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Molecular Foundations of Medicine > Lab Techniques > Flashcards

Lab Techniques Flashcards

1
Q

Polymerase chain reaction (PCR)

A

•amplifies copies of DNA in sample
Uses:
•make more DNA from small amount
•determine if DNA is present (does it amplify?)
•determine amount of DNA (how quickly does it amplify?)

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2
Q

PCR ingredients

A
  • sample DNA
  • DNA polymerase
  • primer: single stranded DNA segment complementary to DNA under evaluation
  • nucleotides
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3
Q

PCR technique

A
  • heat sample (denatures DNA into single strands)
  • cool sample (primer anneals complementary DNA if present)
  • warm sample (DNA pol elongates from primer)
  • repeated in cycles
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4
Q

quantitative PCR

A
  • =real time PCR
  • done in presence of fluorescent dye
  • amount of dye proportional to amount of DNA
  • rapid increase in florescence = more DNA in sample
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5
Q

PCR clinical uses

A
  • herpes simplex virus encephalitis (DNA virus in CSF)

* HIV viral load

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6
Q

southern blot is for

A

identifies DNA

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7
Q

northern blot identifies

A

RNA

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8
Q

western blot identifies

A

protein

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9
Q

probe

A
  • aka cDNA
  • single stranded DNA molecule
  • carries radioactive or chemical markers
  • binds complementary sequences
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10
Q

hybridization

A

when the probe binds to the DNA you wish to identify

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11
Q

steps of southern blot

A
  • DNA sample mixed with restriction nucleases -> enzymatic cleavage
  • cleaved pieces are placed on gel electropohresis -> size separation
  • transfer to filter paper
  • add probe
  • wash away unbound probe
  • filter paper exposed to film -> bound DNA revealed
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12
Q

Restriction fragment length polymorphisms

A
  • a clinical use for southern blotting
  • restriction nucleases to cut base at specific base sequence
  • different genes = different lengths of fragments
  • can be used to determine genotypes
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13
Q

sickle cell anemia

A
  • southern blotting can be used for diagnosis

* looking at β-globin gene

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14
Q

northern blot technique

A

same as southern blot, but for RNA
•RNA sample mixed with restriction nucleases -> enzymatic cleavage
•cleaved pieces are placed on gel electropohresis -> size separation
•transfer to filter paper
•add probe
•wash away unbound probe
•filter paper exposed to film -> bound RNA revealed

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15
Q

northern blotting useful for

A

assessing mRNA levels -> gene expression

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16
Q

western blot technique

A

•separate proteins by size with electrophoresis
•mix antibody specific to protein of interest
•

17
Q

western blot clinical uses

A

look for antibodies
•IgG of IgM in lyme disease
•IgG of HIV-1

18
Q

Southwestern blot

A
  • used to study DNA protein interactions, especially transcription factors
  • proteins separated by electrophoresis
  • DNA probe added
19
Q

flow cytometry

A

flow=motion of fluid
cytometry = measurement of cells
flow cytometry = analysis of cells as they flow in a liquid through a narrow stream

20
Q

key of flow cytometry

A
  • used to analyze cells

* can be based on size or surface proteins

21
Q

forward scatter of light in flow cytometry indicates

A

size of cells

22
Q

side scatter of light in flow cytometry indicates

A

granularity of cells

23
Q

antibody staining and flow cytometry

A
  • add antibodies that bind to surface or intracellular proteins on cells
  • tag with unique florochrome
  • flow cytometer can detect the florochrome if it’s bound, which indicates a specific protein in those cells
24
Q

fetal maternal hemorrhage

A
  • fetal red cells cross placenta to maternal blood (seen with placental failure/trauma)
  • flow cytometry can be used -> monoclonal antibodies to hemoglobin F
  • detects fetal hemoglobin in red cells of maternal circulation
25
Q

paroxysmal noctural hemoglobinuria (PNH)

A
  • flow cytometry
  • fluorescently labeled monoclonal antibodies
  • you’ll see reduced/absent binding on red blood cells with PNH
26
Q

CRISPR stands for

A

clustered regularly-interspaced short palindromic repeats

27
Q

cas genes

A
  • =crisper associated genes
  • make proteins, specifically helicases and nucleases
  • make proteins that make CRISPR RNA that can break apart bacterial dna
28
Q

spacer DNA

A

unique segments of DNA found between CRISPRs that indicate a history of infections

29
Q

CAS9

A

looks at S. pyogenes

30
Q

ELIZA

A
  • enzyme linked immunosorbent assay

* detects antigens and antibodies in serum based on color change reaction

31
Q

direct ELIZA

A
  • add serum to well plate that is designed to secure antigen, wash away fluid leaving only the antigen
  • add enzyme labeled antibody
  • wash away unbound antibodies
  • add substrate -> color change by enzyme
32
Q

indirect ELIZA

A

first antibody is not enzyme linked

secondary antibody is enzyme linked and binds to the first antibody

33
Q

sandwich ELIZA

A
  • plate coated with capture antibody
  • sample added -> antigen will bind
  • add detecting antibody that also binds antigen
  • then indirect or direct ELIZA
34
Q

advantages of sandwich ELIZA

A

high specificity
works with complex samples
can use secondary antibody like indirect

35
Q

competitive ELIZA

A
  • primary antibody incubated with sample
  • antigen-antibody complex
  • more antigen -> more binding -> less free antibody
  • add to antigen coated plates
  • more color change -> less antigen present in initial sample
36
Q

ELIZA uses

A
  • diagnose HIV antibodies in serum

* HIV p24 antigen detection

37
Q

SNoW DRoP

A 
Southern = DNA
Northern = RNA
Western = Protein
(Southwestern = DNA binding proteins)

Molecular Foundations of Medicine (21 decks)

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