Analysis of DNA sequence variation in single cells

Whole genome amplification from a single cell: implications for genetic analysis.

“…The DNA sequences in individual sperm cells were copied by multiple rounds of primer extension using a collection of 15-base oligonucleotides in which any one of the four possible bases could be present at each position. Theoretically, the primer was composed of a mixture of 415 (1 X 109) sequences. Single human sperm were sorted by flow cytometry into 96-well Falcon microtiter dishes containing 5 ,l of an alkaline lysis solution (200 mM KOH/50 mM dithiothreitol) as described (17,18). After a 10-min incubation at 65°C, 5 Specific Gene Analysis.…”

“…A total of 12 loci were studied. We used a hemi-nesting strategy that enhances yield and specificity when starting with one target DNA molecule and allows the product to be detected by ethidium bromide staining (17,18). The first round of PCR utilizes a pair of primers that flank the target sequence.…”

Fine-Mapping of a Region of Variation in Recombination Rate on BTA23 to the D23S22–D23S23 Interval Using Sperm Typing and Meiotic Breakpoint Analysis

“…Sperm were prepared for flow sorting and PEP as described by Li et al (1991) with minor modifications. Briefly, sperm were sorted into 96-well V-bottom plates (MJ Research Inc., Cambridge, MA), lysed in 10 l of 100 mM KOH and 25 mM dithiothreitol for 20 min at 65°C and neutralized with 5 l of 300 mM KCl, 900 mM Tris-HCl (pH 8.3), and 200 mM HCl.…”

Detection of bovine leukemia virus proviral DNA in individual cells.

“…A person skilled in flow cytometry and cell sorting is needed, and precautions are required to eliminate sources of DNA c o n t a m i n a t i o n . (17) From the time of blood sampling t h r o u g h sorting, PCR, and statistical analysis, it takes one skilled person using one 96-well PCR m a c h i n e -3 days to complete the analysis of one-half plate (40-40,000 cells, including controls).…”