Lab techniques

Question 1

CHIP

Answer 1

take cell and treat with formaldehyde, lyse cell breaking DNA into small pieces, precipitate regulatory protein that is of interest, remove protein of bound DNA, use of PCR to amplify target and sequence

Question 2

DNA Foot-printing

Answer 2

looking at presence of DNA binding proteins in a particular cell lysate, does this protein bind to particular DNA? radio label DNA, incubate with cell lysate allowing to protein to bind, cleave with nuclease (ladder formation); if a portion binds it will protect the series of bases of DNA

Question 3

Electrophoretic Mobility Shift Assay

Answer 3

radio active DNA with cell extract, proteins added will make DNA heavier so it will run slower along gel, DNA with nothing bound will be a blob at the bottom

Question 4

Reporter Assay

Answer 4

Insert gene into plasmid (not present in mammalian cells), use restriction enzymes to cleave and input enhancer of interest into plasmid, see if gene turns on or not (only one that tells functional activity)

Question 5

Cytogenetic analysis: commonly used cell types

Answer 5

white blood cells
amniotic cells
cells of the choronic villi
fibroblasts
cancer cells
bone marrow cells

Question 6

Indications for Cytogenetic analysis

Answer 6

1. problems in early childhood growth or development
2. still births/ neonatal births appearing to have cytogenetic abnormalities
3. history of infertility or recurrent miscarriage
4. suspected chromosome abnormality in first degree relative
5. maternal age greater than 35
6. cancer

Question 7

Routine banding

Answer 7

5-15,000,000 bp

Question 8

High resolution banding

Answer 8

1-3,000,000 bp

Question 9

Comparative genome hybridization

Answer 9

50-250,000 bp

Duplication within a chromosome

Question 10

FISH analysis

Answer 10

50-250,000 bp

Spectral karyotyping- staining each chromosome different colors, probe mixture, hybridize and analyze; used to detect aneuploidy (ex. cancer cells), rearrangements such as translocations in nonhomologous chromosomes

can detect abnormalities as well as small deletions or duplications that can not be detected by high resolution chromosome banding techniques

Question 11

Chromosomal microarray

Answer 11

50-250,000 bp

detection of gain or loss of chromosome in the genome