Recombinant DNA and Biotechnology (1B) - Bio & Biochem

1. What is the purpose of a multiple cloning site (MCS) in a plasmid?

   It provides many unique restriction sites for inserting DNA. Clusters rare-cutting restriction sites, offering flexibility for inserting foreign DNA without disrupting vector functions.

2. Identify the correct order of PCR steps within one cycle.

   Denaturation, annealing, extension. Separates strands, allows primer binding, and enables synthesis, exponentially amplifying DNA each cycle.

3. What is the approximate temperature range used for primer annealing in PCR?

   About $50$ to $65\,^{\circ}\text{C}$. Optimizes hydrogen bonding between primers and template, ensuring specific amplification without non-specific binding.

4. What is the primary function of a restriction endonuclease in cloning?

   It cleaves DNA at specific recognition sequences. Recognizes and cuts at palindromic sequences, generating fragments with sticky or blunt ends for cloning.

5. What is the key distinction between a genomic DNA library and a cDNA library?

   Genomic: all DNA; cDNA: reverse-transcribed mRNA (no introns). Genomic libraries include introns and non-coding regions, while cDNA libraries represent expressed genes for protein studies.

6. Which enzyme synthesizes DNA from an RNA template during cDNA library construction?

   Reverse transcriptase. Uses RNA as a template to synthesize complementary DNA, capturing expressed sequences without introns.

7. What is the role of a selectable marker gene on a cloning vector?

   It allows selection of cells that carry the vector. Confers a survival advantage, like antibiotic resistance, to identify and isolate successfully transformed cells.

8. What does a Western blot specifically detect on a membrane?

   A specific protein using antibodies. Uses primary and secondary antibodies to detect and quantify proteins separated by SDS-PAGE.

9. What is the defining function of DNA polymerase in PCR?

   It extends primers to synthesize new DNA strands. Adds nucleotides to the $3'$-end of primers, replicating the template strand during amplification.

10. Which option best explains why primers are required for PCR amplification?

    DNA polymerase cannot initiate synthesis de novo; it needs a $3'$-OH. Provides the necessary $3'$-OH group for DNA polymerase to extend, as it requires an existing chain to build upon.

11. What is the approximate temperature used for DNA denaturation in PCR?

    About $95\,^{\circ}\text{C}$. Disrupts hydrogen bonds between strands, separating double-stranded DNA for primer annealing.

12. What does a Southern blot specifically detect after transferring DNA to a membrane?

    A specific DNA sequence using a labeled nucleic acid probe. Hybridizes probes to immobilized DNA, detecting specific sequences via autoradiography or fluorescence.

13. What is the formula for ideal PCR amplification after $n$ cycles starting from one template?

    $2^n$ copies (ideal, assuming $100\%$ efficiency). Represents exponential doubling per cycle, assuming perfect efficiency and no limiting factors.

14. What is the defining feature of recombinant DNA?

    DNA constructed by joining sequences from different sources. Combines genetic material from multiple organisms or sources to create novel DNA molecules for cloning or expression.

15. Which enzyme covalently joins DNA fragments by forming phosphodiester bonds?

    DNA ligase. Catalyzes the formation of phosphodiester bonds between adjacent nucleotides, sealing nicks in the DNA backbone.

16. What is the approximate temperature used for extension with Taq polymerase in PCR?

    About $72\,^{\circ}\text{C}$. Matches the optimal activity temperature of Taq polymerase for efficient nucleotide incorporation.

17. What is transformation in the context of bacterial cloning?

    Uptake of exogenous DNA (often a plasmid) by bacteria. Introduces recombinant plasmids into competent bacteria, enabling propagation of cloned DNA.

18. What does a Northern blot specifically detect on a membrane?

    A specific RNA transcript using a labeled nucleic acid probe. Measures RNA abundance and size by probe hybridization after gel separation and membrane transfer.

19. What is transfection in the context of eukaryotic molecular biology?

    Introduction of nucleic acids into eukaryotic cells. Delivers DNA or RNA into animal or plant cells using methods like electroporation or liposomes for gene expression studies.

20. Which selectable marker is most commonly used for bacterial plasmid selection?

    An antibiotic resistance gene (for example, ampicillin resistance). Provides resistance to antibiotics, allowing growth of only transformed bacteria on selective media.

21. What is the key difference between sticky ends and blunt ends after restriction digestion?

    Sticky ends have ss overhangs; blunt ends have no overhangs. Sticky ends enable complementary base pairing for efficient ligation, while blunt ends require direct joining.

22. What is the primary basis for separation of DNA fragments in agarose gel electrophoresis?

    Fragment size; smaller fragments migrate farther toward the anode. Applies an electric field where negatively charged DNA moves through the gel matrix, with smaller pieces traveling faster.

23. What is the function of an origin of replication (ori) on a plasmid vector?

    It enables plasmid replication in the host cell. Serves as the starting point for DNA replication machinery, ensuring the plasmid is copied during cell division.

24. Which DNA polymerase is classically used in PCR because it is heat-stable?

    Taq polymerase. Withstands high denaturation temperatures without losing activity, enabling automated thermal cycling.

25. What is a palindromic restriction site in double-stranded DNA?

    A sequence that reads the same $5'\to 3'$ on both strands. Allows restriction enzymes to recognize and cleave symmetrically, facilitating precise DNA cutting.

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