Module induction Flashcards
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
What are the conditions of high stringency? (3)
- High temperature
- Low salt concentration
- DNA more likely to be in ssDNA form so probes can bind
What is the importance of salt in stringency? (3)
- Salts (e.g. NaCl) dissociate into positive and negative ions
- Positive ions bind to the negatively charged DNA backbone to cancel the electrostatic repulsion between the strands
- Therefore more salt lowers the stringency because reduces the repulsion between DNA strands keeping it in the double stranded form
What information does metaphase FISH provide? (2)
- Positional information
- Low resolution copy number information
What information does interphase FISH provide? (2)
- Higher resolution copy number information
- Presence of fusion of genetic elements
What is a limitation of FISH?
It is possible for 2 signals to physically sit on top of each other by chance rather than be a true colocalisation
What are the main classes of FISH probe used to characterise chromosome abnormalities? (4)
- Gene specific probe
- Centromeric probe
- Telomeric probe
- Chromosome-painting probe
What are gene specific probes?
Reports copy number of disease critical regions e.g. Down syndrome
What are centromeric probes? (2)
- Probes complementary to alpha satellite sequences located in sub-centromeric regions of chromosomes
- Often used to report on copy number of 13, 18, 21, X and Y for PND
What are telomeric probes and whole chromosome paints used for? (2)
- Characterising chromosomes which have been observed to be abnormal in G-banding
- Look for gross structural rearrangements and if the abnormality was inherited from a parent
What is an example of a gene specific probe?
TBX1 probe
What is the relevance of TBX1? (2)
- TBX1 gene is on 22q
- Commonly deleted in DiGeorge Syndrome
What is nuchal translucency? (2)
- Area of fluid behind the foetal neck which can be seen in an ultrasound
- Thickness is an important marker for genetic disease
What is the normal range of nuchal translucency thickness?
1.5-3mm
Which condition is caused by trisomy 13?
Patau Syndrome
What symptoms are associated with Patau Syndrome? (3)
- Holoprosencephaly
- Microcephaly
- Polydactyly
