Day four-Aneuploidy(3)
- 小琳师傅
- Jun 30, 2021
- 2 min read
Previously, I wanted to know whether aneuploidy could be included in list,now two possible directions gradually become clear for me :Impact of DNA methylation on oocyte aneuploidy and pool screen to find more candidate genes for aneuploidy.
To study aneuploidy,one problem may be how to count chromosome numbers accurately, efficiently and economically.So I compared several possible experimental methods:
FISH (fluorescence In situ Hybridization)
FISH is suitable for verifying influence of individual genes on chromosome copy number(both metaphase chromosomes and interphase nuclei), but not suitable for high throughput.
There is a newly developed amplified-FISH,with FITC-conjugated DNA probe first hybridizing with the chromosome, and then labeling with Alexa Fluor 488-conjugated anti-FITC secondary antibody to increase sensitivity.
CGH (comparative genomic hybridization)
CGH is mainly used to detect changes in DNA copy number and to locate these abnormalities on chromosomes.
Flow Cytometry Analysis
A flow cytometry-based method to determine ploidy,determined with PI staining, and suitable for high-throughput experiments.
HAP1 cells
HAP1 cells is a near-haploid human cell line,only one copy for each chromosome and gene, may be a suitable subject for CRISPR/Cas9 editing.
Long-term confocal imaging of chromosome dynamics
3D image reconstruction was used to classify chromosome segregation phenotypes at anaphase-I. Segregation phenotypes were related to spindle dynamics and cell cycle timings.
—that is the question:I believe because I see, or I see because I believe.
MSRE(methylation-sensitive restriction endonuclease digestion) and qPCR
Choose a fetal epigenetic marker, such as hypermethylated HLCS promotor,for diagnosis of DS.
SD-QF-PCR (segmental duplication quantitative fluorescent polymerase chain reaction)
I’m exited to find this method,which enables simply and high throughput detect specific chromosome copy number abnormalities (13, 18, 21, X and Y).
If we focus on DS, this method is just what we need,or we can go further to develop it for detecting more chromosomal abnormalities.
SD-QF-PCR is PCR
Reaction of SD-QF-PCR is PCR ,and my understanding of this method is:primers is not to detect genes,but duplicated sequences, which have homology between tested chromosome(such as ch21) and another normal chromosome(such as ch11).One pair of primers can amplify two fragments from two chromosomes in one reaction,but the fragment length is different in the two chromosome. one primer (FW or RV)was labeled with FAM (6-carboxyfluorescein).Then PCR products were run electrophoretic analysis,the electrophoretic machine name is unfamiliar for me( ABI 3130×l Genetic Analyzer).If PCR product ratio is 1.5:1 between ch21 and ch11, a diagnosis of DS could be made.
Why not qPCR?
In 2009,someone wanted to known if qPCR could be used as a sensitive technique for prenatal diagnosis of…
sounds promising! what's the differene between SD-QF-PCR and qRT-PCR?