Allele Drop-Out Can Occur in Alleles Differing by a Single Nucleotide and Is Not Alleviated by Preamplification or Minor Template Increments

Abstract

The ability to analyze the genetic material of single cells by the PCR opens up new prospects for diagnostics. Because only two copies of the genetic template are available for amplification, a problem that frequently arises when examining heterozygous loci in single cells is allele drop-out (ADO). ADO results from the preferential amplification of one of a pair of heterozygous alleles, in which the other allele is totally under-represented. In examining single cells from carriers heterozygous for beta-thalassemia mutations, we have found ADO to occur in alleles differing by a single nucleotide, where either the normal or the mutant genotype was absent. We have found that ADO is not overcome by either increasing the amount of DNA template to 20 pg or by primer extension preamplification (PEP), but rather that the best diagnostic accuracy is obtained by examining multiple single cells and basing a diagnosis on the combined results of such an examination.

Molecular Diagnostics in Preimplantation Genetic Diagnosis

“…55 However, a significant proportion of true ADO exists even using fluorescent PCR. 33,42 Such observations from single cell fluorescent PCR reinforce the need for cut-off values to distinguish background noise, contamination, extremely low amplification, preferential amplification, and allele dropout. Examples of preferential amplification and allele dropout from samples of single heterozygous cells are shown in Figure 1.…”

“…39 -41 ADO is only detectable when heterozygous alleles are present but appears to be indiscriminate, in that the allele successfully amplified is random (even when only differing by a single nucleotide). 42 ADO remains the biggest obstacle to accurate and efficient PGD for single gene disorders and the severity of its consequences is closely linked to the mode of inheritance of the disorder under test. For autosomal recessive conditions when both partners are carrying the same mutation, ADO should not, in the absence of contamination, result in the transfer of an affected embryo.…”

“…63 In a mouse embryo model, dual blastomere biopsy combined with independent blastomere analysis improved preimplantation diagnostic reliability dramatically for a dominant 68 condition but only slightly when the inheritance was recessive. 69 Whereas increasing the DNA template threefold is not effective in reducing ADO, 42 the risk of error due to ADO can be virtually eliminated if more than four cells are taken and independently analyzed. 70 Although blastocyst biopsy could make this approach feasible, the routine removal of four cells from cleavage stage embryos would be unacceptable in terms of the negative impact on subsequent embryo development.…”

Fetal Gender Determination and BclI Polymorphism Using Nucleated Erythrocytes in Maternal Blood

“…Nevertheless, Hahn et al (1998) and Garvin et al (1998) have recommended examination of multiple single cells and multiplex PCR for simultaneous identification using microsatellite loci known to be informative for solving the problem of ADO. Accordingly, we performed PCR in 47 cells and performed PCR for the amelogenin gene and BclI polymorphism sequentially to avoid ADO.…”

Toward Efficient Analysis of Mutations in Single Cells from Ethanol-Fixed, Paraffin-Embedded, and Immunohistochemically Stained Tissues

“…ADO rates between 0% and 83% have been reported and are dependent on the source and quality of the template DNA amplified (Findlay et al, 1995(Findlay et al, , 1998Garvin et al, 1998;Hahn et al, 1998;Rechitsky et al, 1996;Snabes et al, 1994;Wells et al, 1999). As a result of ADO, mutation analysis may fail to detect heterozygous mutations in a significant number of cases.…”