ANALYSIS OF RANDOMLY AMPLIFIED FLOW-SORTED CHROMOSOMES USING THE POLYMERASE CHAIN-REACTION

Bivariate fluorescence-activated sorting is a method for obtaining rel atively pure fractions of chromosomal DNA, Unfortunately, the yields ( < 0.25 mu g/day) frequently limit the types of molecular analysis that can be performed. The polymerase chain reaction (PCR) is capable of a mplifying unique sequences from scant amounts of template DNA. The pur pose of this study was to determine whether the sensitivity of the PCR could be used to detect sequences specific to chromosomes discriminat ed and purified by flow cytometry. Flow-sorted chromosomal DNA was pre pared by collecting similar to 10(5) chromosomes onto a nitrocellulose filter and eluting the DNA by boiling. Amplification products were no t detected when different amounts of chromosomal DNA were used in a si ngle 30 to 40-cycle PCR assay. However, when the eluted DNA was primed with degenerate 15-bp oligonucleotides and randomly amplified prior t o performing the PCR assay, sequence-tagged sites (STSs) were detected after gel electrophoresis and ethidium bromide staining. This random amplification step eliminated the need for both reamplification with n ested primers and detection by DNA hybridization. Furthermore, the ran dom amplification scheme provided enough template DNA from a single so rt (10(5) chromosomes) to perform > 1000 PCR assays. Representational analysis of one chromosome type revealed that > 74% of 70 STSs were de tected. Moreover, the technology could be used to identify and delinea te the breakpoint region of a marker chromosome. This amplification sc heme should simplify greatly the molecular analysis of normal and aber rant chromosomes. (C) 1995 Academic Press, Inc.