4.1 PROBES Flashcards
What identifies sequences of interest in Southern and Northern blots?
Probe
What are examples of probes used in blots?
RNA, denatured DNA, modified nucleic acids
What are nucleic acids with normal nitrogen bases but non-phosphodiester backbones?
Peptide nucleic acids (PNAs)
What nucleic acids are resistant to nuclease degradation and hybridize more efficiently?
Peptide nucleic acids (PNAs)
What are modified nucleic acids designed to hybridize more strongly to target sequences?
Locked nucleic acids (LNAs)
What modified nucleic acids facilitate stronger binding to target sequences?
Peptide nucleic acids (PNAs) and locked nucleic acids (LNAs)
What type of probes are used in Western blots?
Specific binding proteins or antibodies
What is used for visualization of the protein band in a Western blot?
Labeled secondary antibody
What binds to the protein of interest in Western blotting?
Primary binding protein (or primary antibody)
What is directed against the primary binding protein for visualization?
Secondary antibody
What is a frequent application of the Western blot method?
Confirmation of ELISA results for HIV and HCV
What is separated and transferred to a membrane in this Western blot procedure?
Known viral proteins
What is overlaid on the membrane in this Western blot application?
serum
What detects the presence of viral antibodies in the patient’s serum in ELISA testing for HIV and HCV
Labeled antihuman immunoglobulin antibody
How were DNA probes traditionally created?
Cloning a gene fragment on a bacterial plasmid
What are other sources of DNA probes?
Viral genomes and in vitro organic synthesis
What method is used to synthesize DNA probes for short, oligomeric sequences?
Organic synthesis
What is another modern method to synthesize DNA probes?
Polymerase chain reaction (PCR)
What determines the specificity of a probe in hybridization reactions?
Length of the probe
How are double-stranded DNA probes denatured before use?
heating or treating with formamide/SSC
How are RNA probes commonly made?
Transcription from a synthetic DNA template in vitro
How does RNA/DNA helix compare to DNA/DNA in binding affinity?
RNA/DNA helix is stronger, offering more sensitivity
What are common sources for synthesizing RNA probes?
Plasmid templates or DNA produced by PCR
Which polymerases are used for RNA probe synthesis?
SP6, T3, or T7 RNA polymerases
Which strand is used for Southern blots with RNA probes?
Coding strand transcript
Which transcript is required for northern blots with RNA probes?
Antisense transcript (complementary to the coding strand)
How are RNA probes labeled for detection?
incorporating radioactive or modified nucleotides during transcription
How is nonhybridized RNA probe removed after hybridization?
Digestion with RNase, such as RNase A
What advantage do modified nucleic acid probes have over DNA and RNA?
Resistance to nuclease degradation
Chemically synthesized modified nucleic acids examples
peptide nucleic acids (PNA),
locked nucleic acids (LNA), and unlocked nucleic acids (UNA)
They lack the C2′–C3′ ribose bond, making them highly flexible
unlocked nucleic acids (UNA)
What type of probes are used in western blotting for detecting immobilized target proteins?
Antibodies
What are antibodies produced by a generalized response to an antigen, directed at multiple epitopes?
Polyclonal antibodies
What are antibodies produced from cloned hybridomas, directed at a single epitope?
Monoclonal antibodies
What technique involves fusing spleen cells from immunized mice with mouse myeloma cells?
Hybridomas
What technique isolates specific immunoglobulins from sera?
Affinity chromatography
What enhances the antibody response by slowing antigen degradation?
Adjuvants
What type of antibodies provide a more robust signal in western blotting?
Polyclonal antibodies
What happens if a targeted epitope is lost during electrophoresis and transfer when using monoclonal antibodies?
No signal generated
What is the dilution range for primary antibodies in western blotting?
1/100 to 1/100,000
