Glossary
Molecular Diagnostics
Double-stranded DNA 'product' created during PCR.
The binding of complementary DNA or RNA sequences via hydrogen bonding between bases.
The temperature at which a length of single-strand (heat denatured) DNA or RNA will anneal to a complementary strand. Lower temperatures may permit non-specific binding.
Adenine (A), cytosine (C), guanine (G) and thymine (T) are constitutive bases of DNA. Bases in DNA form two pairs of complementary molecules; hydrogen bonds can link adenine to thymine and guanine to cytosine. If the sequence of bases matches a complementary sequence in a second strand of DNA the bonds can hold the two strands together; thus forming double-stranded DNA. See nucleotides.
A matching pair of bases e.g. adenine/thymine or guanine/cytosine. Used to describe the length of strands of nucleic acid. I.e. 1kb = 1000 base pairs. The human genome contains approximately 3x109 bp.
A group of three nucleotides that encodes for an amino acid. The 20 different amino acids form the building blocks of proteins.
Extraneous DNA present in the sample or reaction that will be amplified and give a false positive result. Contamination problems in PCR most often arise from amplicons that were produced in previous PCR assays.
Heat or chemical treatment to break the bonds between double-stranded nucleic acid molecules thus forming single strand molecules lacking secondary structure.
DNA complementary to messenger RNA, synthesised from an RNA template by reverse transcriptase enzyme.
Genomic DNA. Double helix containing the genetic blueprint of the organism. DNA sugar phosphate 'backbone' molecules are joined by the pairing of bases, thymine with adenine and cytosine with guanine. Hydrogen bonds form between the complementary bases holding the two strands together.
An enzyme that synthesizes a double-stranded DNA molecule using single strand DNA and a primer as template.
Phase of PCR cycle following annealing of primer during which the Taq polymerase manufactures a strand of DNA. The optimum temperature depends on the enzyme used but is usually between 68-72°C.
PCR with two or more independent sets of primers in the same reaction tube.
A second PCR is performed on the product of an earlier PCR using primers which are internal to the originals. This improves sensitivity without impairing specificity.
If a full nested PCR (using two internal primers) cannot be performed, sensitivity and specificity can be improved by using just one 'inner' primer in conjunction with one of the 'outer' primers from the first reaction.
Deoxynucleoside triphosphates are the building blocks of nucleic acids. Nitrogenous bases are linked to a five carbon sugar and a phosphate group. The mixture of nucleotides used in PCR to create a new strand of DNA includes 2'-deoxyadenosine 5'-triphosphate (dATP), 2'-deoxycytidine 5'-triphosphate(dCTP), 2'-deoxyguanosine 5'-triphosphate (dGTP) and 2'-deoxythymidine 5'-triphosphate (dTTP).
Short sequences of nucleotides. They can be used as primers or probes. They may be chemically 'labelled' during synthesis.
Polymerase chain reaction. Amplification of specific lengths of DNA by repeated 'thermal cycling' reactions using polymerase enzyme. The basic PCR process is covered by patents owned by Hoffman-La Roche Inc and Hoffman-La Roche Ltd. A range of diagnostic kits is available.
Oligonucleotide that will bind to its target DNA and 'prime' the manufacture of a new strand by DNA polymerase. They are usually between 15 and 30 bases in length.
An oligonucleotide that has been labelled. When the probe binds or hybridizes to a complementary strand of DNA or RNA (its target), the molecule becomes labelled and can then be detected.
Ribonucleic acid. Single strand nucleic acid composed of a phosphate ribose 'backbone' and the nucleotides: adenine, guanine, cytosine and uracil.
Messenger RNA, carries genetic information from DNA to the ribosome where it is translated into the corresponding protein.
Detection of messenger RNA. Using reverse transcriptase enzymes cDNA is synthesised from the mRNA. The cDNA is then amplified by PCR.
An RNA-dependent DNA polymerase used to make cDNA from mRNA.
The specific piece of DNA or RNA to be amplified by the PCR. The sensitivity of an assay can be enhanced if multiple copies of the target are present. e.g. a repeated sequence or mRNA molecules.
Thermostable DNA polymerase, originally isolated from Thermus aquaticus, an organism that resides in hot springs.
For simple and rapid detection of PCR products. An oligonucleotide probe that is specific for the target to be amplified is labelled with a fluorescent tag and a quenching molecule. During the extension step of a PCR the Taq enzyme will disrupt probe bound to the target separating the fluorescent tag from its quencher molecule thus permitting fluorescence.
Flow Cytometry and Cell Cycle Analysis
Having an abnormal number of chromosomes ,thus aneuploid cells also have an abnormal DNA content.
The mean value, which is the total amount divided by the number of contributors.
Percent coefficient of variation is a measure of peak distribution. The percent coefficient of variation is the standard deviation of the peak divided by the mean channel number of the peak, times 100.
Having the normal number of chromosome pairs for non-reproductive, mammalian cells. The amount of DNA in diploid cells defines the normal DNA content for the species.
The ratio of GO/G1 peak channel in a DNA histogram of the experimental sample to the GO/G1 peak channel of the reference sample, when normal human diploid cells or nuclei are the reference. This is a measure of DNA aneuploidy, or abnormal DNA content.
A two parameter data graph used for acquisition and analysis. Each dot on the display represents one event that the flow cytometer analyzed.
Signal received by the photomultiplier tube (PMT), FL1. The range of the signal detected is dependent on the filters associated with the PMT. For FACScans and FACSCaliburs, FL1 measures light in the green range of the spectrum (515 to 545 nm).
Signal received by the photomultiplier tube, FL2. For FACScans and FACSCaliburs, FL2 measures orange-red light (564-606 nm).
Signal received by the photomultiplier tube, FL3. For FACScans, FL3 measures red light (above 650 nm). For FACSCaliburs, FL3 measures red light (above 670 nm).
Signal received by the photomultiplier tube, FL4. For FACSCaliburs, FL4 measures red-orange light (653-669 nm).
A property of molecules to absorb light at one wavelength and emit light at a longer wavelength. Flow cytometers detect Fluorescence emission at a 90 degree angle to the exciting light beam.
A parameter measuring light scattered less than 10 degrees as a cell passes through the laser beam. The FSC measurement is related to cell size.
Phase of cell cycle designating cells that are quiescent and have not yet entered the growth cycle. Normal cells in this phase have exactly one set of chromosome pairs.
Phase of cells cycle in which cells are committed to division. These cells have the same number of chromosomes and the same amount of DNA as GO phase cells. They are about to enter S phase where they synthesize DNA.
Phase of cells cycle in which proliferating cells have duplicated their DNA and formed two sets of chromosome pairs, in preparation for division. G2 follows the S phase and precedes the M (mitosis) phase.
Having one set of single chromosomes. Reproductive cells, like eggs and sperm, are haploid.
A data plot of a single parameter. This displays either the relative fluorescence intensity value or the channel number on the x axis and the number of events on the y axis.
Mitosis, during which a cell divides into two. This precedes GO/G1 and follows G2. M phase and G2 cells contain the same amount of DNA and the same number of chromosomes. In normal cells, this is the tetraploid number (4N) of chromosomes.
The number of chromosomes present in the cell. The amount of DNA in a cell gives an indication of the ploidy, but is not directly proportional. See Haploid, Diploid, and Aneuploid.
DNA synthesis, in which the cell duplicates its DNA.
The maximum number of events displayed for a channel in a histogram. A low scale number magnifies the data.
Also called 90o scatter or right angle scatter. Light scattered at a 90 degree angle as a cell passes through the laser beam. This measurement is related to the internal granularity or complexity of a particle.