In Situ Hybridization in Diagnostic Pathology
Ricardo V. Lloyd and Arie Perry
Abundance - the prevalence of a particular RNA, or class of RNA molecules, in the cell. The relative amounts of a particular messenger RNA (mRNA) species in different samples is frequently expressed in terms of relative abundance.
Acrocentric – A type of chromosome with the centromeres near one end. The human acrocentric chromosomes (13,14,15,21, and 22) have satellited short arms that carry the genes for ribosomal RNA.
Allele - one of several genetically inherited alternate forms of a gene positioned at a specific locus on the chromosome.
Annealing - the coming together of complementary single-stranded nucleic acids (opposite of denaturation).
Amplification - the production of additional copies of a gene sequence.
Anticoding strand - the portion double-stranded DNA used as template to direct the synthesis of a complementary mRNA.
Antisense RNA - a RNA which is complementary to a mRNA sequence.
Autoradiography - method to detect radioactively labeled molecules as an image on a photographic emulsion.
Base - one of five molecules that make up the information content of DNA and RNA. In DNA, bases pair across the two chains of the double helix: adenine with thymine, and guanine with cytosine. RNA is single-stranded and contains uracil instead of thymine.
Base-pairing - the formation of hydrogen bonds between the nitrogenous bases of two nucleic acid molecules.
cDNA - a single-stranded DNA complementary to, and synthesized from, a specific mRNA.
Chemiluminescence - a nonisotopic hybridization detection technique. Chemiluminescence is the production of visible light by chemical reaction.
Chromosome - a morphologic unit of the genome containing genes and is composed of DNA and proteins, and is most easily recognized during mitosis.
Codon - a set of three adjoined nucleotides (triplet) that codes for an amino acid or a termination signal.
Complementarity - specific base pairing of nucleotide bases in nucleic acids.
Denaturation - represents conversion of nucleic acids from the double-stranded into single-stranded forms, usually by heating or changes in pH.
Dot blotting - a process where DNA or RNA is extracted from cells and dotted onto nitrocellulose filters. A labeled probe is added and hybridization is visualized by autoradiography or other techniques. Dot blots do not utilize electrophoretic separations as do Southern and Northern blot procedures.
End labeling - the addition of an isotopically or nonisotopically label to the 5' or 3' end of a DNA molecular probe.
Endonucleases - enzymes that cleave bonds within a nucleic acid chain and may be specific for RNA or single-stranded or double-stranded DNA.
Exon - any segment of an interrupted gene that is present in the transcribed final mRNA.
Exonucleases - enzymes that cut nucleotides one at a time from the 5' or 3' ends of a polynucleotide chain.
Filter hybridization - a method performed by incubating a denatured DNA or RNA immobilized on a membrane with a solution of labeled molecular probe.
Fragile site – Nonstaining gap in the chromatin of a metaphase chromosome.
Gene - the segment of DNA that codes for a transcribed RNA molecule.
Hybridization - the joining or annealing of complementary strands of RNA and/or DNA to give RNA-DNA, RNA-RNA, or DNA-DNA hybrids.
In-situ hybridization - a hybridization reaction performed on intact chromosomes, cells or tissues for direct visualization of morphologic sites of specific DNA or RNA sequences.
Intron - a DNA sequence that interrupts the coding sequences (exons) for a gene product. After information from the genes is transcribed into new strands of hnRNA, the introns are spliced out of the RNA molecule, and are not represented in the mature mRNA. Although the functions of introns are unknown, it has been postulated that some introns have a role in regulating gene expression.
Kinasing - the addition of a radioactively labeled nucleotide to the 5' end of a hybridization probe by means of a reaction catalyzed by the enzyme T4 polynucleotide kinase.
Liquid (solution) hybridization - a reaction between two complementary nucleic acid strands performed in solution.
Melting temperature (Tm) - the temperature point in a hybridization reaction at which 50% of the nucleotides are annealed (double strands) and 50% are denatured (single strands). The melting temperature is dependent on a set of conditions which are referred to as stringency.
mRNA (messenger RNA) - the ribonucleic acid molecule that transmits the genetic information from genomic DNA to the translation machinery where it directs protein synthesis.
Mutation - any change in the sequence of genomic DNA.
Nick - the absence of a phosphodiester bond between two adjacent nucleotides on a single strand in double-stranded DNA.
Nick translation - a probe labeling technique where DNA polymerase I nicks a starting point from which one strand of a duplex DNA can be degraded and replaced by resynthesis with new labeled precursor nucleotides.
Northern blot analysis - a technique for transferring electrophoretically chromatographed RNA from a gel matrix, usually agarose, onto filter paper, for subsequent immobilization and hybridization. The information gained from Northern blot analysis is used to qualitatively and quantitatively assess the expression of specific genes.
Nucleoside - a nucleotide with the phosphate group removed, leaving only a sugar and linked base remaining.
Nucleotide - building blocks of nucleic acids; made up of a nucleotide base, a pentose sugar and one to three phosphate groups.
Oligonucleotide - a short sequence (usually between 10-100 nucleotides) of DNA.
Palindrome - a segment of duplex DNA in which the base sequences of the two strands exhibit a two-fold rotational symmetry about the central axis. Restriction endonucleases often recognize and cut the DNA at a variety of such palindromic sites (the same sequence in both directions).
PCR (polymerase chain reaction) - a systematic, primer mediated enzymatic process for the geometrical amplification of a target DNA sequence. PCR product can be generated from as little as one molecule of target material (DNA or RNA) under optimal conditions.
Plasmid - an autonomous self-replicating circular DNA.
Point mutation - a substitution of single base pair in nucleic acid sequences.
Polyadenylation - the addition of a sequence of polyadenylic acid to the 3' end of a mRNA after transcription.
Polycistronic - pertaining to an mRNA molecule that contains coding regions for more than one gene. A single polycistronic mRNA can direct the translation of more than one polypeptide.
Primer - a short, usually 20-50 base sequence complementary to one strand of DNA providing a free 3'-OH at its terminus, where DNA polymerase of a deoxyribonucleotide chain can be initiated.
Probe - a portion of single-stranded nucleic acid (DNA or RNA) labeled isotopically or nonisotopically and used to hybridize to specific nucleic acids sequences.
Pseudogenes - faulty replicates of normal genes that normally occur in a genome which are stable, inherited, but are unexpressed components of the human genome.
Renaturation - the reassociation of complementary single strands of nucleic acids into double-stranded helical forms.
Restriction endonuclease - a class of enzymes which recognize a specific base sequence (usually four to six base pairs in length) in a double-stranded DNA molecule and cuts both strands of the DNA at every site where this sequence occurs.
Reverse transcriptase - a class of enzymes which catalyze the formation of DNA strands from RNA templates. Once the first DNA strand has been synthesized, it serves as the template for the enzymatic synthesis of the second complementary DNA strand.
Reverse transcription - synthesis of DNA on a template of RNA by reverse transcriptase.
Ribonuclease (RNAse) - a family of resilient enzymes that rapidly degrade RNA molecules. Control of RNase activity is a key consideration in all manipulations involving RNA.
Ribozyme - an enzyme made of RNA rather than protein. Ribozymes can be produced to cut RNA molecules at specific points.
RNA (ribonucleic acid) - a single-stranded nucleic acid which consists of ribose sugar, phosphate groups, and the bases adenine, uridine, guanine, and cytosine. Common forms of RNA include messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA) - all involved in protein synthesis - as well as several small RNA species, the functions of which are still under investigation.
Splicing - an intranuclear process of splicing out of introns and joining together exons in RNA.
Stringency - the set of conditions under which hybridization of a nucleic acid probe with its complementary target sequence occurs. These conditions include the concentration of salt and formamide, temperature, length, and GC content of the probe, and percentage of mismatch of the probe with its target. These variables can be adjusted to either increase or decrease stringency, and thus the specificity of the hybridization reaction.
Southern blot analysis - a technique for transferring electrophoretically chromatographed DNA from a gel matrix, usually agarose, onto a filter paper, for subsequent immobilization and hybridization. The information gained from Southern blot analysis is used to qualitatively and quantitatively assess the organization of specific genes or other loci.
Specific activity - the amount of radioactivity per unit mass of radioactive material. It is most frequently expressed in curies per millimole of material (Ci/mmol).
Tailing - the addition of radioactively or non-radioactively labeled nucleotide to the 3' end of a hybridization probe by means of a reaction catalyzed by the enzyme TdT (terminal deoxynucleotidyl transferase).
Transcription - the process of RNA synthesis on a DNA template.
Translation - the process of synthesis of protein on the mRNA template.
Triploid – A cell with three copies of each chromosome or an individual made up of such cells.
Trisomy – Having three representatives of a given chromosome instead of the usual pair, as in trisomy 21 (Down syndrome).