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Rotation in Molecular Diagnostics

    Questions

    HPV
    1. Explain the process of junctional diversification. What are P- and N-nucleotides and what is their significance for clonality testing?
    2. What is the difference between somatic recombination and somatic hypermutation? What is the physiological function of somatic hypermutation?
    3. What regions contain the highest variability in the TCR and the IGH molecules?
    4. What is the advantage of utilizing the TCRß rather than the TCR? locus for clonality testing?
    5. What role does somatic hypermutation play in the generation of TCR and IGH diversity and antigen recognition?

    Direct Sequencing
    1. When is a missense mutation significant?

    K-ras
    1. Find the genomic sequence for the kras1 gene.
    2. Check for presence of a pseudogene.
    3. How could you design primers that do not amplify the pseudogene?
    4. Locate codons 12 and 13.
    5. Design intronic primers for amplification of a segment containing codons 12 and 13.
    6. Prepare a protocol for how to PCR-amplify this segment.
    7. Compare different approaches for detection of codon 12 and 13 mutations?
      8. - FRET: Approaching Real-Time Molecular Diagnostics: Single-Pair Fluorescence Resonance Energy Transfer (spFRET) Detection for the Analysis of Low Abundant Point Mutations in K-ras Oncogenes. J Am Chem Soc. 2003 Jun 11;125(23):6937-45
      - PNA clamp: Detection of rare mutant K-ras DNA in a single-tube reaction using peptide nucleic acid as both PCR clamp and sensor probe. Nucleic Acids Research. 2006, Vol. 34, No. 2, e12
      - ARMS: Comparison of the novel quantitative ARMS assay and an enriched PCR-ASO assay for K-ras mutations with conventional cytology on endobiliary brush cytology from 312 consecutive extrahepatic biliary stenoses. J Clin Pathol. 2005 Dec;58(12):1315-20
      K-ras point mutation detection in lung cancer: comparison of two approaches to somatic mutation detection using ARMS allele-specific amplification. Clin Chem. 2000 Dec;46(12):1929-38
      - Scorpion primers: Duplex Scorpion primers in SNP analysis and FRET applications. Nucleic Acids Res. 2001 Oct 15;29(20):E96
      Detection of PCR products using self-probing amplicons and fluorescence. Nat Biotechnol. 1999 Aug;17(8):804-7

    JAK2
    1. What strategies can be used to detect one specific point mutation?
    2. What are advantages and pitfalls of the different strategies with regard to sensitivity/specificity, susceptibility to false positive/false negative results and contamination, hands-on time, potential for automatization, and cost?

    Fragile X
    1. What are problems with PCR-based tests for Fragile X syndrome?

    Prader-Willi/Angelman
    1. List different genetic mechanisms for a Prader-Willi/Angelman phenotype.