Testing Modalities and Genetic Diseases Flashcards
Steps of PCR
Denaturation, Annealing, Elongation
What is single-base primer extension used for? How does it work?
To identify known single nucleotide mutation; mutant and wild type nucleotides are labeled with different fluorescent colors- gives yes mutation or no mutation
What is Sanger sequencing used for? How does it work?
Dead-end, fluorescent nucleotides creates ladder which is organized in size order and read out to sequence and compare to normal, tells you if mutations and where
How is next generation sequencing different?
Can use heterogeneous samples- tumor cells mixed with stromal cells- and still find mutations, vs single homogeneous template DNA with Sanger
What is amplicon length analysis used for?
Mutations that make strands longer or shorter than usual
What is restriction fragment length analysis used for?
Mutations at restrictions sites make strands longer than usual because they arent cleaved at the mutated site
What are hybridization based techniques used for? Examples?
Complex rearrangements like CNVs; ISH, cytogenomic microarray
What is cytogenomic microarray used for?
Don’t need know region where mutation is, measures differences between normal and abnormal DNA based on color or intensity
What kind of epigenetic alteration causes genetic disorders?
Increased methylation (silencing) of DNA
How do you identify epigenetic alterations?
Sodium bisulfite technique, to discriminate unmethylated from methylated cytosine
CpG islands in normal cells are; in neoplastic cells are
Unmethylated (housekeeping genes)
Hypermethylated
Difference between a missense and a nonsense mutation
Both point mutations, missense alters the code and a different AA is inserted, nonsense changes to a stop codon, so truncated
What happens with a point mutation in a non-coding sequence?
May prevent transcription, so product not synthesized
Germ line cell mutations are ______________; Two types
Heritable; Autosomal or X linked
Features of autosomal dominant mutations; example
Incomplete penetrance, no sex predilection, usually LOF of non enzyme structural proteins; PCKD
Features of autosomal recessive mutations; example
Complete penetrance, no sex predilection, usually LOF of enzyme; LSD
Almost all X-linked disorders are; example?
Recessive; hemophilia
What is base excision repair for?
Replacing damaged bases
What is nucleotide excision repair for? What is the most common example?
Replacing damaged DNA; adducts (covalently bound chemical)
What is DNA mismatch repair for?
Switching out erroneous bases
What mode of inheritance occurs with the MDR1 gene mutation? What is the result?
Autosomal dominant; can’t transport drugs out of brain due to defect in membrane P-glycoprotein
How are lysosomal enzymes trafficked in the cell?
Synthesized in ER, moved to golgi, tagged with mannose-6-phosphate, bind mannose receptors on inner surface of golgi, pinch off, fuse with lysosomes
What accumulates in Pompe disease? In Tay Sachs?
Glycogen; gangliosides (sphingolipids)
What is glycogen?
The storage form of glucose
Enzyme that degrades glycogen into glucose in lysosomes?
Alpha glucosidase
What causes hepatic glycogenosis?
Deficiency in Glucose-6-phosphatase (converts G-6-P back into glucose)
What causes myopathic glycogenosis?
Deficiency in phosphorylases (converts glycogen to glucose) or phosphofructokinase (glycolysis)
What is familial hypercholesterolemia usually caused by?
Mutation in LDL receptor gene or ApoB
Why are trinucleotide repeats non-classic inheritance? What disease do they cause?
Expansions occur during oo or spermatogenesis; Huntington’s
Why are mitochondrial DNA mutations non-classic inheritance?
All comes from female, so maternal inheritance
