ASCP MB key terms

The flow of genetic information from DNA to RNA to protein: DNA is replicated, transcribed into messenger RNA, and translated into protein. Molecular assays exploit each step, for example by measuring RNA through reverse transcription.

An RNA-dependent DNA polymerase that copies an RNA template into complementary DNA (cDNA). It is used in RT-PCR to convert viral RNA or messenger RNA into a DNA target that can then be amplified.

An enzyme that cuts double-stranded DNA at a specific recognition sequence. It is used to fragment DNA for cloning and fragment analysis, and to digest contaminating plasmid or genomic DNA before amplification.

A technique that separates nucleic acids by size and charge as they migrate through agarose or polyacrylamide under an electric field. It is used to check extraction quality and confirm amplicon or digest sizes.

Dideoxy chain-termination sequencing that incorporates fluorescently labeled chain-terminating nucleotides, generating fragments of every possible length. Capillary separation yields the base sequence and remains the reference method for confirming variants.

Massively parallel sequencing in which millions of DNA fragments are read simultaneously, producing short reads aligned to a reference genome. It supports gene panels, exome and transcriptome studies, and metagenomic pathogen detection.

The temperature at which half of a DNA duplex separates into single strands. It depends on length, GC content, and salt concentration, and it guides primer and probe design and annealing conditions.

A salt such as guanidinium thiocyanate that disrupts hydrogen bonding and protein structure, lysing cells and inactivating nucleases. It forms the basis of silica column and silica bead nucleic acid purification.

Archival pathology tissue preserved in formalin and embedded in wax. Formalin cross-links and fragments nucleic acids, so extraction must reverse cross-links and assays often use short amplicons.

Short, degraded DNA fragments circulating in plasma, released mainly by dying cells including tumor and placental cells. It is the analyte for liquid biopsy and noninvasive prenatal testing and requires collection that prevents leukocyte lysis.

The absorbance ratio used to estimate nucleic acid purity. Pure DNA is about 1.8 and pure RNA about 2.0; lower values suggest protein or phenol contamination that can inhibit amplification.

A tube containing the calcium chelator EDTA, which prevents clotting and helps preserve nucleic acids. EDTA is preferred for molecular testing because heparin inhibits amplification enzymes.

An in vitro method that uses thermal cycling and a thermostable DNA polymerase to copy a target sequence millions of times through repeated denaturation, primer annealing, and extension steps.

PCR monitored during each cycle by a fluorescent signal, allowing quantification of starting target and detection without post-amplification gel analysis, which shortens turnaround and reduces contamination risk.

The cycle number at which fluorescence crosses a defined threshold. It is inversely proportional to the amount of target present and is used for quantification and for qualitative interpretive cutoffs.

A short single-stranded oligonucleotide that anneals to a complementary target and supplies a free 3' end for polymerase extension. A primer pair defines the boundaries of the amplicon.

A sequence-specific oligonucleotide carrying a reporter and quencher that is cleaved by the polymerase exonuclease activity only after hybridizing to the exact target, releasing fluorescence as amplification proceeds.

A fluorescent molecule that inserts between stacked bases of double-stranded DNA and emits light only when bound. It reports any double-stranded product without sequence specificity and enables melting analysis.

PCR in which several primer pairs are combined in one reaction to amplify multiple targets or loci at once, conserving specimen and reagents but requiring careful design to limit competition and nonspecific products.

A two-stage PCR in which the product of a first amplification is reamplified with primers located inside the original amplicon, raising sensitivity and specificity but increasing contamination risk.

An isothermal method in which reverse transcriptase and RNA polymerase generate many RNA copies of a target through transcription. It is highly sensitive and widely used for viral and bacterial detection.

An isothermal method using four to six primers and a strand-displacing polymerase to produce large amounts of concatemeric DNA within about an hour at constant temperature, often detected by turbidity or fluorescence.

A signal-amplification assay, not a target-amplification method, in which branched DNA molecules and labeled probes bind a captured target and generate signal proportional to target amount, used for viral load measurement.

The base pairing of a labeled probe with a complementary nucleic acid sequence under controlled temperature and salt conditions. It underlies blotting, FISH, microarrays, and probe-based detection.

Labeled DNA probes hybridize to complementary sequences within intact cells or tissue, and fluorescence microscopy reveals gene amplifications, deletions, and rearrangements in their structural context.

A method that partitions a reaction into thousands of droplets or wells containing zero or a few target molecules, then counts positive partitions by endpoint fluorescence to give absolute quantification without a standard curve.

The quantity of viral nucleic acid in a specimen, usually reported as copies or international units per milliliter of plasma. It guides diagnosis, treatment monitoring, and resistance assessment for HIV, HBV, HCV, and CMV.

Characterization of an organism's sequence at specific loci to distinguish strains, subtypes, or resistance markers, as in hepatitis C genotype, HPV typing, and sequence-based outbreak comparison.

Broad-range amplification and sequencing of the bacterial ribosomal RNA gene, whose conserved and variable regions allow genus- or species-level identification directly from specimens, including organisms that do not grow in culture.

A one-base variation at a specific genomic position present in a population. SNPs serve as markers for disease association, pharmacogenetics, identity testing, and somatic mutation analysis.

Length changes in short tandem repeat sequences caused by defective DNA mismatch repair. It is tested in colorectal and other tumors to screen for Lynch syndrome and to predict immunotherapy response.

Loss of one allele at a heterozygous locus in tumor DNA, often reflecting deletion of a tumor suppressor gene. It is shown by comparing tumor and normal genotypes at polymorphic markers.

The small number of malignant cells remaining after treatment, detected by highly sensitive molecular or flow cytometric methods. It predicts relapse and helps guide therapy in leukemia and lymphoma.

An increase in the number of copies of a short tandem repeat within a gene, causing disorders such as Huntington disease and fragile X syndrome. It is detected by PCR sizing or Southern blot for very large expansions.

A template added to or co-extracted with each specimen and amplified in the same reaction to confirm that extraction and amplification worked and to flag inhibitors; its failure invalidates the result.

The lowest analyte concentration detected with a stated probability, usually 95%, in a given assay. It defines analytical sensitivity and must be verified by each laboratory before reporting patient results.

An enzyme that destroys uracil-containing DNA from earlier reactions to prevent carryover contamination. Assays use dUTP in master mixes so prior amplicons, but not the new target, are degraded before amplification.

Arranging laboratory space and workflow so that reagents, specimens, and amplicons move in one direction only, from clean to contaminated areas, preventing amplicon contamination of specimens and master mixes.

External assessment in which a laboratory tests blinded samples of known but undisclosed value and compares results with peer laboratories. Acceptable performance is required for accreditation and ongoing competency.

The ability of an assay to detect only the intended target and not related organisms, alleles, or nontarget sequences. It is documented by in silico analysis, testing of near neighbors, and interference studies.