The single cell as a tool for genetic testing: credibility, precision, implication

Abstract

Purpose: To investigate the influence of amplicons size and cell type on allele dropout and amplification failures in single-cell based molecular diagnosis.

Methods: 730 single lymphocytes and amniotic cells were collected from known heterozygotes individuals to one of the common Ashkenazi Jewish mutations: 1278+TATC and IVS12+1G>C which cause Tay Sachs Disease, IVS20+6T and 854A>C which underlie Familial Dysautonomia and Canavan Disease. DNA was extracted and analyzed by our routine methods.

Results: Reduced rates of allele dropout and amplification failure were found when smaller amplification product were designed and in amniotic cultured cells compared to peripheral lymphocytes. Cultured lymphocytes, induced to divide, demonstrated significantly less allele dropout than non induced lymphocytes suggesting the role of division potential on amplification efficiency.

Conclusion: Single cell based diagnosis should be designed for each mutation. Minimal sized amplicons and cell having division potential should be preferred, as well as sensitive techniques to detect preferential amplification.

Fig. 1

Diagnosis by PCR and restriction digest in single cells. a. Detection of the mutation IVS20+6T→C in the IKBCAP gene. Lane 1—DNA ladder, 50–100 bp. Lanes 2–9—Single culture cells of heterozygote embryos. Lane 10—A single lymphocyte of a heterozygote individual. Lane 11—A single lymphocyte of a heterozygote individual that presents ADO of the mutant allele. Lane 12—A single lymphocyte of a non carrier. Lane 13—A single lymphocyte showing PCR failure. Lane 14—Normal control DNA, 1 ng/μl. Lanes 15—Control of heterozygote DNA, 1 ng/μl. Lanes 16–17—Controls of heterozygote DNA, 100 pg/μl. Lane 18—Blank sample (no DNA). Lane 19—Control of mutant homozygote DNA 1 ng/μl. b. Detection of the mutation 854A→C in the ASPA gene. Lane 1—Control of normal DNA 100 pg/μl. Lane 2—Control of Heterozygote DNA 100 pg/μl. Lane 3—A single amniotic cell, homozygote norml. Lanes 4–5—Single amniotic cells of heterozygote embryos. Lane 6—A single lymphocyte of a heterozygote individual showing ADO of the Mutant. Lane 7—Blank sample (no DNA). Lane 9—DNA ladder, 50–100 bp