DLA Genomics Flashcards
Measurement of triplet repeat expansions 3
- PCR and size separation by electrophoresis
- PCR and size separation by column chromatography
- Southern blot using a probe outside of the region of the repetitive repeat
G-band limitation
Only for whole chromosome changes (Down syndrome)
Very large deletions, duplications, or rearrangements (typically > 5Mb), but depends
FISH limits
> 0.5 Mb
FISH probe has to be smaller than the deletion it is designed to detect
Microarray CDH limits
Resolve 30 kbp deletion/duplication
Depends on array
NGS limits
Bp resolution
Microdeletions
Deletion too small to be seen by G-banding and microscope
Cell treatment for preparation of a standard G-banding karyotype or for FISH (5 steps)
- Isolation of cell from peripheral blood sample
- Culture in phytohemagglutinin to stimulate division
- Arrest at metaphase by colchicine or colcemid
- Swell by hypotonic solution and burst onto a microscope slide
- Fix chromosome and prepare for staining
- stain with Giemsa for G-band
- hybridize with a probe for FISH
Ideogram
Schematic diagram of the set of normal human metaphase-stage chromosomes
2 types of FISH (fluorescent in situ hybridization)
- Chromosome-specific unique sequence probes/gene specific probes
- hybridize to a specific locus, only 1
- know what to look for, useful for submicroscopic deletion - Chromosome painting or SKY (spectral karyotyping)
- use whole chromosome specific probes to paint each chromosome a different fluorescent color
- useful for chromosomal rearrangement, look at entire genome
VCFS 22q11.2 deletion syndrome
Immunodeficiency symptoms
Cardiac anomaly
Cleft lip, palate
Due to malformation during fetal development
Rapid FISH
FISH can be done during interphase
Rapid FISH using probes for chromosome 13, 18, 21, X, and Y
SKY FISH can detect
Translocation
Generation of whole chromosome FISH probes for whole chromosome analysis 4 steps
- Sequence unique to each chromosome are created and pooled into individual tubes
- Probes unique to each chromosome are created by labeling with a different colored fluorescent molecule
- Labeled DNA probes are mixed, and hybridized to metaphase chromosomes
- Analyzed by fluorescence microscopy
Used most in cancer genetics to visualize genome instability
Array CGH
Microarray comparative genomic hybridization
Co-hybridization of 2 differentially labeled DNAs to a microarray containing DNA sequences that represent the genome
Compare between: patient and reference or tumor isolates to normal
Detect duplications and deletions: CNV=copy number variants
May not detect balanced rearrangements
Create a array CGH
Immobilize unique chromosomal sequences of closed locations on an array surface on a glass slide, thousands of sequential fragments represent locus close to each other on a chromosome
24 chromosomes: 22 autosomal and 1 X and 1 Y
Green light if gain DNA copies
Red light if loss DNA copies
