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RESET - MOLBIO PRELIMS > 4.1 LEC > Flashcards

4.1 LEC Flashcards

1
Q

What identifies sequences of interest in Southern and Northern blots?

A

Probe

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2
Q

What are examples of probes used in blots?

A

RNA, denatured DNA, modified nucleic acids

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3
Q

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

A

Peptide nucleic acids (PNAs)

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4
Q

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

A

Peptide nucleic acids (PNAs)

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5
Q

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

A

Locked nucleic acids (LNAs)

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6
Q

What modified nucleic acids facilitate stronger binding to target sequences?

A

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

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7
Q

What type of probes are used in Western blots?

A

Specific binding proteins or antibodies

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8
Q

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

A

Labeled secondary antibody

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9
Q

What binds to the protein of interest in Western blotting?

A

Primary binding protein (or primary antibody)

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10
Q

What is directed against the primary binding protein for visualization?

A

Secondary antibody

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11
Q

What is a frequent application of the Western blot method?

A

Confirmation of ELISA results for HIV and HCV

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12
Q

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

A

Known viral proteins

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13
Q

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

A

serum

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14
Q

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

A

Labeled antihuman immunoglobulin antibody

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15
Q

How were DNA probes traditionally created?

A

Cloning a gene fragment on a bacterial plasmid

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16
Q

What are other sources of DNA probes?

A

Viral genomes and in vitro organic synthesis

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17
Q

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

A

Organic synthesis

18
Q

What is another modern method to synthesize DNA probes?

A

Polymerase chain reaction (PCR)

19
Q

What determines the specificity of a probe in hybridization reactions?

A

Length of the probe

20
Q

How are double-stranded DNA probes denatured before use?

A

heating or treating with formamide/SSC

21
Q

How are RNA probes commonly made?

A

Transcription from a synthetic DNA template in vitro

22
Q

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

A

RNA/DNA helix is stronger, offering more sensitivity

23
Q

What are common sources for synthesizing RNA probes?

A

Plasmid templates or DNA produced by PCR

24
Q

Which polymerases are used for RNA probe synthesis?

A

SP6, T3, or T7 RNA polymerases

25
Q

Which strand is used for Southern blots with RNA probes?

A

Coding strand transcript

26
Q

Which transcript is required for northern blots with RNA probes?

A

Antisense transcript (complementary to the coding strand)

27
Q

How are RNA probes labeled for detection?

A

incorporating radioactive or modified nucleotides during transcription

28
Q

How is nonhybridized RNA probe removed after hybridization?

A

Digestion with RNase, such as RNase A

29
Q

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

A

Resistance to nuclease degradation

30
Q

Chemically synthesized modified nucleic acids examples

A

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

31
Q

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

A

unlocked nucleic acids (UNA)

32
Q

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

A

Antibodies

33
Q

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

A

Polyclonal antibodies

34
Q

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

A

Monoclonal antibodies

35
Q

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

A

Hybridomas

36
Q

What technique isolates specific immunoglobulins from sera?

A

Affinity chromatography

37
Q

What enhances the antibody response by slowing antigen degradation?

A

Adjuvants

38
Q

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

A

Polyclonal antibodies

39
Q

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

A

No signal generated

40
Q

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

A

1/100 to 1/100,000

RESET - MOLBIO PRELIMS (16 decks)

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