DLA Genomics

Question 1

Measurement of triplet repeat expansions 3

Answer 1

1. PCR and size separation by electrophoresis
2. PCR and size separation by column chromatography
3. Southern blot using a probe outside of the region of the repetitive repeat

Question 2

G-band limitation

Answer 2

Only for whole chromosome changes (Down syndrome)

Very large deletions, duplications, or rearrangements (typically > 5Mb), but depends

Question 3

FISH limits

Answer 3

> 0.5 Mb

FISH probe has to be smaller than the deletion it is designed to detect

Question 4

Microarray CDH limits

Answer 4

Resolve 30 kbp deletion/duplication

Depends on array

Question 5

NGS limits

Answer 5

Bp resolution

Question 6

Microdeletions

Answer 6

Deletion too small to be seen by G-banding and microscope

Question 7

Cell treatment for preparation of a standard G-banding karyotype or for FISH (5 steps)

Answer 7

1. Isolation of cell from peripheral blood sample
2. Culture in phytohemagglutinin to stimulate division
3. Arrest at metaphase by colchicine or colcemid
4. Swell by hypotonic solution and burst onto a microscope slide
5. Fix chromosome and prepare for staining
   - stain with Giemsa for G-band
   - hybridize with a probe for FISH

Question 8

Ideogram

Answer 8

Schematic diagram of the set of normal human metaphase-stage chromosomes

Question 9

2 types of FISH (fluorescent in situ hybridization)

Answer 9

1. Chromosome-specific unique sequence probes/gene specific probes
   - hybridize to a specific locus, only 1
   - know what to look for, useful for submicroscopic deletion
2. 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

Question 10

VCFS 22q11.2 deletion syndrome

Answer 10

Immunodeficiency symptoms
Cardiac anomaly
Cleft lip, palate
Due to malformation during fetal development

Question 11

Rapid FISH

Answer 11

FISH can be done during interphase

Rapid FISH using probes for chromosome 13, 18, 21, X, and Y

Question 12

SKY FISH can detect

Answer 12

Translocation

Question 13

Generation of whole chromosome FISH probes for whole chromosome analysis 4 steps

Answer 13

1. Sequence unique to each chromosome are created and pooled into individual tubes
2. Probes unique to each chromosome are created by labeling with a different colored fluorescent molecule
3. Labeled DNA probes are mixed, and hybridized to metaphase chromosomes
4. Analyzed by fluorescence microscopy

Used most in cancer genetics to visualize genome instability

Question 14

Array CGH

Answer 14

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

Question 15

Create a array CGH

Answer 15

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

Question 16

CGH will not detect

Answer 16

Triplet repeat expansion or SSR (too small)
Balanced translocation
SNP

Question 17

SNP microarray = SNP chips

Answer 17

Obtain individual ssDNA and shredded into small lengths
Obtain 2 ssDNA, 1 from father 1 from mother as probes on the chip
Hybridization
Results:
1 spot: homozygous
2 spots: heterozygous

Question 18

cDNA microarray: expression arrays

Answer 18

1 identification: a set of genes or all genes expressed in a cell
2. Comparative: gene expression between cell or tissue samples

Question 19

cDNA microarray steps 5

Answer 19

1. Isolation of 2 tissues
2. Isolation of RNA
3. Production of labeled cDNA
4. Hybridization of cDNA to microarray
5. Results
   Red: more cDNA from this cell (cancer)
   Green: more cDNA from this cell (normal)
   Yellow: equal abundance