Module induction Flashcards
(39 cards)
What is aneuploidy?
The gain or loss of one or a few chromosomes
What is the maximum resolution for G-banding?
3-5 megabases
Which condition is caused by trisomy 18?
Edwards Syndrome
Which phase of the cell cycle must cells be in for karyotype analysis?
Metaphase because the chromosomes are condensed and the nucleus has broken down so they are free in the cytoplasm
What is required for PND?
DNA from the foetus
How can DNA from the foetus be harvested for PND? (4)
- Amniocentesis
- Chorionic villus sampling
- Foetal blood sample from umbilical cord
- Cell Free Foetal DNA Sampling (cffDNA)
What is amniocentesis?
Taking a sample of amniotic fluid which contains epithelial cells from the foetus which fall off during development
What is chorionic villus sampling (CVS)?
Taking a sample of placental tissue
How are cells processed for PND after collection? (6)
- Stimulate growth with additional hormones
- Microtubule inhibitor used to prevent entry into anaphase
- Swell the nuclei via osmosis to increase the space between the chromosomes
- Cells are fixed using acetic acid and methanol
- Adhere chromosomes to glass slide using fixative
- Ageing period of chromosomes being exposed to sunlight for 48 hours
What is the purpose of fixing cells for analysis? (2)
- Kills the cells and blocks cellular processes including chromosome condensation to maintain a minimum resolution of analysis
- Kills possible contaminants (i.e. pathogens)
What is the purpose of the ageing period? (4)
- Denature proteins
- Remove residual fixative
- Enhance adherence to the slide
- Remove water from the chromosomes to improve banding quality
What is required to generate the banding structure for karyotyping? (2)
- Partial digestion with trypsin
- Stain with Leishman’s dye
What is euchromatin? (2)
- Highly transcribed regions of the genome with an open conformation
- Typically rich in GC sequences
What is heterochromatin? (2)
- Highly condensed regions of the genome with little/no transcriptional activity
- Typically rich in AT sequences
Which form of chromatin corresponds to the dark bands in G-banding chromosomes?
Heterochromatin (AT-rich)
Which form of chromatin corresponds to the pale bands in G-banding chromosomes?
Euchromatin (GC-rich)
How is staining done using trypsin and Leishman’s dye? (2)
- Trypsin degrades histones and causes chromatin collapse
- Chromatin collapse excludes Leishman’s dye from its binding pocket so it is unable to access the chromatin
What is the explanation of why heterochromatin (more highly condensed) stains darker than euchromatin? (3)
- Trypsin digestion causes partial collapse of all chromatin structures
- Regions that start off relatively open (i.e. euchromatin) are more accessible to trypsin digestion so suffer greater collapse
- Euchromatin collapses more than heterochromatin so euchromatin corresponds to pale bands and heterochromatin corresponds to dark bands
What is mosaicism?
When a person has two or more genetically different sets of cells in their body
What kind of DNA is in the p arm of acrocentric chromosomes?
Repetitive DNA and ribosomal DNA genes (rDNA)
How does FISH work?
Uses fluorescently labelled oligonucleotide probes which are complementary to regions of genomic interest to report on copy number and positional information
What is stringency in FISH?
A measure of the experimental conditions that govern how likely 2 nucleic acid sequences are to anneal
What are the major experimental parameters that can change the stringency conditions of a reaction? (2)
- Temperature
- Salt concentration
How is FISH performed? (5)
- Nuclei from patient samples are adhered to microscope slides
- Chromosomal DNA is denatured to generate single stranded DNA (ssDNA) by increasing the temperature to 73 degrees (increasing stringency)
- DNA is single stranded which allows the probes to bind to their complementary sequences
- Temperature is cooled
- Samples are washed in different stringency buffers to remove probe bound to off target sequences and reduce background signalling
