Methods Of Chromosome Analysis Flashcards
What is chromosome analysis?
Banding or manipulating techniques to look for changes in chromosomes, including DNA based methods (array, PCR) and microscopy (karotype/gram and FISH)
Describe array-based genomic hybridisation as a DNA-based method of chromosome analysis.
Molecular technique to measure DNA copy across genome
Can identify gains and losses not visible by karyotyping but doesn’t detect balanced translocations
Can use comparative genomic hybridization (array-CGH) or analysis of signal strength within each sample
Extract and label DNA from cells in normal control (red) and patient (green) with fluorochromes – Mix in equal quantities and hybridise to microarray of clones – read red and green fluorescence – work out red-green ratio for each cell and identify to database of clones
SNP array hyb - when we know something causes specific disease outcome; amplification, fragmentation, labelling, hybridisation with probes containing SNPs, detection of binding
Short Tandem Repeat Assay - know individuals have different numbers of specific chromosomes (e.g. trisomy 21); might see 3 peaks of equal height of two peaks with one double the other
Describe Karotype Analysis as a Microscopy based method of chromosome analysis.
Can identify chromosome translocations (if one C is shorter than it should be) - often causes problems with meiosis as translocated part of C tries to pair up with corresponding C but can’t be in 2 places at once
Advantageous if you don’t know what you’re looking for; quick/easy to do but miss changes smaller than around 5Mb
Chromatids aren’t separated so don’t see X structures
See banding patterns relating to how densely packed DNA is
Describe Giesma method of stimulating cells to divide
0.5ml blood in culture medium
Add Phytohaemagglutinin
Culture 48-72 hours
Add Colcemid (arrests cells in metaphase)
Add Hypotonic KCl (cell swell) and fix in methanol with acetic acid
Drop onto slide from great height (burst)
Brief trypsin digestion stain with Giesma
Describe Paris nomenclature of numbering human chromosomes.
Numbered according to size (21 and 22 wrong way round; 23 other exception)
Metacentric, sub-metacentric or acrocentric
Short arms labelled p
Long arms labelled q
Labelled outwards from centromere
Satllites (short arms 13,14,15,21,22) contain rDNA and repetitive sequences
Describe FISH (Fluorescent in-situ hybridisation) as a Microscopy based method of chromosome analysis.
Get DNA of region of interest and fluorescently label (endonuclease take some out)
Make preparation of C and fix onto slide (formaldehyde based fixative)
Complementary probe can be short dsDNA or RNA
Binds to their DNA and can be detected as a fluorescent signal
Need to know what you’re looking for
Important that you also have control probes to bind onto the same chromosome, expected to see 2 copies
If any sort of mutation has occured, no hybridisation occurs and probe won’t hybridise
What is Chromothripsis?
Chromosome shattering and reassembling leading to rearrangement and loss of genetic information
Most examples seen in cancer genomes
C scrambled
Can occur in normal cells - rearrangement of C2 led to cure of WHIM syndrome
Describe how meiosis and gamete formation differs between males and females.
Male meiosis occurs in one go from puberty.
Female meiosis starts in female fetal germline at 11-12 weeks gestation
Arrests after recombination with homologues (remain connected by crossovers)
At ovulation, oocyte resumes, completes meiosis1
Cells arrest again in meiosis 2
Fertilisation triggers completion of meiosis2 (second small polar body produced and discarded)
Ovulated egg has most of cell’s contents and polar DNA with other DNA discarded
Sometimes polar body doesn’t get any genetic material and remains in egg that is ovulated (non-dysjunction)
What is aneuploidy?
Where organism/cell has only one/few chromosomes added or missing
Gene dosage effect - normal function needs 2 copies
Examples include the 3 compatible with life trisomies (Down21, Patau13, Edwards18)
Describe cause and diagnosis of Down syndrome (trisomy 21).
Usually due to error in either stage of meiosis
70% due to non-dysjunction in 1st stage mother
Increased maternal age is risk factor
Association with Alzheimer disease - 100% age 70 - extra copy of gene for amyloid precursor protein
Diagnosis through traditional karyotype (distinguished translocation Down’s), FISH, Array analysis, STR typing
What are microdeletion syndromes?
Distinct clinical syndromes caused by chromosomal microdeletions (1 to 5Mb)
Causes haploinsufficiency - 1 not enough
Not visible on karyotype but detected by molecular techniques
Arise due to unequal cross-over events in meiosis
E.g. VCFS (DiGeorge Syndrome), WIlliams-Beuren Syndrome (WBS), Angelman and Prader-Willi syndromes
