Molecular Diagnostics Hybridization-Based Techniques

Question 1

Terminology:
When 2 single-stranded nucleic acid molecules form a double-stranded hybrid

Answer 1

Hybridization

Question 2

Which of the following can hybridization occur between?:
- 2 ssDNA molecules
- 2 ssRNA molecules
- 1 ssDNA and 1 ssRNA
- All of the above

Answer 2

• All of the above

Question 3

TRUE or FALSE:
For hybridization to occur, nucleic acid populations must be single stranded

Answer 3

TRUE

Question 4

Terminology:
Hybridization of complementary sequences

Answer 4

Annealing

Question 5

Terminology:
Loss of hydrogen bonding between complementary nucleic acid strands

Answer 5

Denaturation

Question 6

Terminology:
The reversal of denaturation under certain conditions

Answer 6

Renaturation

Question 7

Terminology:
A nucleic acid with a detectable signal designed to bind to a specific target sequence

Answer 7

Probe

Question 8

Terminology:
A short, single-stranded DNA fragment designed to prime DNA synthesis of a specific DNA region

Answer 8

Primer

Question 9

Terminology:
Specific sequences or regions of interest that are detected in an analytical procedure

Answer 9

Target

Question 10

Fill in the blank:
a. Southern Blot
b. Northern Blot
c. Western Blot

Answer 10

a. Southern Blot

Question 11

Fill in the blank:
a. DNA
b. RNA
c. Proteins

Answer 11

a. DNA

Question 12

Fill in the blank:
a. DNA
b. RNA
c. Antibodies
d. Both a and b

Answer 12

d. Both a and b

Question 13

Fill in the blank: (more than one selection)
a. Analyze gene structure
b. Detection of sickle cell mutation
c. Analyzing gene expression
d. Detecting splicing abnormalities (beta-thalassemia)
e. HIV, HCV, and Lyme disease confirmation testing

Answer 13

a. Analyze gene structure
&
b. Detection of sickle cell mutation

Question 14

Fill in the blank:
a. Southern Blot
b. Northern Blot
c. Western Blot

Answer 14

b. Northern Blot

Question 15

Fill in the blank:
a. DNA
b. RNA
c. Proteins

Answer 15

b. RNA

Question 16

Fill in the blank:
a. DNA
b. RNA
c. Antibodies
d. Both a and b

Answer 16

d. Both a and b

Question 17

Fill in the blank: (more than one selection)
a. Analyze gene structure
b. Detection of sickle cell mutation
c. Analyzing gene expression
d. Detecting splicing abnormalities (beta-thalassemia)
e. HIV, HCV, and Lyme disease confirmation testing

Answer 17

c. Analyzing gene expression
&
d. Detecting splicing abnormalities (beta-thalassemia)

Question 18

Fill in the blank:
a. Southern Blot
b. Northern Blot
c. Western Blot

Answer 18

c. Western Blot

Question 19

Fill in the blank:
a. DNA
b. RNA
c. Proteins

Answer 19

c. Proteins

Question 20

Fill in the blank:
a. DNA
b. RNA
c. Antibodies
d. Both a and b

Answer 20

c. Antibodies

Question 21

Fill in the blank: (more than one selection)
a. Analyze gene structure
b. Detection of sickle cell mutation
c. Analyzing gene expression
d. Detecting splicing abnormalities (beta-thalassemia)
e. HIV, HCV, and Lyme disease confirmation testing

Answer 21

c. Analyzing gene expression
&
e. HIV, HCV, and Lyme disease confirmation testing

Question 22

Who developed Southern Blot?

Answer 22

Edwin Southern

Question 23

James Alwine, David Kemp and George Stark developed which hybridization-based molecular technique?

Answer 23

Northern Blot

Question 24

This hybridization-based molecular technique uses SDS-PAGE instead of standard gel electrophoresis

Answer 24

Western Blot

Question 25

4 types of labels for probes

Answer 25

-Radioactive 32P
-Fluorescent
-Chemiluminescent
-Enzyme

Question 26

Longer probes = (less/greater) specificity

Answer 26

Greater specificity

Question 27

Design and optimal hybridization of the ________ are the most critical parts of any hybridization procedure

Answer 27

Probe

Question 28

Terminology:
Conditions under which the target is exposed to the probed

Answer 28

Stringency

Question 29

What happens if stringency is too high?

Answer 29

Probe will not bind to the target

Question 30

What happens if stringency is too low?

Answer 30

Probe will bind to non-specific/unrelated targets

Question 31

Factors that affect stringency (5)

Answer 31

• Temperature
• Salt concentration in the buffer
• Length of the probe
• Type of probe
• Concentration of denaturant in the buffer

Question 32

Name the type of probe:
-Hybridizes with the target
-Nitrocellulose is exposed to x-ray film to visualize the probes
-Known as autoradiography

Answer 32

Radioactive 32P probe

Question 33

Name the type of probe:
-Produces fluorescent light

Answer 33

Non-radioactive Fluorescent tags

Question 34

Name the type of probe requires a substrate?

Answer 34

Non-radioactive Chemiluminescent enzyme

Question 35

This probe will develop color directly on the membrane

Answer 35

Non-radioactive Chromogenic enzyme

Question 36

The following are common uses for which type of hybridization?:
- Gene expression
- Mutations
- Amplification/deletion analysis
(this hybridization technique cannot determine target size)

Answer 36

Array-based hybridization