chromosomal analysis Flashcards
karyotype preparation
For analyzing a karyotype: can be any tissue w living nucleated cells. Usually use WBC but samples from skin, bone marrow, chorionic villi, and amniotic fluid can be used.
- Sample added to small volume of nutrient medium (stimulates division)
- Cells are cultured for 3 days and colchicine added to prevent formation of spindle+ arresting the cell in Metaphase where chromosomes maximally condensed=most visible
- Hypotonic saline (1st processing)= cause cells to lyze and spreads chromosomes
- Fixed and mounted on slide
- Visualization of banding methods
- Stain w Giemsa
- Analyse using light microscope
G banding vs R banding vs Q banding
G BANDING
DNA binding dye, Giemsa stain results in Monochromatic chromosomes
- Gives chromosomes light and dark bands due to digestion of proteins in chromosomes eg trypsin
- 400-500 bands per haploid set=6,000-8,000 kilobases
Ideal karyotype ideogram=specific banding of each chromosome
- # of stripes depends on the degree of condensation
- Metaphase: 400-450 stripes and prometaphase 850 stripes
Bright stripes=euchromatin (rich in G-C)
Dark stripes=heterochromatin (rich in T-A)
R BANDING
Reverse of G stain
- Incubate slides in hot phosphate buffer
- treatment of Giemsa dye
Useful for analyzing genetic deletions+ translocations that involve telomere of chromosome
alkaline reagents + high temp treatments
Result- darkly stained R bands (complement of G band)
Are G-C rich; A-T rich areas easily denatured by head
Q BANDING
Q= quinacrine
- Oldest banding method (1972) stains w fluorescent dye
- Unstable: needs fluorescent microscope+ photograph taken
- Result is G like bands
selective staining (3)
C-banding
Constitutive heterochromatin found at pericentric regions- on centromere
-Composed of high copy of tandem repeats = satellite, minisatellite, microsatellite, and transposon repeats
-Regions account for 6.5% of genome but repeat composition tough to sequence so small regions are sequenced
-Visualization of constitutive heterochromatin is possible using C banding (stain darker due to highly condensed nature of DNA)
Staining after denaturation in alkaline phosphatase
Labels centromere+ other constitutive heterochromatic regions
AgNOR staining
Tracks variation of second constriction of acrocentric chromosomes I.e the NOR (nucleolar organizing region) on the stalks of satellites in acrosomal chromosomes.
- Has argyrophilic proteins (stained by silver methods). Seen as black dots throughout nucleolar area called AgNOR. Silver nitrate precipitated in NOR.
sister chromatid exchange SCE
Type of selective staining for BrdU during cultivation, differential staining of sister chromatids
FISH
flourescent in situ hybridization
Use fluorescence probes that bind only to part of chromosome w high degree of sequence complementary
I.e targets specific DNA using fluorescent labelled DNA probe that have the complementary segments of the chromosomal DNA
- Used to detect presence/absence of specific DNA sequences on chromosomes.
steps
protease to get rid of proteins, only mRNA.
overnight probe hybridization- (shows location of Mrna)
washing to remove probes that did not bind
Probed using enzyme-linked antibody which bind to substrate= color reaction to clearly see location of Mrna
Color reaction based on enzyme reaction and antibody attached to it has alkaline phosphatase which binds to substrate to make insoluble dye=blue-purple
CGH
comparative genetic hybridization
Detect quantitative unbalanced genomic changes gain/loss to detect balanced rearrangements
For analysis of solid tumours
Analyse insertion+ deletion of genome of patient and compared to reference DNA (healthy)
Slide preparation
- Cells brought to halt in metaphase using colchicine
- Hypotonic KCl to take in water causing lysis=burst so DNA could be removed
- Fixed to slides
- Both DNA labeled w fluorescent dye
hybridization of both DNA sample put in 1:1 ratio into the metaphase slide
- Both compete to bind
- Reference dna (green) bound to slide causes deletion to occur in DNA of patient
- Sick patient binds to slide (red) , insertion has occurred w DNA of patient
No hybridization (no deletion.insertion) so red+green hybridize in same ratio=yellow
microarray
Highest resolution level 10-100kb (karyotype only uses 5-10kb) . Whole genome analysis but only targeted on unbalanced changes
- To detect microdeletions+ microduplications in patients w unexplained mental retardation
Performed on special slides=chips w thousands of holes (pits) which have short specific chromosomal fragments (complementary mRNA strands to the specific gene)
compares the genes between cancer and normal cell to see which is being overproduced and underproduced thorough fluorescent dye to be able to differentiate between normal and cancerous
Molecular cytogenetic methods (See in slides below)
Array- CGH: based on CGH but higher resolution
SNP-array: identify single nucleotide polymorphism (changes in DNA- deletions/insertions)
karyotype vs microarray
karyotype
1. numerical+structural abnormalities
2. balanced rearrangements
3. detect triploid
4. particular appearance of rearrangment
5. only rough estimation of size+genome localization
6. more clear interpretation
microarray
1. submicroscopic abnormalities
2. unbalanced rearrangements
3. no triploidy
4. summarized result, no chromosomal fashion
5. precise estimation
6. interpretation doubtful
