ASCP Molecular Biology – Advanced Physical Assessment (2024) Comprehensive Study Notes

Histone–DNA Architecture

  • Type of bond between histone proteins and the DNA phosphate backbone

    • Ionic electrostatic attraction (basic histone → acidic DNA)

  • Histone amino-acid composition

    • Enriched in Lysine and Arginine → positively charged side-chains enhance DNA binding

  • Biological significance

    • Ionic interaction allows rapid, reversible chromatin remodeling during replication, repair, transcription

    • Post-translational modifications (acetylation of Lys, methylation of Arg/Lys) modulate bond strength / gene expression

Hematologic Malignancies & Cytogenetics

  • T-cell lymphoma with T-cell receptor rearrangements: Sezary Syndrome (leukemic variant of cutaneous T-cell lymphoma)

  • Acute Promyelocytic Leukemia (APL)

    • Classic reciprocal translocation t(15;17)(q24;q21)t(15;17)(q24;q21) → PML-RARα fusion → blocks promyelocyte maturation; responds to ATRA/arsenic

  • FLT3 mutations (ITD or TKD) in Acute Myeloid Leukemia (AML)

    • Constitutive tyrosine-kinase signaling → poor prognosis; candidate for FLT3 inhibitors

DNA & RNA Polymerases (Eukaryotic)

  • Leading-strand DNA synthesis: Polymerase δ\delta

  • Lagging-strand (Okazaki fragments): Polymerase α\alpha (initiating) followed by δ\delta

  • Repair polymerases: β\beta or ε\varepsilon (base-excision / mismatch)

  • Mitochondrial DNA polymerase: γ\gamma (POLG)

Next-Generation Sequencing (NGS)

  • Coverage (depth): number of independent reads containing a given base; higher coverage ↑ accuracy, ↑ rare variant detection

  • Paired-end sequencing

    • Reads are generated from both ends of a DNA fragment; superior for detecting small indels, structural rearrangements

  • Types of library chemistries

    • Pyrosequencing – visible light (luciferase-generated photons)

    • Molecular beacon general design: 5’-Fluorophore–loop (R sequence)–quencher-3’ → hairpin opens only on perfect hybridization

Human Genomic Variation

  • Single Nucleotide Polymorphisms (SNPs)

    • Genome-wide estimate ≈ 10610^{6} (book) – 10710^{7} (exam key) unique loci across population

Sample Storage & Stability

  • RNA (long-term ∼6 mo): store at −70 ∘C-70\,^\circ \text C in ethanol to limit RNase activity / hydrolysis

  • Whole blood for DNA extraction: stable 72 h at 4 ∘C4\,^\circ \text C (EDTA tube)

  • FFPE tissues: expect degraded / cross-linked DNA → shorter amplicon design (<150 bp)

  • Glassware RNase decontamination: bake 250 ∘C250\,^\circ \text C, 4 h

Virus & Transplant / Infectious Disease Markers

  • BK polyomavirus → viral nephropathy in renal transplant recipients (immunosuppressed)

  • HIV viral-load clinical significant change: ≥0.5 log⁡10\ge 0.5\,\log_{10} copies ⁄ mL

  • HCV genotypes

    • Genotype 2 → typically lower baseline viral load, best sustained virologic response to DAAs vs Genotype 1

Antibiotic & Drug Resistance Mechanisms

  • MRSA resistance: altered target Penicillin-Binding Protein 2a (PBP2a) encoded by mecA gene → reduced β-lactam affinity

  • Vancomycin resistance hierarchy (Enterococcus): vanA (highest MIC), vanB, vanC etc.

Pharmacogenomics & Drug Metabolism

  • Warfarin dosing: polymorphisms in VKORC1 (vitamin K epoxide reductase) and CYP2C9 (warfarin hydroxylation)

  • Irinotecan toxicity: reduced glucuronidation when UGT1A1*28 allele present → ↓ metabolism, ↑ SN-38 levels, neutropenia

  • Carbamazepine hypersensitivity (SJS/TEN) in HLA-B∗15:02*15:02 carriers (esp. Asian ancestry)

  • Trastuzumab (Herceptin): targets HER2/neu over-expression in breast / gastric cancers

Chromatin / DNA Forms & Melting

  • Canonical DNA in vivo: B-form, right-handed helix, 10.4 bp/turn

  • Formamide effect: lowers TmT_m by disrupting hydrogen bonds (destabilizes duplex)

  • Optimal PCR primer GC content: 50–60 % (balances annealing specificity & melting temperature)

PCR Fundamentals & Ordering of Reagents

  • Typical master mix: buffer (Mg2+^{2+}), dNTPs, polymerase, stabilizers; add to reaction tubes first to minimize pipetting error, then template DNA

  • Nested PCR > Real-time PCR > conventional: ascending specificity (two successive primer sets decrease off-target amplification)

  • Primer-dimer formation: 3’ complementarity between primers → non-specific low-m.w. bands / fluorescence background

PCR Troubleshooting & Optimization

  • Multiplex PCR low yield across all targets

    • Extend annealing time, fine-tune annealing TaT_a, adjust Mg2+^{2+} or primer concentrations

  • Two probes with disparate TmT_m (41 °C vs 53 °C)

    • Increase GC content / length of lower-TmT_m probe to equalize hybridization kinetics

  • Sample & positive-control both negative after spike → presence of broad-spectrum PCR inhibitor (e.g.

    • Hemoglobin, Heparin, Xylene, Phenol, Cyanol, Bromophenol Blue)

Laboratory QA / QC & Regulatory

  • Obsolete SOP retention (CAP): 2 years minimum

  • Thermal-device calibration traceable to NIST (National Institute of Standards and Technology)

  • Test complexity (CLIA): screening / validation differs for waived, moderate, high complexity → performance metrics required before implementation (accuracy, precision, reportable range, reference intervals, analytical sensitivity/specificity)

Analytical Performance Terms

  • Sensitivity: ability to detect disease when present (True-Positive Rate)

  • Specificity: ability to exclude disease when absent (True-Negative Rate)

  • Accuracy: closeness of measurement to true value

  • Precision (repeatability): closeness of replicate measurements to each other

  • Positive Predictive Value (PPV): proportion of positive results that are true positives PPV=TPTP+FP\text{PPV}=\frac{\text{TP}}{\text{TP+FP}}

Genetic Code Essentials

  • Start codon: AUG (Methionine)

  • Stop codons: UAA, UAG, UGA

Oncogenes & Tumor-Suppressor Genes

  • TP53 (p53) – guardian of genome, mutation common in diverse cancers; exam list mis-labels as “oncogene” (functionally tumor suppressor)

  • bDNA (branched DNA) signal-amplification assays

    • Does not amplify target nucleic acid, uses branched probes + alkaline phosphatase chemiluminescence to amplify signal; resistant to contamination

HLA & Immune Genetics

  • HLA-DR gene structure: polymorphic exon 2 encodes β-chain peptide-binding groove → sequenced for transplant matching/autoimmunity

DNA / RNA Structural Bonds & Enzymes

  • Intrastrand bonds: covalent phosphodiester within single strand (strong)

  • Interstrand bonds: hydrogen bonding between complementary bases (weak); denaturation disrupts interstrand H-bonds

  • Helicase vs Topoisomerase

    • Helicase separates parental strands (breaks H-bonds)

    • Topoisomerase relieves supercoils, untwists duplex ahead of fork

  • Primase: synthesizes RNA primer, providing 3’-OH to join 5’ → 3’ direction; functionally “connects” 5’ end of new RNA to 3’ end of DNA template during initiation

Triplet-Repeat Expansion Disorder

  • Fragile X syndrome: CGG repeat in FMR1 5’-UTR; >200 repeats → full mutation, hypermethylation, transcriptional silencing → intellectual disability

Electrophoresis Media Selection

  • 200–250 bp fragments: 6 % polyacrylamide gel (higher resolving power than agarose)

High-Performance Liquid Chromatography (HPLC)

  • Principle: liquid mobile phase pumped under high pressure through column containing stationary phase; separates analytes by polarity/affinity

    • Widely used for nucleoside quantitation, drug monitoring, oligo purity assessment

Miscellaneous Numerical / Laboratory Facts

  • GC clamp or 5’ cap on mRNA: 7-methyl-guanosine cap ↑ half-life & translation efficiency

  • Formamide inclusion in hybridization buffer decreases TmT_m ≈ ∼0.6 ∘C\sim 0.6\,^{\circ}\text C per % formamide (rule-of-thumb)

  • Oven-baking glassware at 250 ∘C250\,^\circ \text C destroys RNase; autoclaving alone insufficient

  • Coverage goals in NGS

    • Germline variant calling: ≥30×30\times; somatic cancer panels: 200–500×; mtDNA heteroplasmy, MRD require >1000×1000\times



What kind of bond do histones have with DNA? - Ionic

What amino acids compose histones? - Lysine and Arginine

Which lymphoma has t-cell rearrangements? - Sezary Syndrome

Acute Promyelocytic leukemia translocation? - t(15,17)

Leading strand polymerase: - Delta

DNA repair polymerase(s): - Beta or Epsilon

Mitochondrial polymerase: - Gamma

Molecular beacon design: - (R-sequence-Q)

Which method uses visible light? - Pyrosequencing

Lagging strand polymerase: - Alpha

In NGS what is coverage referring to - # of times a base is read

Which type of NGS is best used for small indels? - paired-end

Number of SNPs in human genome? - 10^7 book answer

10^6 exam answer (closest answer available)

FLT3 is involved in what disease? - AML

RNA storage condition for 6 months? - -70C in Ethanol

How long to keep obsolete document in lab according to CAP? - 2 years

HIV viral load difference? - 0.5 log 10

Antibiotic resistance mechanism in MRSA? - altered drug target

PBP2a

mecA

What is the effect of Irinotecan and UGT1A1? - Decrease drug metabolism

What part of RNA increases half-life and stability? - G cap or 5' cap

Formamide decreases Tm by: - altering H-bonds

Common form of DNA is: - B form, Right-Handed

Order to add to a PCR reaction - Master mix

Patient sample

Positive control

Negative control

Primer dimers are formed due to: - 3' Complementarity

Virus affecting patients after kidney transplant is - BK Virus

Warfarin is associated with? - VKORC1 and CYP2C9

Which is most specific PCR? - Nested PCR then Real Time PCR

What is the proper GC content of primer? - 50-60%

Which sample type to expect degraded DNA from? - FFPE

Highest vancomycin resistance? - vanA

HCV genotype 1 vs 2 : Which has a low viral load and best sustained response to

drugs? - HCV Genotype 2

To avoid RNAse at what temp should you bake the glassware? - 250*C for 4 hours

Aldehyde dehydrogenase question - Alcohol dependence

Metabolizing alcohol