Pre-natal genetic diagnosis Flashcards
2 methods of chromosome analysis
Karyotyping+ FISH
Karyotyping use what cells?
Proliferating cells cultured with Colchicine
karyotyping method
1) Arrest cells in metaphase =condensed chromatin
2)cells in hypotonic solution = cells swell
3) fix in methanol or acetic acid
4) cells burst and Chromosomes stick on slide= metaphase spread
5) Brief digestion with Trypsin
6) Staining ( Giemsa/ Leishman’s) = Bands
Why arrest cells in metaphase?
Chromatin condensed + chromosomes visible
2 types or bands
Major measured from centromere
Minor bands within major
Karyotyping + FISH example diagnosis
Di George Syndrome (deletion) loss of red, retention of green
What FISH use
fluorescent labelled DNA probes bind + highlight specific chromosomes
FISH: cell in what cycle
metaphase spread or Inter phase
FISH highlights?
whole chromosome or localized region
Counterstain
DAPl= blue to visualise total chromatin
Metaphase v interphase FISH cells
M= cultured cells
I= fresh or archival material
FISH method
1) chromosomes denatured = SSDNA
2) DNA probe labelled with fluorescent molecule e.g nucleotide
3) probes denatured
4) Probes hybridized
5) wash off excess
6) counterstain with DAPI
M- FISH/ Spectral Karyotyping difference
chromosomes have diff colours
Fluorochrome labels are
chemically bound to nucleotides
stokes Shift
shift in peak excitation to emission
