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Questions and Answers
A researcher is performing a Southern blot to identify a specific DNA sequence. After electrophoresis and transfer, they find that the probe hybridizes non-specifically to multiple fragments. Which adjustment to the procedure would most likely improve the specificity of the hybridization?
A researcher is performing a Southern blot to identify a specific DNA sequence. After electrophoresis and transfer, they find that the probe hybridizes non-specifically to multiple fragments. Which adjustment to the procedure would most likely improve the specificity of the hybridization?
- Using a probe with a lower melting temperature.
- Adding formamide to the hybridization buffer. (correct)
- Increasing the salt concentration in the hybridization buffer.
- Decreasing the temperature of the hybridization reaction.
A scientist is using a restriction enzyme that normally produces sticky ends but observes that under the current reaction conditions, it is producing blunt ends. What is this phenomenon known as, and what factor might be causing it?
A scientist is using a restriction enzyme that normally produces sticky ends but observes that under the current reaction conditions, it is producing blunt ends. What is this phenomenon known as, and what factor might be causing it?
- Star activity, caused by a high glycerol concentration. (correct)
- Southern blotting, caused by improper transfer to a membrane.
- RFLP, caused by variations in the DNA sequence.
- Comparative Genomic Hybridization, caused by unequal DNA concentrations.
In comparative genomic hybridization (CGH), a researcher observes a region on a chromosome where the test DNA signal is significantly weaker than the reference DNA signal. What does this observation likely indicate?
In comparative genomic hybridization (CGH), a researcher observes a region on a chromosome where the test DNA signal is significantly weaker than the reference DNA signal. What does this observation likely indicate?
- The melting temperature of the reference DNA is higher than that of the test DNA.
- The stringency of the hybridization conditions was too low.
- There is a deletion of the corresponding sequence in the test DNA relative to the reference DNA. (correct)
- There is an amplification of the corresponding sequence in the test DNA relative to the reference DNA.
A molecular biologist is designing a DNA probe for a Southern blot experiment. Which of the following factors is most critical in determining the specificity of the probe for its target sequence?
A molecular biologist is designing a DNA probe for a Southern blot experiment. Which of the following factors is most critical in determining the specificity of the probe for its target sequence?
A researcher digests a DNA sample with a restriction enzyme that recognizes a 6-base pair sequence. After electrophoresis, they observe a smear of DNA fragments instead of distinct bands. Which of the following could be a likely cause of this result, assuming the enzyme is functioning correctly?
A researcher digests a DNA sample with a restriction enzyme that recognizes a 6-base pair sequence. After electrophoresis, they observe a smear of DNA fragments instead of distinct bands. Which of the following could be a likely cause of this result, assuming the enzyme is functioning correctly?
Flashcards
Restriction Enzymes
Restriction Enzymes
Enzymes that cut DNA at specific sequences, defending bacteria against foreign DNA.
Sticky Ends
Sticky Ends
Overhanging DNA cuts that are complementary, which allows efficient rejoining.
Blunt Ends
Blunt Ends
Straight DNA cuts, requiring adaptors for efficient rejoining.
RFLPs
RFLPs
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Melting Temperature
Melting Temperature
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Study Notes
- Restriction enzymes protect bacteria by cutting DNA at specific sites, particularly against foreign DNA like bacteriophages.
Types of Cuts
- Sticky ends feature overhanging cuts (5' or 3') and are complementary, which facilitates efficient rejoining.
- Sticky ends can be converted to blunt ends using nucleases or polymerases.
- Blunt ends result from straight cuts and are less efficient to rejoin.
- Blunt ends can be modified to create sticky ends using synthetic adaptors.
Restriction Enzyme Mapping
- Restriction Fragment Length Polymorphisms (RFLPs) are DNA variations for identification, tracking inheritance, and diagnosing diseases.
- Star activity is the altered enzyme specificity under abnormal conditions.
Southern Blot Procedure
- Southern blotting detects specific DNA sequences in a sample through a series of steps.
- Restriction enzymes cut DNA into fragments.
- Gel electrophoresis separates DNA fragments by size.
- DNA is denatured into single strands and transferred to a membrane.
- A labeled probe hybridizes (binds) to complementary DNA sequences.
- Detection methods include radioactive, chemiluminescent, or chromogenic techniques.
Key Concepts
- Stringency refers to the factors like temperature, salt concentration, and formamide levels that affect the specificity of hybridization.
- Melting temperature is when 50% of DNA separates into single strands.
- Comparative Genomic Hybridization (CGH) uses fluorescent probes to compare test DNA against reference DNA.
- In CGH, a stronger test signal indicates amplifications in the test DNA.
- In CGH, a stronger reference signal indicates deletions in the test DNA.
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