Preimplantation single-cell analysis of multiple genetic loci by whole-genome amplification.

Abstract

ABSTRACTsplice-junction mutation, an inner set of primers, TS12F and TS12R, was used for secondary PCR. Thermocycling conditions were similar to those used in the secondary PCR for exon 11, except that primer annealing occurred at 60TC. Four microliters of a solution containing 5x buffer (500 mM NaCl/250 mM Tris.HCl/50 mM MgCl2/5 mM dithiothreitol, pH 7.9) and 20-25 units ofDde I restriction endonuclease was added to 16 p1 of amplified 176-bp product, with digestion at 370C for 90 min and electrophoresis in a 10%o polyacrylamide minigel (Bio-Rad) ZFX and ZFY (11), using 2 p1 of PEP product for the primary PCRs. The CFTR AF508 locus was analyzed by Abbreviation: PEP, primer extension preamplification.

Multiple Mutation Analyses in Single Tumor Cells with Improved Whole Genome Amplification

“…Because MSI and LOH studies must be done with multiple markers, preamplification of the entire DNA by WGA would be very helpful. Although this technique has already been shown useful in intact sperm cells, 13 blastomeres, [15][16][17] and fetal nucleated erythocytes, 18 the method has not yet been optimized for samples routinely used in tumor pathology. Our intention was to establish a protocol that allows multiple DNA analyses of single cells or small cell groups in a single-round locus-specific PCR after preamplification.…”

“…Whole genome amplification (WGA) methods have been introduced as powerful tools, initially for sperm typing 13,14 and DNA analysis in single or few blastomeres [15][16][17] and nucleated erythrocytes from maternal blood 18 for the diagnosis of fetal inherited diseases. These techniques have recently been extended for the use in tumor pathology where preneoplastic and neo-plastic fluorescence-activated cell sorter (FACS)-sorted aneuploid esophageal tumor cells were analyzed for microsatellite instability.…”

Genotypic analysis of multiple loci in somatic cells by whole genome amplification

“…PEP was originally developed for amplifying the genome of single sperm cells for haplotype analyses (9) and more recently has been used for germline analysis in single diploid cells (10). The use ofPEP samples in haplotyping requires that a single allele be scored in locus-specific PCR assays.…”

“…Multiple loci in a single PEP sample PEP has been shown to be effective in single cell analysis for gernline mutations in both haploid and diploid genomes (9,10). To determine whether we could accurately screen multiple loci in somatic diploid and aneuploid cells from the same PEP samples we selected markers for separate chromosomal regions, 17p, 5q, 13q and 8p, that are often associated with tumor development.…”

“…PEP uses a degenerate pentadecanucleotide primer, consisting of 415 different combinations ofthe four possible bases, that anneals randomly throughout the genome to prime amplification. PEP has been used in combination with nested PCR analysis to detect germline abnormalities at different loci in single cell analysis (9,10). However, the presence of low levels of normal tissue in biopsy samples could obscure the detection of somatic lesions in neoplastic tissue, especially when using multiple steps of locus-specific PCR amplification.…”