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REI Written Boards > Lab Techniques/Genetics > Flashcards

Lab Techniques/Genetics Flashcards

1
Q

What does a Southern blot do?

A

Hybridizes DNA

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2
Q

What does a Western blot do?

A

Hybridizes protein

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3
Q

What does a Northern blot do?

A

Hybridizes RNA

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4
Q

What does a Southwestern blot do?

A

Hybridizes DNA binding proteins (southern=DNA, western=protein)

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5
Q

What is cDNA?

A

DNA synthesized from a single-stranded RNA template in reaction catalyzed by reverse transcriptase

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6
Q

What is PCR?

A

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)

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7
Q

What does H&E stain?

A

Haematoxylin stains nuceli of cells blue (does not require nucleic acids, stains arginine-rich nucleoproteins like histones)

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8
Q

What does Eosin Y stain?

A

Stains eosinophic structures red/pink (extracellular connective tissue, cytoplasm)

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9
Q

Describe the process of frozen section processing

A

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

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10
Q

What is a TUNEL assay?

A

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

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11
Q

What is PCR used (and not used) for?

A

o Used for single gene defects

o Cannot be used for large deletions

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12
Q

What are the steps of PCR and what is the exception?

A

o Steps: denaturation, annealing, extension

o Real-time PCR (qPCR) does not require whole genome amplification step

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13
Q

How does FISH work?

A

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.

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14
Q

What are the common uses of FISH?

A

Used for sex selection, structural anomalies, and aneuploidy screening

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15
Q

What is the downside of using FISH for polyploidy?

A

Polyploidy is labor intensive also to look for polyploidy due to limitation in number of fluorochromes and overlapping signals (-> aCGH)

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16
Q

How does Array CGH work?

A

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

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17
Q

How does NGS work?

A

Creates short DNA fragments that allow samples to hybridize to and determine sequences in parallel

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18
Q

What can NGS detect?

A

Segmental aneuploidy (down to 14MB), haploidy/polyploidy, and unbalanced translocations

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19
Q

Advantages of NGS?

A

No need to use control sample to co-hybridize; can detect mosaicism

20
Q

What is the secretion rate (with regards to hormones)?

A

Amount of hormone secreted by individual organ per unit time

21
Q

What is the production rate (with regards to hormones)?

A

Amount of hormone secreted by the organ + the amount of hormone produced through peripheral conversion per unit time

22
Q

What is the equation for production rate?

A

PR = concentration x MCR (metabolic clearance rate)

23
Q

Production rate of estradiol?

A

100-300 ug/day

24
Q

Production rate of androstenedione

A

3mg/day

25
Q

How does a radioimmunoassay (RIA) work?

A

o A mixture is prepared of:
• Radioactive antigen: radioactive isotopes 125I or 131I are often used
• Antibodies against that antigen
o Known amounts of unlabeled (“cold”) antigen are added to samples of the mixture (unknown = patient), which competes for the binding sites of the antibodies
o At increasing concentrations of unlabeled antigen, an increasing amount of radioactive antigen is displaced from the antibody molecules
o The antibody-bound antigen is separated from the free antigen in the supernatant fluid, and the radioactivity of each is measured
o From these data, a standard binding curve (see fig in notes) can be drawn
o The samples to be assayed (the unknowns) are run in parallel
o After determining the ratio of bound to free antigen in each unknown, the antigen concentrations can be read directly from the standard curve

26
Q

Other types of antigen excess (competitive) assays (other than RIA)

A
  • Flourimmunoassay (FIA)
  • Chemiluniscent assay (CIA)
  • Enzyme immunoassay (EIA)
27
Q

Immunoradiometric assays (IRMA) and hook effect?

A

Labeled antibodies (affected by hook effect)

28
Q

How does ELISA work?

A

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”

29
Q

What is the difference between older and newer ELISAs?

A

o Older ELISAs utilize chromogenic substrates, newer assays use fluorogenic substrates for higher sensitivity (chemical reaction)

30
Q

What are two major considerations for accuracy of ELISA?

A

o Need good specificity of antigen to antibody

o Need to periodically wash plate to remove non-specific binding proteins

31
Q

How does Mass Spec (LC/MS) work?

A

o LC permits separation of a mixture of liquid analytes into separate analyte fractions. This is achieved with the use of a column packed with a stationary phase which retains the analytes
o Steroids are retained on the column stationary phase at differing degrees based on their polarity, increasing the polarity of the mobile phase elutes the steroids sequentially.
o MS offers positive compound identification to enable the unequivocal identification of a compound free from interference, thus enabling accurate quantitation.

32
Q

If you want to make an assay for a hormone but can’t get the protein to accept radioactive iodine, why might that be?

A
  • No phenylalanine

* No tyrosine residue

33
Q

What is specificity with regards to an assay?

A

Capacity to measure only the substance in question

34
Q

How can specificity be quantified?

A
  • Compare cross reactivity of an antibody

* Compare to GC-MS (assay after purification step)

35
Q

How can specificity be improved?

A

Add dry milk to decrease nonspecific antibody binding

36
Q

What is sensitivity with regards to an assay?

A

Smallest amount of a substance that can be measured

37
Q

How can one measure sensitivity?

A

Use known antigen

38
Q

Example of low sensitivity with hormonal assay?

A

Testosterone assay = low sensitivity in female range

39
Q

What is assay drift?

A

Changes in signal level at a given concentration across the assay (put on ice?)

40
Q

What is a Scatchard Plot?

A

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

41
Q

What is a missense mutation?

A

Point mutation in which single nucleotide results in a codon that codes for a different amino acid

42
Q

What is a nonsense mutation?

A

Point mutation in a sequence of DNA that results in a premature stop codon

43
Q

What is a silent mutation?

A

Mutation that does not result in a change in the amino acid sequence of the protein

44
Q

What is an example of an activating (gain-of-function) mutation?

A

MEN2 – gain-of-function variant in the RET proto-oncogene

45
Q

What is an example of an inactivating (loss-of-function) mutation?

A

MEN1

REI Written Boards (20 decks)

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