Lab Techniques

Question 1

Polymerase chain reaction (PCR)

Answer 1

•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?)

Question 2

PCR ingredients

Answer 2

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

Question 3

PCR technique

Answer 3

• 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

Question 4

quantitative PCR

Answer 4

• =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

Question 5

PCR clinical uses

Answer 5

• herpes simplex virus encephalitis (DNA virus in CSF)

* HIV viral load

Question 6

southern blot is for

Answer 6

identifies DNA

Question 7

northern blot identifies

Answer 7

RNA

Question 8

western blot identifies

Answer 8

protein

Question 9

probe

Answer 9

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

Question 10

hybridization

Answer 10

when the probe binds to the DNA you wish to identify

Question 11

steps of southern blot

Answer 11

• 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

Question 12

Restriction fragment length polymorphisms

Answer 12

• 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

Question 13

sickle cell anemia

Answer 13

• southern blotting can be used for diagnosis

* looking at β-globin gene

Question 14

northern blot technique

Answer 14

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

Question 15

northern blotting useful for

Answer 15

assessing mRNA levels -> gene expression

Question 16

western blot technique

Answer 16

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

Question 17

western blot clinical uses

Answer 17

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

Question 18

Southwestern blot

Answer 18

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

Question 19

flow cytometry

Answer 19

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

Question 20

key of flow cytometry

Answer 20

• used to analyze cells

* can be based on size or surface proteins

Question 21

forward scatter of light in flow cytometry indicates

Answer 21

size of cells

Question 22

side scatter of light in flow cytometry indicates

Answer 22

granularity of cells

Question 23

antibody staining and flow cytometry

Answer 23

• 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

Question 24

fetal maternal hemorrhage

Answer 24

• 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

Question 25

paroxysmal noctural hemoglobinuria (PNH)

Answer 25

* flow cytometry * fluorescently labeled monoclonal antibodies * you'll see reduced/absent binding on red blood cells with PNH

Question 26

CRISPR stands for

Answer 26

clustered regularly-interspaced short palindromic repeats

Question 27

cas genes

Answer 27

* =crisper associated genes * make proteins, specifically helicases and nucleases * make proteins that make CRISPR RNA that can break apart bacterial dna

Question 28

spacer DNA

Answer 28

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

Question 29

CAS9

Answer 29

looks at S. pyogenes

Question 30

ELIZA

Answer 30

* enzyme linked immunosorbent assay | * detects antigens and antibodies in serum based on color change reaction

Question 31

direct ELIZA

Answer 31

* 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

Question 32

indirect ELIZA

Answer 32

first antibody is not enzyme linked | secondary antibody is enzyme linked and binds to the first antibody

Question 33

sandwich ELIZA

Answer 33

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

Question 34

advantages of sandwich ELIZA

Answer 34

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

Question 35

competitive ELIZA

Answer 35

* 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

Question 36

ELIZA uses

Answer 36

* diagnose HIV antibodies in serum | * HIV p24 antigen detection

Question 37

SNoW DRoP

Answer 37

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