Molecular Pathology Rotation Notes

UNIT 1

List three differences between DNA and RNA.
DNA (double stranded, thymine, deoxyribose, more stable; RNA single stranded, ribose, uracil

What are the three stop codons?
[UAA, UGA, UAG]]

Where does transcription begin?
promoters at the 5' end before the coding region

List 2 enzymes necessary for transcription and their function.
helicase, polymerase

List and describe three post transcription modifications of RNA.
Splicing, cappping, 3'polyadenylation,

Why is alternative splicing important?
Using the basic construction blocks of coding sequences allows a large variety of recombinations, more efficient coding (e.g. creating functions to call))

List three differences between somatic and germline mutations.
Somatic: not passed on to progeny, only tumour or particular tissue cells with mutation, Germline: passed onto progeny, all cells have mutation * unless mosaicism or chimerism

What is the difference between a missense and a non-sense mutation?
Missense the new base pair does not change the amino acid found in the protein at that location, non-sense changes the amino acid in the protein at that location

Define a frameshift mutation.
deletion of a non-multiple of 3 which causes all further trinucleotide combinations to no longer code for the correct amino acid, often results in a premature stop codon

Why are inversion mutations difficult to detect?
When the are smal, e.g. only a few base pairs

Describe the potential sequelae of a translocation mutation.
when a segment on one chromosome is transferred to another, make a gene non-functional or can result in a fusion gene

UNIT 2

Translate the following: c.1524_1527delCGTA.
a small deletion of CGTA between the 1524 and 1527 base pairs

List 5 features of SNPs.
[[Most common DNA sequence variation in humans, must occur in >=1% of a particular population, frequency of SNPs varies by groups, but responsible for >90% of human genetic variation, an can be found in any region of genome]]

Define a regulatory SNP versus a synonymous SNP?

What is the difference between a microstalellite and a minisattelite?
Microsatellite = stretches of DNA with sequences of 2-4 base pairs repeated a few dozen times (STRP), minisatellite = variable number of tandem repeats 10-100bp in lenght

Describe Hardy-Weinberg Equilibrium?
Mathematical probability function to describe allelic and genotype frequency in a random mating scenario

What factors can disrupt the H-W equilibrium?
non random mating, migration, genetic drift, founder effects, mutation, natural selection

What is linkage disequilibrium?
The closer two genes are together on the chromosome the more likely they are to be found toghether in a population, during meiosis some exchange of material happens between the two chromosomes

UNIT 3

What are the three major steps of PCR?
denaturing, primer annealing, strand extending

What is the hallmark of PCR?
The cycling at different temperatures, in the presence of key reaction components to traget and exponentially amplify a specific DNA target sequence

What factors affect the method of genotyping chosen?
throughput, type of variants that can be genotyped, equipment and costs, TAT, technical expertise, and multiplex ability

Define sensitivity, specificity, positive predictive value and negative predictive value.
Sensitivity = probability of a positive test in a disease, specificity = probability of a negative dest in a non-diseased patient

Define reproduciblity and accuracy of an analytical test.
Reproducability = probability of the test repeatedly producing the same reults in the same person, Accuracy = the degree to which the observed genotype matches the true genotype

Describe briefly Sanger sequencing.
DIdeoxynucleotides are used in a mix with deoxynucleotides, the Di*** terminate the chain, and so you get all possible lengths of chains so then you put them all in order and you can read (based on weight) which one is at each position

Describe briefly how Taqman automated genotyping is used for allele detection.
Microsatellite instability

How are DNA microarrays used to identify drug disposition or responses?
Microsatellite instability

UNIT 4

Describe the procedure for submitting FFPE slides for KRAS for colorectal cancer.
Microsatellite instability

Compare and contrast uniplex versus multiplex genotyping.
Microsatellite instability

Compare and contrast conventional vs massively parallel sequencing.
Microsatellite instability

What is multiplex ligation-dependent ligation (MLPA)?
Microsatellite instability

What is fragment analysis?
Microsatellite instability

Compare and contrast RT-PCR vs qRTPCR.
Microsatellite instability

What is MSI?
Microsatellite instability

What is methylation analysis?
Microsatellite instability

UNIT 5

What are the four test features required to be documented by the CLIA?
Microsatellite instability

What are "in vitro diagnostics" vs "laboratory developed tests"?
Microsatellite instability

What does validation mean?
Microsatellite instability

What are the four performance characteristics that need to be verified for FDA cleared/approved tests?
Microsatellite instability

What are the six performance characteristics that need to be verified for FDA cleared LDTs or modified FDA cleared/approved tests?
Microsatellite instability

UNIT 6

List the components of a molecular pathology report.
Microsatellite instability

Define analytical sensitivity and clinical sensitivity.
Microsatellite instability

What should be said in a report of a molecular test on a patient for residual disease if no previous positive assay was confirmed?
Microsatellite instability

Define ammended report versus addendum report.
Microsatellite instability

Whose responsibility is it to sythesize the test results with other clinico-pathological information?
Microsatellite instability

How long are cytogenetic reports required to be kept by CAP?
Microsatellite instability

What is the recommended process to use test results if an assay is not yet validated for clinical use?
Microsatellite instability

Give three examples of "grey areas" which warrant discretion of professionals involved to use a non-validated test?
Microsatellite instability

What reference standard is available for gene nomenclature?
Microsatellite instability

Create a table of the most common gene rearrangements associated with heme and soft tissue diseases.
Microsatellite instability

What is a "DNA fingerprint" and what can it be used for?
A method that examines multiple areas of short tandem repeats to identify paternity, mosaicism, chimerism, and identity in forensics cases