4.4 DETECTION SYSTEMS + INTERPRETATION OF RESULTS

Question 1

What detection system uses 32P-labeled probes to detect hybridization through autoradiography?

Answer 1

Radioactive detection

Question 2

What must be done after hybridization to ensure that only completely hybridized probes remain on the blot?

Answer 2

Wash under stringent conditions

Question 3

What are two common nonradioactive labels used in detection systems?

Answer 3

Digoxigenin and Biotin

Question 4

Which enzyme is typically conjugated to anti-digoxigenin or streptavidin for nonradioactive detection?

Answer 4

Alkaline phosphatase (AP)

Question 5

What detection system involves the release of light by phosphorylation of dioxetane?

Answer 5

Chemiluminescent detection

Question 6

Which detection method allows direct observation of color developing on the membrane?

Answer 6

Chromogenic detection

Question 7

What is a key factor in achieving a successful blotting method?

Answer 7

High signal-to-noise ratio

Question 8

What agents are used to prevent nonspecific binding in nonradioactive detection systems?

Answer 8

Blocking agents

Question 9

What type of enzyme conjugates, in addition to alkaline phosphatase, may also be used for signal generation?

Answer 9

Horseradish peroxidase (HRP)

Question 10

What are two common substrates used in nonradioactive detection that produce chemiluminescent or color signals?

Answer 10

Dioxetane and Tetrazolium dye derivatives

Question 11

What can a Southern blot detect by comparing restriction patterns of samples?

Answer 11

Gene locus restriction patterns

Question 12

What type of control is included in Southern blot analysis to ensure accuracy despite low expression levels?

Answer 12

Internal standardized control

Question 13

What issue can arise in Southern blot assays when a probe binds to multiple similar sequences?

Answer 13

Cross-hybridization

Question 14

What can Southern blot analysis not detect unless it affects a restriction enzyme site?

Answer 14

Tiny deletions, insertions, or single-nucleotide differences

Question 15

Which blotting technique is used for analyzing gene expression, transcript size, and protein modifications?

Answer 15

Northern or Western blot

Question 16

What is the importance of including an internal control in Northern and Western blot analyses?

Answer 16

To correct for errors in isolation, gel loading, and transfer

Question 17

What indicates the amount of expression in a Northern or Western blot?

Answer 17

The relative amount of the target transcript or protein product compared to the internal control