Q: Which of the following is the correct recognition sequence for BamHI, and where does it cut?

$$\text{5'-GGATCC-3'}$$; cuts after the first G, leaving a 4-base 5' overhang.

Question 1

Which of the following best explains why a eukaryotic gene must be converted to cDNA before being expressed in *E. coli*?

Accepted Answer

Eukaryotic genes contain introns that *E. coli* cannot splice out.

Question 2

A researcher wants to express a human enzyme in *E. coli* with an N-terminal His-tag that can later be removed. Which vector features and primer modifications are necessary?

Accepted Answer

A vector with a His-tag sequence, a thrombin cleavage site, and primers adding restriction sites for cloning.

Question 3

Which of the following is the correct recognition sequence for BamHI, and where does it cut?

Accepted Answer

$$	ext{5'-GGATCC-3'}$$; cuts after the first G, leaving a 4-base 5' overhang.

Question 4

Why is Taq polymerase used in PCR instead of a typical DNA polymerase?

Accepted Answer

It is thermostable and can withstand repeated heating to $$95^\circC.$$

Question 5

Which of the following best describes the role of IPTG in recombinant protein expression in *E. coli*?

Accepted Answer

It binds to LacI, causing it to dissociate from the operator and allowing transcription.

Question 6

What is the main chemical difference between lactose and IPTG that makes IPTG resistant to hydrolysis by β-galactosidase?

Accepted Answer

IPTG contains a sulfur atom in the glycosidic linkage instead of an oxygen.

Question 7

Which three amino acid residues form the core of the GFP fluorophore?

Accepted Answer

Ser65, Tyr66, Gly67

Question 8

A Sanger sequencing reaction uses dideoxynucleotides (ddNTPs). What feature of ddNTPs causes chain termination?

Accepted Answer

They lack a 3'-OH group, preventing further elongation.

Question 9

Why is agarose gel electrophoresis used for DNA, while SDS-PAGE is used for proteins?

Accepted Answer

Agarose gels have larger pores suitable for large DNA molecules, and DNA is already negatively charged.

Question 10

Which of the following best explains why ampicillin resistance is used as a selectable marker in plasmid transformation?

Accepted Answer

Only cells that have taken up the plasmid with the bla gene can survive in the presence of ampicillin.

Question 11

A researcher observes many small colonies (satellites) around larger colonies on an ampicillin plate after transformation. What is the most likely explanation?

Accepted Answer

Ampicillin was degraded by β-lactamase secreted from transformed cells, allowing non-transformed cells to grow nearby.

Question 12

Which of the following steps is NOT required for the polymerase chain reaction (PCR)?

Accepted Answer

Addition of dideoxynucleotides (ddNTPs)

Question 13

Which of the following best describes the function of the lacI gene in an expression plasmid?

Accepted Answer

It encodes a repressor protein that blocks transcription until an inducer is present.

Question 14

Which of the following is a reason why two plasmids in the same *E. coli* cell must have compatible origins of replication and different antibiotic markers?

Accepted Answer

To ensure both plasmids are maintained and selected for in the cell population.

Question 15

Which of the following best explains why Sanger sequencing reads degrade after about 800–1000 bases?

Accepted Answer

Polymerase processivity and resolution of fragment separation decrease with length.

Question 16

Which of the following is a key advantage of next-generation sequencing (NGS) over Sanger sequencing for environmental DNA surveys?

Accepted Answer

NGS can sequence millions of DNA fragments in parallel, allowing detection of many species at once.

Question 17

Which of the following best describes the role of the T7 promoter in the pET-15b vector?

Accepted Answer

It recruits T7 RNA polymerase to initiate transcription of the inserted gene.

Question 18

Which of the following is a correct sequence of steps for cloning a gene into a plasmid vector using restriction enzymes?

Accepted Answer

PCR amplification with restriction sites, double digestion, gel purification, ligation.

Question 19

Which of the following best explains why the small excised fragment from a double-digested vector is often hard to see on an agarose gel?

Accepted Answer

It binds much less ethidium bromide than the larger fragment, making it less visible.

Recombinant DNA Technology: Study Notes for Biochemistry

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