Fluorescence in situ hybridization (FISH)
DDx diffuse signal (speckling)
o
Optimal size of probe and blocking DNA is 200-400 bp
o
Probe types:
§
Shorter probes may require amplification of the signal
§
Longer probes may require more blocking DNA to suppress the
repeat sequences (ex. interspersed repetitive elements) within the probe
§
Cosmids
·
Modified plasmids with capacity for ~40 kbp
inserts
§
P1 and P1-derived artificial chromosomes (PACs)
·
P1 vector: 70-100 kbp inserts
·
PAC vector:130-150 kbp inserts
o
More stable and lower frequency of chimeras than P1 libraries
·
Commercially available
·
Bright signal without amplification
§
Bacterial Artificial Chromosomes (BACs)
·
Up to 300 kbp inserts
·
Clones are very stable and easily manipulated
·
Commercially available
·
Bright signal without amplification
§
Oligonucleotide probes (oligos)
·
Synthetic oligomers
·
20-25 bp
·
Used for detection of tandemly repeated
satellite DNA sequences
o
Centromeres – can be
chromosome specific
o
Telomeres – can be chromosome specific
§
Yeast artificial chromosome (YAC) clones
·
1 Mbp
·
Used for genome mapping studies
·
Bright signal without amplification
o
Target slides should be:
§
Lacking in cytoplasm
§
Used within a few days or carefully stored
o
Denaturation:
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heat
§
formamide
§
alkali
§
additional DNA to block nonspecific binding may be used:
·
carrier DNA (from a species other than that undr
study)
·
blocking DNA (derived from the species being tested)
·
amount of blocking DNA required depends on the probe
o
amount and distribution of repeat sequences within the probe
o
hybridization:
o
washing:
§
stringency of the wash is critical in determining how much signal
will remain
o
secondary detection step (if necessary)
o
o
counterstain:
§
DAPI
§
Propidium iodide (PI)
§
Antifade solution
o
References: