4.1 PROBES

Question 1

What identifies sequences of interest in Southern and Northern blots?

Answer 1

Probe

Question 2

What are examples of probes used in blots?

Answer 2

RNA, denatured DNA, modified nucleic acids

Question 3

What are nucleic acids with normal nitrogen bases but non-phosphodiester backbones?

Answer 3

Peptide nucleic acids (PNAs)

Question 4

What nucleic acids are resistant to nuclease degradation and hybridize more efficiently?

Answer 4

Peptide nucleic acids (PNAs)

Question 5

What are modified nucleic acids designed to hybridize more strongly to target sequences?

Answer 5

Locked nucleic acids (LNAs)

Question 6

What modified nucleic acids facilitate stronger binding to target sequences?

Answer 6

Peptide nucleic acids (PNAs) and locked nucleic acids (LNAs)

Question 7

What type of probes are used in Western blots?

Answer 7

Specific binding proteins or antibodies

Question 8

What is used for visualization of the protein band in a Western blot?

Answer 8

Labeled secondary antibody

Question 9

What binds to the protein of interest in Western blotting?

Answer 9

Primary binding protein (or primary antibody)

Question 10

What is directed against the primary binding protein for visualization?

Answer 10

Secondary antibody

Question 11

What is a frequent application of the Western blot method?

Answer 11

Confirmation of ELISA results for HIV and HCV

Question 12

What is separated and transferred to a membrane in this Western blot procedure?

Answer 12

Known viral proteins

Question 13

What is overlaid on the membrane in this Western blot application?

Answer 13

serum

Question 14

What detects the presence of viral antibodies in the patient’s serum in ELISA testing for HIV and HCV

Answer 14

Labeled antihuman immunoglobulin antibody

Question 15

How were DNA probes traditionally created?

Answer 15

Cloning a gene fragment on a bacterial plasmid

Question 16

What are other sources of DNA probes?

Answer 16

Viral genomes and in vitro organic synthesis

Question 17

What method is used to synthesize DNA probes for short, oligomeric sequences?

Answer 17

Organic synthesis

Question 18

What is another modern method to synthesize DNA probes?

Answer 18

Polymerase chain reaction (PCR)

Question 19

What determines the specificity of a probe in hybridization reactions?

Answer 19

Length of the probe

Question 20

How are double-stranded DNA probes denatured before use?

Answer 20

heating or treating with formamide/SSC

Question 21

How are RNA probes commonly made?

Answer 21

Transcription from a synthetic DNA template in vitro

Question 22

How does RNA/DNA helix compare to DNA/DNA in binding affinity?

Answer 22

RNA/DNA helix is stronger, offering more sensitivity

Question 23

What are common sources for synthesizing RNA probes?

Answer 23

Plasmid templates or DNA produced by PCR

Question 24

Which polymerases are used for RNA probe synthesis?

Answer 24

SP6, T3, or T7 RNA polymerases

Question 25

Which strand is used for Southern blots with RNA probes?

Answer 25

Coding strand transcript

Question 26

Which transcript is required for northern blots with RNA probes?

Answer 26

Antisense transcript (complementary to the coding strand)

Question 27

How are RNA probes labeled for detection?

Answer 27

incorporating radioactive or modified nucleotides during transcription

Question 28

How is nonhybridized RNA probe removed after hybridization?

Answer 28

Digestion with RNase, such as RNase A

Question 29

What advantage do modified nucleic acid probes have over DNA and RNA?

Answer 29

Resistance to nuclease degradation

Question 30

Chemically synthesized modified nucleic acids examples

Answer 30

peptide nucleic acids (PNA), locked nucleic acids (LNA), and unlocked nucleic acids (UNA)

Question 31

They lack the C2′–C3′ ribose bond, making them highly flexible

Answer 31

unlocked nucleic acids (UNA)

Question 32

What type of probes are used in western blotting for detecting immobilized target proteins?

Answer 32

Antibodies

Question 33

What are antibodies produced by a generalized response to an antigen, directed at multiple epitopes?

Answer 33

Polyclonal antibodies

Question 34

What are antibodies produced from cloned hybridomas, directed at a single epitope?

Answer 34

Monoclonal antibodies

Question 35

What technique involves fusing spleen cells from immunized mice with mouse myeloma cells?

Answer 35

Hybridomas

Question 36

What technique isolates specific immunoglobulins from sera?

Answer 36

Affinity chromatography

Question 37

What enhances the antibody response by slowing antigen degradation?

Answer 37

Adjuvants

Question 38

What type of antibodies provide a more robust signal in western blotting?

Answer 38

Polyclonal antibodies

Question 39

What happens if a targeted epitope is lost during electrophoresis and transfer when using monoclonal antibodies?

Answer 39

No signal generated

Question 40

What is the dilution range for primary antibodies in western blotting?

Answer 40

1/100 to 1/100,000