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Questions and Answers
What type of sugar is found in deoxyribonucleic acid (DNA)?
Which of the following bases is a purine?
What is the primary function of restriction endonucleases in molecular biology?
What is a key difference between DNA and RNA?
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What is the purpose of gel electrophoresis in molecular biology?
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Which nucleobase is present in RNA but not in DNA?
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In the process of PCR, why is it necessary for DNA polymerase to have a free 3'-OH group?
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Which component is not essential for the standard PCR reaction?
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What is the primary function of a vector in DNA cloning?
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What is a characteristic feature of plasmids used as cloning vectors?
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Which enzyme is responsible for covalently ligating the sugar-phosphate backbone during the cloning process?
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What role do antibiotic resistance genes play in the selection of positive transformants?
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What is the purpose of using restriction endonucleases in the cloning process?
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In the context of cloning, what does transformation refer to?
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Which method can be used for screening positive transformants in DNA cloning?
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What does the term 'chimeric plasmid' refer to in the cloning context?
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What distinguishes ribose in RNA from deoxyribose in DNA?
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Which bases are classified as pyrimidines?
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What is the primary function of restriction endonucleases in molecular biology?
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Which statement correctly describes a difference between DNA and RNA?
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In gel electrophoresis, what is the primary factor determining the migration rate of nucleic acid fragments?
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What is a molecular weight ladder used for in gel electrophoresis?
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Which restriction enzyme recognizes the sequence G↓AATTC and cuts the DNA at that site?
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How do smaller nucleic acids behave in a gel electrophoresis setup compared to larger ones?
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Study Notes
Polymerase Chain Reaction
- PCR amplifies specific DNA sequences (up to 6 kb)
- PCR requires:
- Heat-stable DNA polymerase (e.g., Taq polymerase) for nucleotide addition (5'→3')
- Deoxynucleoside triphosphates (dNTPs): dATP, dCTP, dGTP, dTTP
- Two oligonucleotide primers flanking the DNA segment of interest
- Denaturation: Separating dsDNA into single strands using heat to break hydrogen bonds
- DNA polymerase requires a free 3'-OH group on the template strand for nucleotide addition.
- Primers bind to the 3' end of the template strand, allowing DNA polymerase to add nucleotides.
- Amplified strand is complementary to the template strand and synthesized in the 5'→3' direction.
- Each PCR cycle doubles the amount of target DNA.
- At the end of 1 cycle, there are 2² strands of DNA.
- Number of DNA molecules after 'n' cycles = 2^(n+1)
- PCR applications:
- Sequencing (e.g., forensics, infectious disease diagnosis, mutation detection)
- Cloning (e.g., studying models, therapeutics)
Recombinant DNA Technology
- Isolates, amplifies, and modifies specific DNA sequences.
- Also known as molecular cloning or genetic engineering.
- Cloning involves producing multiple identical organisms from a single ancestor.
- A clone is a collection of cells containing a vector carrying the DNA of interest.
- Cloning aims to:
- Produce large amounts of DNA
- Express large amounts of protein
Overview of Cloning
- Steps:
- Generate DNA fragment of interest using restriction enzymes, PCR, or chemical synthesis.
- Ligation: Insert fragment into a vector (another DNA molecule) containing sequences for replication.
- Transformation: Introduce the vector (with DNA of interest) into host cells for replication.
- Selection/screening: Identify and select cells with the desired DNA.
Cloning: Vectors
- Small, autonomously replicating DNA molecules.
- Plasmids are circular DNA molecules (up to 200 kb) found in bacteria or yeast.
- Present in hundreds of copies within the cell
- Small and replicate easily
- Carry genes encoding antibiotic resistance for selection.
- Carry restriction endonuclease sites for foreign DNA insertion.
- Can clone up to 10 kb.
- Example: pUC18
Cloning: Ligation
- A restriction fragment is inserted into a cut made in a cloning vector by the same restriction enzyme (sticky ends).
- Complementary ends of the DNA fragment and vector base pair (anneal).
- The sugar-phosphate backbone is covalently joined by DNA ligase.
- The inserted fragment of foreign DNA can be excised using the same restriction enzyme.
Cloning: Transformation
- A chimeric plasmid is taken up by a host bacterium (transformation).
- The plasmid becomes permanently established and replicates indefinitely in the host cell.
- This produces large amounts of recombinant DNA.
- Bacteria are typically cultured on semi-solid growth medium at low density to enable the formation of single colonies.
- Assumption: One colony arises from a single cell.
- Only host cells containing a properly constructed vector are selected for further propagation.
Cloning: Selection
- Methods for selecting positive transformants:
-
Antibiotic resistance: Use antibiotic genes (ampR, tetR, kanR)
- Untransformed cells do not grow on antibiotic media; transformed cells do.
-
Chromogenic substances:
- Nucleic acid gel electrophoresis: Separates nucleic acids by size.
- In an electric field, the velocity of a charged molecule is proportional to charge density, size, and shape.
- Nucleic acids have constant shape and charge density; velocity depends on size.
-
Antibiotic resistance: Use antibiotic genes (ampR, tetR, kanR)
Nucleic Acid Gel Electrophoresis
- Separates nucleic acids based on size using a gel-like matrix (agarose or polyacrylamide).
- Molecules are applied at one end of an electric field and migrate to the other end.
- Smaller molecules move through the pores faster and migrate farther.
- Visualized using stains:
- Dyes binding to DNA
- Radioactive labeling
- Fluorescence
- A molecular weight ladder (set of standards of known sizes) can be added to estimate size.
- Derived from restriction enzyme cleavage of a known DNA sequence, PCR, or DNA ligation.
DNA Fingerprinting
- Used to identify individuals based on their unique DNA profiles.
- Relies on differences in the length of restriction fragment length polymorphisms (RFLPs).
- RFLPs are created by cutting DNA with restriction enzymes.
- Different individuals have different RFLP patterns due to variations in their DNA sequences.
- Can be used for:
- Paternity testing
- Forensic investigations
Restriction Enzymes
- Enzymes that cut DNA at specific recognition sequences.
- Each restriction enzyme recognizes a unique sequence of nucleotides.
- Examples:
- EcoRI: G↓AATTC
- HaeIII: GG↓CC
- BamHI: G↓GATCC
- PvuII: CAG↓CTG
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Description
This quiz covers the fundamentals of Polymerase Chain Reaction (PCR), including its requirements, processes, and applications. Learn about the key components such as heat-stable DNA polymerase and the cycling mechanism that allows for DNA amplification. Test your knowledge on the significance of primers and the role of PCR in various fields like forensics and infectious disease diagnosis.
