Molecular Diagnostics

Question 1

hybridization

Answer 1

ssDNA binds to DNA or RNA making DNA-DNA hybrid or DNA-RNA hybrid

detection and quantification of target DNA or RNA

uses ss oligonucleotides as probes

target DNA is converted to ssDNA then immobilized and then hybridized to complementary sequence

Question 2

examples of hybridization

Answer 2

southern blotting and norther blotting

Question 3

southern blotting

Answer 3

both probe and target nucleic acid are DNA

Question 4

norther blotting

Answer 4

probe is single stranded DNA and target is mRNA

Question 5

PCR

Answer 5

dsDNA obtained, subjected to denaturation

primers comlement each end of the DNa in the 3-5’ direction and anneal

dNTPs are added (all four)

Taq polymerase synthesizes copies of FNA by extending primers on both ends. DNA doubles each cycle and amplifies

advantage: small amount of DNA needed
disadvanetage: ned to know sequence of dlanking DNA at the end for primer design, error prone, could aplify contaminants

Question 6

cell-free cloning PCR

Answer 6

uses DNA samples, primers, heat stable DNA polymerase, dNTPS, thermocycler

amplifies isolated DNA regions

useful for early detection of HIV, specific mutations, CF, thalassmeia, fragile X (is this what Progenity uses?)

Question 7

qPCR

Answer 7

real time

quanitify copy number of specific gene in two or more samples

add primers and fluorecscnt probe that shows up in presence of PCR product

detects levels of infectious agent or gene expresion

Question 8

Restriction Fragment Length Polymorphism

Answer 8

individual geneomes differ by 1 in every 1000 bases and some of these ifferences occur in recognition sequences for restriction enzymes

used in DNA fingerprinting, forensic analysis, paternity testing, disease detection

Question 9

RLFP detection of mutations

Answer 9

normal B globulin allele has 3 Ddel restriction sites while pts with sick cell only have two restriction sites

cleave DNA followed by Ddel and do electrophoresis and southern blotting with specific probe to detect mutation

Question 10

Variable number of tandem repeats

Answer 10

pattern of short tandem repeats within the genome that varies in individuals

useful in ID and severity of inherited diseases like Huntington’s, fragile X, fredircik ataxia

Question 11

recombinant proteins

Answer 11

insulin
growth hormone
erythropoietin 
clotting factors 
vaccines for flu and malaria

Question 12

to make recombinant proteins

Answer 12

cDNA of the protein is inserted into an expression vector to allow for high levels of replication, trascriptiona nd translation which can then be purified in a lab

Question 13

recombinant proteins can make

Answer 13

modified forms of insulin with bettwe biolpgical activity

Question 14

normal human insulin has

Answer 14

proline at position 28 and lysin at position 29 at the C terminus of the B chain

Question 15

Lispro

Answer 15

reverses the position of 28proline and 29lysine

Question 16

both lispro and insulin apart are

Answer 16

fast acting, more readily absorbed

normal insulin mized with Lispro provides longer range of glycemic control

Question 17

production of antibodies

Answer 17

used as drugs and for research purposes

Question 18

monoclonal Ab are

Answer 18

specific for a single epitope on an antigen

Question 19

single cones of B lymphcytes are fused with a tumor cell to

Answer 19

immortalize it

Question 20

humanization minimizes

Answer 20

immunogenisty of antibodies to prolong half life in pt

Question 21

ELISA

Answer 21

immunological test for the levels of specific antigens or antibody concentrations

Question 22

indirect Elisa measures

Answer 22

amount of antibody in a sample

Question 23

Sandwich Elisa measures

Answer 23

amount of an antigen in a sample

Question 24

HIV and Elisa

Answer 24

specific antibodies to HIV are produced in humans 4-6 weeks after infection

Elisas are used for detection because they are cheap and effective

can produce false positives and false negatives

needs confirmation with western blot

INDIRECT ELisa

Question 25

MI detection and Elisa

Answer 25

toponin in heart has three subunits

cardiac forms of T and I increase in MI

measured with SANDWICH Elisa

Question 26

Elisa and Pregnancy Tests

Answer 26

reaction site is the free HCG antibodies which binds to the HCG in urine and complex moves to test site

test site has HCG ab and the complex of AbACG from reaction site moves to test site to immonbilized HCG ab

SANDWICH complete-dye gives color

control site-nonspecigic antibody immobilized and dye gives color regardless of +/- of HCG to confirm that test is working

Question 27

western blot detects levels of

Answer 27

a target protein in a sample containing a mixture of proteins

Question 28

SDS PAGE separates proteins on

Answer 28

gel with electricty based on size, small proteins move faster than larger ones

Question 29

after SDS PAGE, transfer proteins to

Answer 29

nitrocellulose membrane to expose them

add primary antibody and then secondary antibody with an enzyme tag

add substrate and this gives you the color

Question 30

western blotting is useful in the

Answer 30

confirmation of HIV

Question 31

Southern Blot Does DNA to

Answer 31

DNA hybridization

Question 32

Norther blotting does DNA to

Answer 32

RNA hybridization

Question 33

SNOW DROP and E

Answer 33

Eastern Blot-PTM (tau in AZD)

Question 34

Insulin aspart aka Novo Nordisk

Answer 34

Prolin 28 replaced by aspartic acid

Both lispro and insulin aspart faster acting, more readily absorbed

Question 35

Uses of chimeric MAB

Answer 35

Inhibits platelet aggregation

Prevents rejection of transplanted kidney

Treats metastatic colorectal cancer

Treats autoimmune disease (via tumor necrosis factor)

Treats lymphomas and leukemia’s

Question 36

Indirect Elisa measures antibody in a sample so the well has

Answer 36

Antigen in it

Question 37

Sandwich Elisa measures amount of antigen in sample so well has

Answer 37

Antibody in it