Genetic Testing Platforms & PGT Flashcards
What seen on D3 cleavage stage embryo biopsies dramatically improves PCR amplification rates?
Visible intact nucleus
Presence of nucleated cells is not as critical in a trophectoderm biopsy, when multiple cells are removed from a piece of the trophectoderm
What celluar lysis methods are available for biopsied samples prior to molecular analysis for PGT?
- Alkaline lysis
- SDS
- Proteinase K
Alkaline lysis (NaOH, KOH with DTT): loosens cell walls, releases DNA or sheared cellular DNA
- DTT reducing agent disrupts disulfide bonds
SDS (sodium dodecyl sulfate): detergent to remove lipids from cell membranes, increase access to cytoplasm
Proteinase K (may be used with SDS): digests proteins, cleaves peptides, proteinase K rapidly degrades DNAses, allowing isolation of “raw” DNA
PCR steps
Most initial PGT techniques used PCR or RT-PCR to amplify DNA
PCR steps:
1. Denature template DNA
2. Anneal DNA primers
3. Extension of new DNA strands using DNA polymerase and dNTPs
& repeat
How much DNA is in a single cell?
How much is needed for molecular analysis?
What is a method to achieve the amount of DNA needed for analysis?
7-10 pg
At least 1 mcg needed for microarray or NGS
–> Use whole genome amplification (WGA) to increase quantity of template DNA
What is a disadvantage of WGA techniques?
Increased risk of allele drop out (ADO), when 1 allele is preferentially copied over another
What is karyomapping?
- Using single nucleotide polymorphisms (SNPs) to identify both parental alleles
- Family members with known genetic status used as reference to track inheritance pattern
- Able to detect structural chromosomal rearrangements and meiotic aneuploidy
Micro-satellite markers
- How is it used for PGT
- Pros/cons
WGA > multi-site PCR for multiple multi-satellite markers > construct haplotype (chromosomal markers generally inherited as a set)
Pros: can detect less common mutations without developing a specific direct test for mutation analysis; can mitigate ADO by detecting multiple sites
Cons: need affected family member DNA to trace inheritance patterns
What is 1 of the most important factors affecting PGT for single gene defects after WGA?
Allele drop out (ADO): preferential amplification of only 1 allele (random)
Heterozygote: 1 normal allele, 1 affected allele > amplification of only 1 allele > potential mis-diagnosis
When are ADO effects most prominent?
- In single cell (the more cells there are, as in trophectoderm biopsy, the less likely ADO will affect the result)
- In low copy number templates (multiple copy number templates can still have allele dropout, but effects are masked)
- Most problematic when diagnosing compound heterozygotes or autosomal dominant disease in which information re: both alleles is essential
What are some methods that have been developed to mitigate ADO?
- Lysis conditions
- Denaturation temperature
- Use of polymorphic markers
- Biopsy of 2nd PB
- Biopsy-ing 2 cells from cleavage-stage and multiple cells from trophectoderm
What to do when ADO is suspected after embryo biopsy?
Warm, re-biopsy, re-vitrify
Why have methods other than cytogenetics been developed for PGT-A?
Difficult to obtain/stain metaphase chromosomes for a karyotype
What was the initial method of PGT-A?
FISH (only 1 of 12 RCTs demonstrated increase in LBR with PGT-A with this method)
Requires skilled personnel
What is “24 chromosome testing” includes?
- Chromosome copy number analysis
- Comparative genomic hybridization (CGH)
Testing of 22 autosomes + sex (X, Y)
Comparative genomic hybridization (CGH)
- Why is it not widely used for PGT-A?
- WGA to amplify
- Sample vs control labeled with different fluorochromes
- Hybridized and compared to normal metaphase spread
- Analyzed by filtered fluorescent microscope and specialized software
Cons: labor-intensive, needs normal metaphase spread, requires several days, and is not widely used for PGT-A
