Molecular Diagnostics

Question 1

southern blotting

Answer 1

both probe and target nucleic acid are DNA

Question 2

northern blotting

Answer 2

probe is single stranded DNA and target is mRNA

Question 3

western blotting

Answer 3

target is protein or antibody

Question 4

how does PCR work

Answer 4

dsDNA obtained from patient/individual/pathogen is denatured with heat and separated to ssDNA

primers flank each end of DNA and are allowed to anneal in cooler temps

deoxynucleotide triphosphates (dNTPS) are added

Taq polymerase synthesizes copy of DNA

Question 5

Taq polymerase

Answer 5

synthesizes copy of DNA by extending the primers on both ends; DNA doubles in each cycle and becomes greatly amplified

Question 6

advantages of PCR

Answer 6

very small amount of DNA is needed for the template; 10^9 fold increase from original DNA

Question 7

disadvantages of PCR

Answer 7

you need to know the sequence of the flanking DNA for primer design

it’s error prone

you could amplify contaminated DNA

Question 8

quantitative PCR (qPCR)

Answer 8

used to quantify copy number of a specific gene in two or more samples in real time

in addition to primers, it uses a probe which fluoresces only in presence of the PCR product

used for: detecting levels of an infectious agent and determining levels of gene expression

Question 9

restriction fragment length polymorphism (RFLP) steps (5)

Answer 9

DNA fingerprinting; results is bands of DNA on nylon membrane

1. DNA is cleaved by restriction endonucleases
2. resolve DNA fragments on agarose gel
3. transfer DNA to nylon membrane
4. add radioactive DNA probe; wash off excess probe
5. expose x-ray film to nylon membrane

Question 10

how is sickle cell anemia shown on an RFLP

Answer 10

patients with sickle cell only have two restriction sites in their DNA, so their RFLP will only show one fragment on the membrane

normal beta globulin allele has 3 Ddel restriction sites and will show two fragments

Question 11

variable number of tandem repeats (VNTR)

Answer 11

short tandem repeats (STR) occurs in normal genome and varies in individuals

VNTR repeat regions are isolated from genomic samples by flanking restriction sites or through PCR

useful in identification and severity of inherited diseases

ex: huntington disease, fragile X syndrome, frederich ataxia

Question 12

how huntington’s is diagnosed

Answer 12

uses VNTR

PCR is done on healthy individual and patient with huntington’s; alpha-32 probe is attached to dCTP

resolve DNA fragments on urea-polyacrylamide gel

expose x-ray film to dried gel

huntingon’s will show much higher CAG segments

Question 13

recombinant proteins

Answer 13

engineered to allow high levels of replication, transcription, and translation to occur in a host; cDNA of the protein is inserted into expression vectors

large scale production and purification

used to make insulin, growth hormones, erythropoietin, clotting factors, vaccines

Question 14

how human insulin production works

Answer 14

insulin producing gene is isolated from pancreas cells

plasmid DNA is extracted from a bacterium and cut with restriction enzymes, forming plasmid vector

human insulin producing gene is inserted into the bacterial plasmid vector to form the recombinant DNA

Question 15

how does Lispro improve insulin use

Answer 15

reverses the position of proline at position 28 with lysine at position 29 at C terminus of B chain

works in 2-4 hours instead of 6-8 hours

Question 16

how does insulin aspart improve insulin use

Answer 16

the proline at 28 is replaced with aspartic acid

works in 2-4 hours instead of 6-8 hours

Question 17

monoclonal antibody

Answer 17

specific for a single epitope on antigen

used to treat many cancers

Question 18

how antibodies are generated

Answer 18

mouse is immunized with immunogen peptide protein

antibody secreting cells are harvested from the spleen

mouse spleen cells are fused with myeloma cells to form hybridomas

the cells are grown in culture in HAT medium

cells divide infinitely because they are cancer cells, which causes infinite production of the monoclonal antibody

Question 19

abciximab

Answer 19

chimeric monoclonal antibody

inhibits platelet aggregation

Question 20

baciliximab

Answer 20

chimeric monoclonal antibody

prevents rejections of transplanted kidney

Question 21

cetuximab

Answer 21

chimeric monoclonal antibody

treats metastatic and colorectal cancers

Question 22

infliximab

Answer 22

chimeric monoclonal antibody

treats autoimmune disease

Question 23

retuximab

Answer 23

chimeric monoclonal antibody

treats lymphomas, leukemias

Question 24

ELISA

Answer 24

immunological tests which tests for the levels of specific antigen or antibody concentrations in biological samples using a corresponding antibody or antigen; quantitative

uses fluorescence to determine quantitative results

Question 25

indirect ELISA

Answer 25

measures the amount of an antibody in a sample

Question 26

sandwich ELISA

Answer 26

measures the amount of an antigen in a sample

Question 27

how ELISA works

Answer 27

microplate is pre-coated with antibody target protein will be captured (bound) to the antibody when added to the well second antibody conjugated with horseradish peroxidase (HRP) is added and binds to captured protein unbound second antibody is washed away and the HRP substrate TMB is added which develops blue color

Question 28

how ELISA diagnoses HIV

Answer 28

specific antibodies to HIV are produced 4-6 weeks after infection uses indirect ELISA to search for the antibodies in the body

Question 29

how ELISA can detect MI

Answer 29

troponin is the antigen in this case uses sandwich ELISA to find the antigen

Question 30

how ELISA works for a pregnancy test

Answer 30

looking for HCG (the antigen) uses sandwich ELISA to look for HCG the test stick has immobilized hCG antibody complex, which will bind to hCG in urine to complete the sandwich which produces the colored line

Question 31

western blotting (immunoblotting)

Answer 31

used to detect the levels of a target protein in a biological sample

Question 32

mechanism of western blotting

Answer 32

protein samples are placed in SDS polyacrylamide gel proteins are transferred to nitrocellulose membrane where they are exposed primary antibody is added which binds to the target protein tagged secondary antibody is added which binds to primary antibody visualization agents/chemicals are used to detect secondary antibody signal

Question 33

what antigen of HIV does western blotting detect

Answer 33

detects HIV p24 surface antigen other antigens include: - gp160 - gp120 - p66 - p66