Lab Techniques Flashcards
Polymerase chain reaction (PCR)
•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?)
PCR ingredients
- sample DNA
- DNA polymerase
- primer: single stranded DNA segment complementary to DNA under evaluation
- nucleotides
PCR technique
- 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
quantitative PCR
- =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
PCR clinical uses
- herpes simplex virus encephalitis (DNA virus in CSF)
* HIV viral load
southern blot is for
identifies DNA
northern blot identifies
RNA
western blot identifies
protein
probe
- aka cDNA
- single stranded DNA molecule
- carries radioactive or chemical markers
- binds complementary sequences
hybridization
when the probe binds to the DNA you wish to identify
steps of southern blot
- 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
Restriction fragment length polymorphisms
- 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
sickle cell anemia
- southern blotting can be used for diagnosis
* looking at β-globin gene
northern blot technique
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
northern blotting useful for
assessing mRNA levels -> gene expression
western blot technique
•separate proteins by size with electrophoresis
•mix antibody specific to protein of interest
•
western blot clinical uses
look for antibodies
•IgG of IgM in lyme disease
•IgG of HIV-1
Southwestern blot
- used to study DNA protein interactions, especially transcription factors
- proteins separated by electrophoresis
- DNA probe added
flow cytometry
flow=motion of fluid
cytometry = measurement of cells
flow cytometry = analysis of cells as they flow in a liquid through a narrow stream
key of flow cytometry
- used to analyze cells
* can be based on size or surface proteins
forward scatter of light in flow cytometry indicates
size of cells
side scatter of light in flow cytometry indicates
granularity of cells
antibody staining and flow cytometry
- 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
fetal maternal hemorrhage
- 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
paroxysmal noctural hemoglobinuria (PNH)
- flow cytometry
- fluorescently labeled monoclonal antibodies
- you’ll see reduced/absent binding on red blood cells with PNH
CRISPR stands for
clustered regularly-interspaced short palindromic repeats
cas genes
- =crisper associated genes
- make proteins, specifically helicases and nucleases
- make proteins that make CRISPR RNA that can break apart bacterial dna
spacer DNA
unique segments of DNA found between CRISPRs that indicate a history of infections
CAS9
looks at S. pyogenes
ELIZA
- enzyme linked immunosorbent assay
* detects antigens and antibodies in serum based on color change reaction
direct ELIZA
- 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
indirect ELIZA
first antibody is not enzyme linked
secondary antibody is enzyme linked and binds to the first antibody
sandwich ELIZA
- plate coated with capture antibody
- sample added -> antigen will bind
- add detecting antibody that also binds antigen
- then indirect or direct ELIZA
advantages of sandwich ELIZA
high specificity
works with complex samples
can use secondary antibody like indirect
competitive ELIZA
- 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
ELIZA uses
- diagnose HIV antibodies in serum
* HIV p24 antigen detection
SNoW DRoP
Southern = DNA Northern = RNA Western = Protein (Southwestern = DNA binding proteins)
