Cytogenetic analysis Flashcards
(38 cards)
what groups are metacentric chromosomes?
centromere in the middle of the chromosome, group H large metacentrics, group F small
what are acrocentric chromosomes?
centromere at end of chromosome. group D and G
what are submetacentric chromosomes?
centromere neither at end or middle
in g-banding, where cell cycle are the cells arrested?
in metaphase of mitosis
what does killing cells with fixative do?
blocks chromosome condensation and renders sample inert
what bands does the trypsin digest create?
pale bands
what do you stain it with after trypsin digest?
Leishmans dye
what sequences are dark and light bands rich in?
dark= AT rich. light= GC rich
what is open chromatin associated with?
darker bands
why may the banding structure differ between 2 homologues?
due to stage of cell cycle that cell was in when fixative was added, tissue type
what can make your chromosomes longer?
more mature cells
what does a longer trypsin incubation cause?
a paler band as chromosome more collapsed so Leichmans cant get into collapsed chromosome
what else can influence banding resolution?
slide aging, staining time and chromosome spread
difference between direct and indirect FISH
indirect is when the flourscent dies are added after sample and probes hybridised. more sesitive but slower. direct is much quicker but less sensitive so longer probes required
what is DAPI?
intercalating agent and used as counter stain
how is FISH carried out?
heat metaphase spread sample to 75-78 degrees to make DNA ss but not destroy the chromatin structures. then wash to remove unbound probes
what is chromosome enumeration dues for detecting?
common aneuploidies
how is chromosome enumeration carried out?
probes made that are specific to certain alpha satellite sequences of common aneuplodies.
what does a bright signal allow?
allowing rapid hybridisation times and less ambiguity
what are microdeletion probes used for?
used to check an abnormality detected by G-banding eg Cri du chat. used for detection of small translocation, inversion, deletion, insertion, duplication in parent chromosome which causes unbalanced foetal karyotype
what is whole chromosome painting used for?
if unsure of origin of fragment of DNA or if dont know where breakpoint is
how does dTTP excess arrest cell cycle?
inhbits ribonuclease reductase enzyme at high concentration, reduces dCTP conc and becomes rate limiting-arrests DNA synthesis so cells remain in S phase
how is the thymidine block released?
addition of dCTP to bypass the need for ribonucleotide reductase, or by washing and centrifuging and replacing media to dilute out the excess dTTP
what happens once the cells are released from the block?
they proceed through mitosis in a synchronous manner