity and producing a digitized autoradiogram-l ike image that can be used for computer analysis. In an effort to simp lify the procedure and to reduce the cost of fluorescence STRP analysi s, we have developed a technique known as multiplexing STRPs with tail ed primers (MSTP) using primers that have a 19-bp extension, identical to the sequence of an M13 sequencing primer, on the 5' end of the for ward primer in conjunction with multiplexing several primer pairs in a single polymerase chain reaction (PCR) amplification. The banding pat tern is detected with the addition of the M13 primer-dye conjugate as the sole primer conjugated to the fluorescent dye, eliminating the nee d for direct conjugation of the infrared fluorescent dye to the STRP p rimers. The use of MSTP for linkage analysis greatly reduces the numbe r of PCR reactions. Up to five primer pairs can be multiplexed togethe r in the same reaction. At present, a set of 148 STRP markers spaced a t 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 softw are that both detects the STRP banding pattern and determines their si zes. This information can then be exported in a user-defined format fr om a database manager for linkage analysis. (C) 1995 Academic Press, I nc.