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Questions and Answers
What effect do mismatches in a DNA duplex have on thermal stability?
What effect do mismatches in a DNA duplex have on thermal stability?
The Tm of a double-stranded DNA increases with higher salt concentration.
The Tm of a double-stranded DNA increases with higher salt concentration.
True
What is the process of separating the polynucleotide strands of duplex nucleic acid called?
What is the process of separating the polynucleotide strands of duplex nucleic acid called?
What is the approximate formula to calculate Tm for short duplexes (14-20 base pairs)?
What is the approximate formula to calculate Tm for short duplexes (14-20 base pairs)?
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Every 1% mismatch of bases in a DNA duplex reduces the Tm by approximately _____ °C.
Every 1% mismatch of bases in a DNA duplex reduces the Tm by approximately _____ °C.
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The melting temperature (Tm) decreases with increased G+C content in nucleic acids.
The melting temperature (Tm) decreases with increased G+C content in nucleic acids.
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Match the following factors with their effect on thermal stability of DNA:
Match the following factors with their effect on thermal stability of DNA:
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What is the significance of 'hybridization' in the context of nucleic acids?
What is the significance of 'hybridization' in the context of nucleic acids?
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The process of restoring typical base-pairing of two separated complementary sequences is known as ______.
The process of restoring typical base-pairing of two separated complementary sequences is known as ______.
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Match the following terms with their definitions:
Match the following terms with their definitions:
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Study Notes
Denaturation and Renaturation
- Denaturation is the separation of the polynucleotide strands of duplex nucleic acid, also called "melting"
- Renaturation is the restoration of typical base pairing between two fully separated complementary sequences, forming a native duplex structure, also called "annealing"
- Melting temperature (Tm): the temperature where half of the nucleic acid helices are denatured
Factors affecting nucleic acid stability
- Base composition: Tm increases with increased G+C content due to stronger triple hydrogen bonding between G and C bases.
- Length of base-paired sequence: Longer base pairs result in higher stability.
- Mismatches: Unpaired bases decrease the stability of the double helix.
- Salt concentration: Higher cation concentration leads to increased stability.
- Other factors: pH, concentration of nucleic acids themselves, and denaturing agents can also influence stability.
Calculating Tm
- Approximate Tm formula for short duplexes (14-20 base pairs): Tm = 4°C (G + C)+ 2°C (A + T)
- Every 1% mismatch of bases in a duplex reduces Tm by ≈1°C.
Effect of temperature on annealing rate
- Maximum rate of annealing for DNA-DNA re-association occurs at ~25°C below Tm: This temperature is known as the annealing temperature.
Hybridization analysis
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Hybridization analysis is a technique for detecting and mapping genes, studying gene expression using DNA-DNA or DNA-RNA interactions:
- Southern blotting: DNA-DNA hybridization.
- Northern blotting: DNA-RNA hybridization.
- Microarrays: DNA-DNA hybridization.
- Procedure: Nucleic acid samples are fixed to solid surfaces (membranes, glass slides), then "probed" with a nucleic acid sequence of interest.
Labeling Techniques
- Labeling is the attachment of radioactive, fluorescent, or other markers to DNA molecules for detection purposes.
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Random primer labeling: A mixture of random oligonucleotides (typically hexamers) are used as primers to initiate DNA synthesis.
- Klenow fragment of E.coli DNA polymerase I is used along with labeled dNTPs to incorporate radioactive precursors into DNA randomly.
Detection of labelled molecules
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Radioactive label: 32P, 33P, 35S, and 3H can be detected using X-ray sensitive film (autoradiography) or a radiation-sensitive screen (phosphorimaging).
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Non-radioactive label:
- Fluorescence: Molecules labeled with fluorophores are detected with film or fluorescence detectors.
- Chemiluminescence: Uses reactions between label and chemicals to generate light, which is detected with film.
Microarrays
- Microarrays are glass surfaces with thousands of DNA fragments arrayed at discrete sites ("spots").
- Procedure: The DNA spots are hybridized to samples of fluorescently labeled DNA or RNA in solution, and the signals are analyzed and compared.
- Applications: Used to study gene expression, gene discovery, and disease diagnostics.
Key Take-Aways
- Denaturation, renaturation, and melting temperature are crucial concepts in understanding nucleic acid structure and function
- Understanding these concepts is essential for various molecular biology techniques like hybridization analysis and DNA microarrays.
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Description
This quiz covers key concepts related to the denaturation and renaturation of nucleic acids, including the factors that affect their stability. It also explores the melting temperature (Tm) and how to calculate it based on various parameters. Test your understanding of the dynamics of nucleic acid structures with this informative quiz.
