Table 4.
Strategies for the Reduction and Detection of Allele Dropout (ADO)
| Action/measure | Reduce/detect ADO | Mechanism | Potential problems/disadvantages |
|---|---|---|---|
| Use of any lysis buffer | Reduce | Protein removal/DNA accessibility/destroys endogenous nucleases (fewer DNA strand breaks) | Quality control of additional reagents |
| Choice of lysis buffer | Reduce | As above | Quality control of additional reagents |
| Increase denaturation temperature in first ten cycles of PCR | Reduce | Accessibility of DNA, complete denaturation of DNA strands | Taq polymerase failure due to prolonged exposure to high temperature |
| ≥2 cells taken from cleavage stage embryo (analyzed together) | Reduce | Increase starting template reduces probability of ADO | Possible detrimental effects of 2 cell biopsy |
| Reverse-transcriptase PCR | Reduce | Increased starting template | Prone to contamination/ presence of maternal transcripts/imprinted genes will exhibit ADO |
| Restriction enzyme digestion prior to PCR | Reduce | Shortens genomic DNA template strands—facilitating primer-template annealing | Limited data available |
| Use of Taq/Pwo-polymerase mixture | Reduce | Proof-reading activity? | No data available for single cells |
| Blastocyst biopsy (>2 cells) | Reduce/detect | Increase starting template reduces probability of ADO | Reduced embryo cohort at blastocyst stage |
| ≥2 cells taken from cleavage stage embryo (analyzed independently) | Detect | Low probability of two independent analyses both exhibiting ADO | Possible detrimental effects of 2 cell biopsy |
| Fluorescent PCR | Detect | ∼1000 times more sensitive than ethidium. High sensitivity can identify preferential amplification | Equipment and reagent cost |
| SYBR green I fluorescent stain | Detect | ∼25 times more sensitive than ethidium bromide. High sensitivity can identify preferential amplification | Reagent cost |
| Same fragment PCR | Detect | Impossible to have ADO if fragment contains both mutations of interest. Either both alleles are detected or amplification failure is observed | Only a small proportion of compound heterozygous conditions have mutations within several hundred base pairs of each other |
| Diagnosis of two normal alleles | Detect | In the absence of contamination, the presence of two normal alleles indicates that a mutant (expanded) allele cannot be present | Maternal and paternal alleles must be informative |
| Use of linked markers | Detect | Low probability of ADO occurring at a series of different adjacent loci | Requires design of single cell duplex/multiplex PCR |