Q: EcoRI is a restriction enzyme that recognizes the palindromic sequence $$5'\text{-GAATTC-}3'$$ and cuts to produce sticky ends. What is the sequence of the sticky end produced?

$$5'\text{-G}3'$$ and $$3'\text{-CTTAA}5'$$

Question 1

Which of the following best describes the function of restriction enzymes in recombinant DNA technology?

Accepted Answer

They recognize specific DNA sequences and cut both strands of the sugar-phosphate backbone.

Question 2

EcoRI is a restriction enzyme that recognizes the palindromic sequence $$5'	ext{-GAATTC-}3'$$ and cuts to produce sticky ends. What is the sequence of the sticky end produced?

Accepted Answer

$$5'	ext{-G}3'$$ and $$3'	ext{-CTTAA}5'$$

Question 3

A researcher digests the lambda phage genome with ApaI and XhoI. The resulting fragments are 10.1 kb, 4.9 kb, and 33.5 kb. What does this result indicate about the locations of the restriction sites?

Accepted Answer

ApaI and XhoI sites are located at different positions, dividing the genome into three distinct fragments.

Question 4

Which of the following is a key advantage of using sticky ends in molecular cloning?

Accepted Answer

Sticky ends facilitate the annealing of complementary sequences, increasing the efficiency of recombinant DNA formation.

Question 5

In directional cloning, why are two different restriction enzymes used to digest the vector and insert DNA?

Accepted Answer

To ensure the insert is cloned into the vector in only one orientation.

Question 6

Which enzyme is used to synthesize complementary DNA (cDNA) from mRNA during the construction of a cDNA library?

Accepted Answer

Reverse transcriptase

Question 7

A genomic library is constructed from the DNA of an organism. Which of the following statements is TRUE regarding the representation of sequences in this library?

Accepted Answer

All sequences, including genic and intergenic regions, are represented approximately equally.

Question 8

Why are plasmids with an origin of replication and a selectable marker gene used as cloning vectors?

Accepted Answer

To allow independent replication and selection of bacteria containing the plasmid.

Question 9

Which of the following is a limitation of using plasmids as cloning vectors for eukaryotic genome DNA?

Accepted Answer

Plasmids over about 20 kb are difficult to maintain in high copy number.

Question 10

What is the main difference between a genomic library and a cDNA library?

Accepted Answer

Genomic libraries contain all sequences from the genome, while cDNA libraries contain only transcribed sequences from mRNA.

Question 11

Which of the following is required for expression of a heterologous gene in E. coli using an expression vector?

Accepted Answer

A Shine–Dalgarno sequence in the 5' untranslated region of the mRNA.

Question 12

Which of the following best explains why transgenic plants are often engineered for herbicide resistance?

Accepted Answer

To allow farmers to treat fields with herbicides that kill weeds but not the crop plants.

Question 13

Which method is most commonly used to generate transgenic plants?

Accepted Answer

Transformation using Agrobacterium tumefaciens and the Ti plasmid.

Question 14

Why must transgenes introduced into plants contain host regulatory sequences?

Accepted Answer

Because regulatory sequences differ greatly between organisms and are required for proper expression.

Question 15

Which of the following is a concern associated with transgenic plants?

Accepted Answer

Transgenes may escape into the environment through interbreeding with related species.

Question 16

In the creation of transgenic mice using homologous recombination, what is the purpose of including both a positive and a negative selectable marker in the transformation vector?

Accepted Answer

To select for cells that have incorporated the transgene at the target locus and against cells with nonhomologous recombination.

Question 17

Which enzyme is used to convert sticky ends to blunt ends for cloning purposes?

Accepted Answer

DNA polymerase (for 5' overhangs) or DNA exonuclease (for 3' overhangs)

Question 18

A scientist wants to express a human gene in E. coli. Which of the following must be considered to ensure proper expression?

Accepted Answer

The gene must be placed downstream of a bacterial promoter and contain a Shine–Dalgarno sequence.

Question 19

Which of the following best describes the process of positive–negative selection in the creation of knockout mice?

Accepted Answer

Cells are selected for the presence of the positive marker and absence of the negative marker, ensuring targeted integration.

Recombinant DNA Technology and Its Applications: Study Notes

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