7 chromosome analysis p2

in situ hybridization

method that allows to localize and detect nucleic acid sequences within structurally intact cells or morphologically preserved tissues sections

fluorescence in situ hybridization

FISH

FISH

is a kind of ISH which uses fluorescent probes binding parts of the chromosome to show a high degree of sequence complementarity

dsDNA
ssDNA
RNA
synthetic oligonucleotides

4 basic probe types

dsDNA probes

stable, available, easier to obtain

RNA probes

higher thermal stability, better tissue penetration, more specific, low background noise by RNAse

ssDNA probes

stable, easier to work with, more specific, resistant to RNAses, better tissue penetration, without self hybridization

synthetic oligonucleotides probes

economical, stable, available, easier to work with, more specific, resistant to RNAses, better tissue penetration, better reproducibility

radioactive isotopes
non-radioactive labels

probes labeling technique

radioactive isotopes

32p, 35s, 3h

non-radioactive labels

biotin, digoxigenin, fluorescent dye (FISH)

sample prep
probe prep
denaturation and hybridization
detection

protocol outline

single-molecule RNA
FIber
Q
microfluidics-assisted (MA)
hybrid fusion

variations of FISH

single-molecule RNA FISH

detectin and quantifying mRNA
simplex or multiplex
cancer diagnosis

Fiber FISH

alternative technique to interphase or metaphase prep

chromosomes stretched on slides

applying mechanical shear along the length of the slide

Q-FISH

combines DISH with peptide nucleic acid and computer software to quantify fluorescence intensity

routinely used in telomere length research

microfluidics-assisted (MA) FISH

uses microfluidic flow to increase DNA hybridization efficiency

detect HER2 gene in breast cancer tissues

hybrid fusion FISH

primary additive excitation/emission combination of fluorophores to generate additional spectra through a labeling process known as dynamic optical transmission (DOT)

enables highly multiplexed FISH applications that are targeted within clinical oncology panels

morphology

population structure of microorganisms

pathology

pathogen profiling, abnormal gene expression

developmental biology

gene expression profiling in embryonic tissues

karyotyping and phylogenetic analysis

unique FISH patterns on individual chromosomes, chromosomal abberations

physical mapping

mapping clones on chromosomes and direct assignment of mapped clones to chromosomal regions associated with heterochromatin or euchromatin

microarray

use of treated glass instead of nitrocellulose or nylon membranes for the production of arrays

increase versatility of array applications

improved spotting technology

chip

glass carrying the array of targets

DNA, cDNA

PCR products
Oligomers
RNA or protein

targets of microarray