Unit 12 In Situ Hybridization

Purine bases

adenine (A) and guanine (G) which have double ring bases

Discuss detection methods for ISH

after hybridization a visualization system is necessary to locate hybridized probe and target. probe may be labeled by direct methods: signal molecule directly attached to probe -enzyme, fluorochrome or radioisotope probe maybe labeled with indirect method - probe labeled with a specific ligand (biotin, hapten) that has a affinity for a link molecule (streptavidin)

proteolytic digestion

allows probes to penetrate tissue (permeability without affecting structural integrity) - amount depends on level of cross-linking Enzymes: tryspin Pepsin proteinase *specific to the areas they digest

phosphate group

alternating phosphate groups and pentoses form the backbone form the backbone of the DNA strand

Hybrid

anything of mixed origin; crossbreeding

Two sets of hybrid...

are possible! -probe/target and target/probe *increases sensitivity or hybridization signal

nucleoside

base + sugar (no phosphate)

Compare and contrast ISH and IHC

both IHC and ISH can be performed on tissue section- frozen and FFPE Comparative Steps: pretreatment= ISH digestion= both blocking= IHC quenching= IHC HIER= IHC denaturation= ISH hybridization= ISH stringent washes= ISH IHC target= antigen (protein) tool= antibody ISH target= DNA or mRNA tool= probe detection= both- direct and indirect methods IHC= proteins cane be destroyed by heat and lose their reactivity. silent mutation cannot be detected at protein level

Pyrimidine bases

cytosine and thymine (single ring)

Homology

degree of complementarity between the probe and its target. perfectly matched hybrids are 100% homologous As homology is decreased so is Tm -formamide concentration shoul be </= 50% in order to lower the melting point of hybrids -salt concentration should be 2x SSC (sodium chloride, sodium citrate)

process of hybridization

during ISH, labeled probe and the target DNA are denatured using heat and then allowed to hybridize by lowering temp ***probe and target DNA should be denatured together to avoid reannealing of the probe and target with themselves - probe/target hybrids favored*** **Double stranded DNA and probes need to be denatured prior to hybridization. -Single stranded probes and mRNA do not**

Probe labels

fluorochromes biotin haptens (digoxigenin, fluorescein) radio isotypes enzymes (HRP, ALP)

complimentary base pairing

hydrogen bonding between particular bases; in DNA, thymine (T) pairs with adenine (A), and guanine (G) pairs with cytosine (C); in RNA, uracil (U) pairs with A, and G pairs with C

Stringent washes

limit non specific hybridization (non complementary hybridization of probe and chromosomal DNA) -gets rid of mismatched pairs

Define in situ hybridization

localizing cytoplasmic mRNA or nuclear DNA by hybridizing a specific nucleotide sequence within cells and tissue to a complimentary strand of nucleotide probe

Raising the temperature because

non-specific hybrids have lower Tm's than specific hybrids

Prehybridization

optional step to reduce background staining (blocks non-specific probe binding. Equilibrates tissue to hybridization conditions all elements of hybridization solution minus probe

In situ

original position confined to the site of origin

probe size

probe length is identified as the number of bases-mer -500 mer indicates a probe with 500 bases *large probes are able to carry label and are therefore more sensitive *oligonucleotide probe: short (15-100 base pairs) -very specific but less sensitive (less label) -smaller probes can detect point mutations

RNA

ribonucleic acid carries information from DNA to the sites where proteins are manufactured (amino acids assembled) in the cells -single strand; uracil replaces thymine

Nucleotides are made up of

sugar, phosphate, base -form genes, and single DNA molecule may contain thousands of genes.

Transcription

the synthesis of a complementary strand of RNA (mRNA) from a DNA template

stringency

% of nucleotides which must match on two unrelated single-stranded nucleic acids before they will base pair with each other Allows for specificity Allowed fidelity of base pairing

Hybridization

*annealing* lowering of temp or pH opposite of denaturation process of joining two complementary strands of DNA or one each of DNA and RNA to form a double stranded molecule -forms hybrids

Explain the use of controls in ISH

*positive control should always be included -controls testing performance *no hybridization control should be included (non specific negative control) - no probe in hybridization solution antisense probe: complementary to the target gives positive staining sense probe: identical to the target gives negative staining (negative control probe) - probe which has no homology with the sequence of interest -positive control tissue known to have the sequence of interest - negative control- tissues known not to contain the sequence of interes

Denaturation

-aka melting -dissociation of two complimentary nucleic acid strands by breaking the hydrogen bonds between base pairs - heat or chemical used - heat required also= melting temp (Tm) *amount dependent on type of hydrogen bond in the DNA helix *80-95C or by high alkaline pH Cool or acidify methods will restore structure

Detection system will depend on the label on the probe

-biotin label on probe- use avidin/enzyme -enzyme label on probe- use chromogen -digoxigenin label- use anti- dig and then secondary detection (polymer w/ enzyme) -fluorochrome label - use fluorescence microscope

DNA

-deoxyribonucleic acid -forms genetic material in each cell -Double helix

Define FISH and compare and contrast it with ISH

-fluorescence in situ hybridization -fluorescent dyes as labels -can demonstrate multiple probes at the same time using different fluorescent dyes cannot see morphology. looking for specific changes in DNA or RNA

Nucleic acid targets

-genomic DNA or RNA (some viruses use RNA) -mRNA for studying expression of a particular gene -rRNA to identify bacterial species

Viral detection using probes

-viruses contain RNA or DNA - viral nucleic acids are replicated in the nuclei of host cells - probes can be used to detect unique target to a virus or targets shared by a group of viruses

Discuss the use of ISH in detecting diseases

1)identify cells actively undergoing protein synthesis 2)detect the positions of some genes on some types of chromosomes 3)detect viruses in cells

Probes

1. select the probe sequence 2. isolate the nucleic acid fragment of interest 3. DNA digestion and removal of probe region - restriction of enzymes 4.use of the cloning vector - plasmid, bacteriophage or virus replicated DNA fragment inserted into it 5. vector inserted into bacteria to produce large amounts of probe 6.probes purified and labeled

anti-parallel strands

3' end of one strand aligns with 5' end of other

Codons

A three-nucleotide sequence of DNA or mRNA that specifies a particular amino acid or termination signal; the basic unit of the genetic code. AAA, GGC, AUG

Discuss the advantages and disadvantages of ISH

ADV: *nucleic acids are heat stable and fixation resistant - also resist proteolysis *ISH detects blue print or instructions for protein *can detect silent mutations in the nucleic acids *organisms that are difficult to culture can be detected * nonviable organisms can be detected * DNA or RNA can be detected DIS: *DS DNA requires denaturing *nuclease enzyme (cuts sugar- phosphate backbone) could degrade nucleic acids - fixation within 30 minutes to prevent the release of nuclease *more time consuming than IHC *limited # of probes available * not sensitive enough to detect low #s *staffing

Describe the affect of formamide and salt concentrations

Increasing formamide concentration lowers the Tm - an organic denaturant, disrupts hydrogen bonds, and lowers the melting point to allow denaturation at lower temp - increasing F concentration will increase stringency -decreasing concentration will decrease stringency High salt concentration allows for relaxed hybridization with the formation of mismatched, non-homologous hybrids. Low salt concentration disallows mismatches; only perfectly matched hybrids will be allowed to form - high= low stringency -low= high stringency

Translation

Process by which mRNA is decoded and a protein is produced

probe application

a complementary sequence of nucleotide bases specific to RNA or DNA sequence of interest (known sequence)

Genes

-Genes determined traits that are inherited -each gene is a segment of a DNA molecule - hereditary information passed on to daughter cells during cell division