Lab Techniques/Genetics Flashcards
What does a Southern blot do?
Hybridizes DNA with probe
What does a Western blot do?
Hybridizes proteins w/ antibodies
What does a Northern blot do?
Hybridizes RNA with probe
What does a Southwestern blot do?
Hybridizes DNA binding proteins (southern=DNA, western=protein)
What is cDNA?
DNA synthesized from a single-stranded RNA template in reaction catalyzed by reverse transcriptase
What is PCR?
Technique used to amplify the amount of known fragment of DNA; sequence must be known (high temp to denature -> single strand, lower temp to anneal primers (20-30bp), medium temp for DNA polymerase -> dsDNA)
What does H&E stain?
Haematoxylin stains nuceli of cells blue (does not require nucleic acids, stains arginine-rich nucleoproteins like histones)
What does Eosin Y stain?
Stains eosinophic structures red/pink (extracellular connective tissue, cytoplasm)
Describe the process of frozen section processing
Tissue is frozen and sliced thinly using a microtome mounted in a below freezing cryostat. The thin sections are mounted on a glass slide and fixed in a liquid fixative and stained similar to traditional was embedded sections
What is a TUNEL assay?
Method for detecting DNA fragmentation that results from apoptotic signaling cascades. Relies on presence of nicks in DNA which can be identified by terminal deoxynucleotidyl transferase (TdT) an enzyme that will catalyze the addition of dUTPs secondarily labeled with a marker
What is PCR used (and not used) for?
o Used for single gene defects
o Cannot be used for large deletions
What are the steps of PCR and what is the exception?
o Steps: denaturation, annealing, extension
o Real-time PCR (qPCR) does not require whole genome amplification step
How does FISH work?
o Uses larger stretches of DNA (approximately 50 to 200 kb probes) labeled with fluorescence reagents to target specific genome regions
o Labeled probes are hybridized to either metaphase chromosomes or interphase nuclei, used for detection of chromosome number (autosomes and sex-chromosomes), translocations, inversions or deletions.
What are the common uses of FISH?
- sex selection
- structural anomalies
- aneuploidy screening
What is the downside of using FISH for polyploidy?
Polyploidy is labor intensive also to look for polyploidy due to limitation in number of fluorochromes and overlapping signals (-> aCGH)
How does Array CGH work?
o Requires whole genome amplification to increase the template available
o Sample DNA labeled with one fluorochrome, normal control labeled with another
o Sample and control hybridized to DNA microarray (no need for metaphase spread like traditional CGH)
o Microarray slide spotted with segments of DNA, DNA libraries, or bacterial artificial chromosomes (BACs)
o Analyzed by microarray reader and specialized software to determine intensity of fluorochromes/copy number
How does NGS work?
Creates short DNA fragments that allow samples to hybridize to and determine sequences in parallel
What can NGS detect?
- Segmental aneuploidy (down to 14MB)
- haploidy/polyploidy
- unbalanced translocations
Advantages of NGS?
- No need to use control sample to co-hybridize
- can detect mosaicism
How does ELISA work?
o Enzyme used to label either antigen or antibody
o Unknown amount of antigen is affixed to a surface (either specifically - using antibody capture OR non-specifically - using adsorption), and a specific antibody is washed over the surface to bind to the antigen
o Antibody is covalently linked to an enzyme, and in the final step a substrate is added that the enzyme can convert to some detectable signal (light, color, etc) – “sandwich”
What is a Scatchard Plot?
o Ratio of concentrations of bound ligand (B) to unbound (U) ligand versus the bound ligand concentration
o Method for analyzing data for freely reversible ligand/receptor binding interactions.
o Kd= affinity of ligand for receptor, concentration of ligand that occupies 50% receptors
o As ligand concentration increases, specific binding to the receptors increases until all sites filled at Bmax
pic caption: Examples of the Scatchard plot, titration curve, and Hill plot. The Scatchard plot is generally used to determine the affinity of the receptor for its ligand and the number of binding sites; the titration curve best shows how the affinity is determined by points above and below Kd, and shows the whole range of response; the Hill Plot is generally used to determine the cooperativity of the ligand–receptor interaction.
