Analysis of randomly amplified flow-sorted chromosomes using the polymerase chain reaction.
Bivariate fluorescence-activated sorting is a method for obtaining relatively pure fractions of chromosomal DNA. Unfortunately, the yields (< 0.25 microgram/day) frequently limit the types of molecular analysis that can be performed. The polymerase chain reaction (PCR) is capable of amplifying unique sequences from scant amounts of template DNA. The purpose of this study was to determine whether the sensitivity of the PCR could be used to detect sequences specific to chromosomes discriminated and purified by flow cytometry. Flow-sorted chromosomal DNA was prepared by collecting approximately 10(5) chromosomes onto a nitrocellulose filter and eluting the DNA by boiling. Amplification products were not detected when different amounts of chromosomal DNA were used in a single 30 to 40-cycle PCR assay. However, when the eluted DNA was primed with degenerate 15-bp oligonucleotides and randomly amplified prior to 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 nested primers and detection by DNA hybridization. Furthermore, the random amplification scheme provided enough template DNA from a single sort (10(5) chromosomes) to perform > 1000 PCR assays. Representational analysis of one chromosome type revealed that > 74% of 70 STSs were detected. Moreover, the technology could be used to identify and delineate the breakpoint region of a marker chromosome. This amplification scheme should simplify greatly the molecular analysis of normal and aberrant chromosomes.
Hui
S M
SM
Joint Program in Neonatology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Trask
B
B
van den Engh
G
G
Bartuski
A J
AJ
Smith
A
A
Flint
A
A
Lalande
M
M
Silverman
G A
GA
D001483:Base Sequence; D002458:Cell Fractionation; D002461:Cell Line, Transformed; D002869:Chromosome Aberrations; D002877:Chromosomes, Human; D005434:Flow Cytometry; D005819:Genetic Markers; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D016324:Sequence Tagged Sites
D005819:Genetic Markers