ASCP MB cheat sheet

Central dogma: DNA → RNA → protein; reverse transcriptase synthesizes cDNA from an RNA template.

Base pairing: A pairs with T by two hydrogen bonds; G pairs with C by three hydrogen bonds; GC-rich duplexes have higher melting temperatures.

Short-primer Tm estimate: Tm ≈ 2(A+T) + 4(G+C) °C; nearest-neighbor methods are preferred for longer or mismatched primers.

Dilution formula: C1V1 = C2V2, where C is concentration and V is volume.

Copy number: copies/µL = (DNA ng × 6.022×10^23) / (fragment bp × 660 × 10^9).

Restriction endonucleases recognize specific palindromic sequences; CpG methylation can block cleavage.

Sanger sequencing uses dideoxynucleotide chain termination and capillary electrophoresis; each terminated fragment ends at a specific base.

In gel electrophoresis, DNA migrates toward the anode; migration distance increases as fragment size decreases.

Hardy-Weinberg: p² + 2pq + q² = 1 for genotype frequencies at an autosomal locus with two alleles.

EDTA (lavender) chelates calcium and is preferred for plasma or whole-blood molecular tests; heparin can inhibit PCR.

Citrate (light blue) and serum tubes are acceptable only when the assay has been validated for that matrix; plasma viral load assays typically specify EDTA plasma.

Separate plasma from cells promptly, usually within 4–6 hours of collection, and freeze aliquots to limit nucleic acid degradation.

Formalin fixation crosslinks macromolecules and fragments RNA/DNA; FFPE specimens require proteinase K digestion and assays designed for short amplicons.

RNAlater preserves RNA by inactivating RNases; specimens must be kept cold or frozen according to the manufacturer's instructions.

Silica-based extraction: chaotropic salts and alcohol promote nucleic acid binding to silica; washes remove proteins and salts; low-salt buffer elutes nucleic acid.

Phenol-chloroform extraction: at pH 8, DNA partitions into the aqueous phase; acidic phenol retains RNA in the aqueous phase and DNA in the interphase/organic phase.

Purity: A260/A280 ≈ 1.8 for DNA and ≈ 2.0 for RNA; A260/A230 below ~2.0 suggests guanidine or phenol contamination.

Use DNase/RNase-free consumables and separate pre- and post-amplification areas to protect extracted nucleic acid.

PCR phases: exponential (maximal efficiency), linear (reagents limiting), and plateau (product no longer doubles).

Mg²⁺ is a cofactor for DNA polymerase; low Mg²⁺ reduces yield, while excess Mg²⁺ promotes nonspecific amplification.

Annealing temperature is typically set about 5°C below the primer Tm; higher annealing temperatures increase specificity.

Hot-start polymerases prevent extension during reaction setup, reducing primer dimers and nonspecific products.

qPCR Ct is inversely related to starting template: a 1-cycle lower Ct represents approximately a 2-fold higher target amount; 3.32 cycles represents 10-fold.

Standard-curve efficiency: E = 10^(−1/slope) − 1; slope −3.32 ≈ 100% efficiency, and acceptable assays often show 90–110% efficiency.

Hydrolysis probes (TaqMan) depend on 5′→3′ exonuclease activity; SYBR Green detects any double-stranded product and requires melt-curve analysis.

Hybridization stringency increases with higher temperature, lower salt, and formamide; stringent conditions favor perfect matches over mismatches.

Multiplex PCR requires primers with similar Tm and minimal cross-dimerization; each target's amplicon should be distinguishable by size, probe color, or melt peak.

Nucleic acid tests detect pathogens earlier than serology because nucleic acid appears before antibodies, shortening the diagnostic window period.

Quantitative viral load is reported in IU/mL or copies/mL; changes are often expressed as log10, where a 1-log change is 10-fold.

Broad-range bacterial PCR targets conserved 16S rRNA regions flanking variable sequences; sequencing identifies organisms but does not establish viability.

Fungal broad-range PCR often targets ITS regions; mycobacterial assays may target IS6110, rpoB, or 16S rRNA depending on the species and purpose.

Drug-resistance testing detects mutations: HIV reverse transcriptase/protease/integrase, HCV NS5A/NS5B, HBV polymerase, and M. tuberculosis rpoB/katG/inhA.

Contamination control for infectious NAT includes unidirectional workflow, aliquoted reagents, negative controls, and wipe-down with freshly prepared hypochlorite.

A negative NAT does not exclude infection when the specimen is collected too early, contains inhibitors, or is improperly transported.

Respiratory virus multiplex panels use RT-PCR for RNA viruses and PCR for DNA viruses; cycle threshold values are not interchangeable between assays.

CT/NG NAATs are preferred for urogenital screening because they are more sensitive than culture; positive results should be interpreted with assay-specific confirmatory algorithms.

HGVS nomenclature: c. denotes coding DNA, g. genomic, m. mitochondrial, and p. protein; variants are described relative to a reference sequence.

Variant allele fraction (VAF) = variant reads / total reads; germline heterozygous variants usually approximate 50%, whereas somatic variants are often lower.

ACMG/AMP categories are pathogenic, likely pathogenic, variant of uncertain significance, likely benign, and benign; classification combines population, functional, and segregation evidence.

Germline testing uses constitutional DNA, often from blood; somatic testing uses tumor tissue or plasma and may detect acquired mutations.

BCR-ABL1 from t(9;22) is monitored by quantitative RT-PCR; rising transcript levels can indicate loss of treatment response.

JAK2 V617F is common in polycythemia vera, essential thrombocythemia, and primary myelofibrosis; allele burden can be measured quantitatively.

EGFR T790M confers resistance to early-generation tyrosine kinase inhibitors; KRAS codon 12/13 mutations predict resistance to EGFR-directed antibodies in colorectal cancer.

Microsatellite instability reflects mismatch repair deficiency; MSI-high tumors may respond to immune checkpoint inhibitors and can indicate Lynch syndrome.

Cell-free DNA screening estimates trisomy 21, 18, and 13 risk; it is a screening test, and abnormal results require diagnostic confirmation.

Clinical molecular laboratories follow CLIA and, when accredited, CAP requirements; validation is required before clinical use of a new assay.

Each run should include positive and negative controls and a no-template control; a contaminated no-template control invalidates negative results.

Limit of detection is the lowest concentration detected in ≥95% of replicates; precision studies assess repeatability and reproducibility.

Proficiency testing uses blinded external samples; unacceptable performance requires root-cause analysis, corrective action, and documentation.

Unidirectional workflow moves from reagent preparation to specimen processing to amplification to detection; never move amplicons back into pre-amplification areas.

Most clinical molecular work is performed at BSL-2 with gloves, gown, eye protection, and a biosafety cabinet for aerosol-generating procedures.

Decontaminate surfaces with freshly prepared 10% bleach or a validated disinfectant; allow contact time and then remove bleach residue.

Equipment must be calibrated and maintained per schedule; temperature, pipettes, thermocyclers, and biosafety cabinets require documented monitoring.

Document control ensures only current procedures are in use; audit trails and corrected reports must preserve original results and reasons for change.