Molecular Diagnostics Hybridization-Based Techniques Flashcards
Terminology:
When 2 single-stranded nucleic acid molecules form a double-stranded hybrid
Hybridization
Which of the following can hybridization occur between?:
- 2 ssDNA molecules
- 2 ssRNA molecules
- 1 ssDNA and 1 ssRNA
- All of the above
- All of the above
TRUE or FALSE:
For hybridization to occur, nucleic acid populations must be single stranded
TRUE
Terminology:
Hybridization of complementary sequences
Annealing
Terminology:
Loss of hydrogen bonding between complementary nucleic acid strands
Denaturation
Terminology:
The reversal of denaturation under certain conditions
Renaturation
Terminology:
A nucleic acid with a detectable signal designed to bind to a specific target sequence
Probe
Terminology:
A short, single-stranded DNA fragment designed to prime DNA synthesis of a specific DNA region
Primer
Terminology:
Specific sequences or regions of interest that are detected in an analytical procedure
Target
Fill in the blank:
a. Southern Blot
b. Northern Blot
c. Western Blot
a. Southern Blot
Fill in the blank:
a. DNA
b. RNA
c. Proteins
a. DNA
Fill in the blank:
a. DNA
b. RNA
c. Antibodies
d. Both a and b
d. Both a and b
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
a. Analyze gene structure
&
b. Detection of sickle cell mutation
Fill in the blank:
a. Southern Blot
b. Northern Blot
c. Western Blot
b. Northern Blot
Fill in the blank:
a. DNA
b. RNA
c. Proteins
b. RNA
Fill in the blank:
a. DNA
b. RNA
c. Antibodies
d. Both a and b
d. Both a and b
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
c. Analyzing gene expression
&
d. Detecting splicing abnormalities (beta-thalassemia)
Fill in the blank:
a. Southern Blot
b. Northern Blot
c. Western Blot
c. Western Blot
Fill in the blank:
a. DNA
b. RNA
c. Proteins
c. Proteins
Fill in the blank:
a. DNA
b. RNA
c. Antibodies
d. Both a and b
c. Antibodies
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
c. Analyzing gene expression
&
e. HIV, HCV, and Lyme disease confirmation testing
Who developed Southern Blot?
Edwin Southern
James Alwine, David Kemp and George Stark developed which hybridization-based molecular technique?
Northern Blot
This hybridization-based molecular technique uses SDS-PAGE instead of standard gel electrophoresis
Western Blot
4 types of labels for probes
-Radioactive 32P
-Fluorescent
-Chemiluminescent
-Enzyme
Longer probes = (less/greater) specificity
Greater specificity
Design and optimal hybridization of the ________ are the most critical parts of any hybridization procedure
Probe
Terminology:
Conditions under which the target is exposed to the probed
Stringency
What happens if stringency is too high?
Probe will not bind to the target
What happens if stringency is too low?
Probe will bind to non-specific/unrelated targets
Factors that affect stringency (5)
- Temperature
- Salt concentration in the buffer
- Length of the probe
- Type of probe
- Concentration of denaturant in the buffer
Name the type of probe:
-Hybridizes with the target
-Nitrocellulose is exposed to x-ray film to visualize the probes
-Known as autoradiography
Radioactive 32P probe
Name the type of probe:
-Produces fluorescent light
Non-radioactive Fluorescent tags
Name the type of probe requires a substrate?
Non-radioactive Chemiluminescent enzyme
This probe will develop color directly on the membrane
Non-radioactive Chromogenic enzyme
The following are common uses for which type of hybridization?:
- Gene expression
- Mutations
- Amplification/deletion analysis
(this hybridization technique cannot determine target size)
Array-based hybridization
