18. Biotechnology

Steps to DNA Cloning

Mechanism of Recombination

A biologist isolated a gene from a human cell, inserted it into a plasmid, and inserted the plasmid into a bacterium. The bacterium made a new protein, but it was nothing like the protein normally produced in a human cell. Why? (Explain your answer.)
a. The bacterium had undergone transformation.
b. The gene did not have sticky ends.
c. The human gene contained introns.
d. The gene was not synthesized from scratch.

DNA cloning

DNA cloning and recombinant DNA | Biomolecules | MCAT | Khan Academy

A biochemist hopes to find a gene in human cells that codes for an important blood-clotting protein. She knows that the nucleotide sequence of a small part of the blood-clotting gene is CTGGACTGACA. Briefly outline a possible method she might use to isolate the desired gene.

Which of the following sequences in double-stranded DNA is most likely to be recognized as a cutting site for a restriction enzyme?
a. AAGG
TTCC
b. GGCC
CCGG
c. ACCA
TGGT
d. AAAA
TTTT

BioInteractive: Genetic Engineering

An organism which has foreign genes incorporated into its genomes is known as a:

Expression of a cloned eukaryotic gene in a bacterial cell involves many challenges. The use of mRNA and reverse transcriptase is part of a strategy to solve the problem of
a. post-transcriptional processing.
b. post-translational processing.
c. nucleic acid hybridization.
d. restriction fragment ligation.

Each restriction enzyme has a specific sequence of nucleotides where it cuts the DNA. These sequences of DNA are unique to each restriction enzyme and are known as:

Explain how you might engineer E. coli to produce human growth hormone (HGH) using the following: E. coli containing a plasmid, DNA carrying the gene for HGH, DNA ligase, a restriction enzyme, equipment for manipulating and growing bacteria, a method for extracting and purifying the hormone, and an appropriate DNA probe. (Assume that the human HGH gene lacks introns.)

Animation: Restriction Enzymes

1b) Use Ligation Enzymes

Introduction to Molecular Cloning

No. However, this choice is partially correct.

No. Neither of these is required for the production of recombinant DNA.

Review recombinant DNA technology.

What two enzymes are needed to produce recombinant DNA?

The single-stranded ends of DNA molecules can be joined together by:

When a typical restriction enzyme cuts a DNA molecule, the cuts are uneven, giving the DNA fragments single-stranded ends. These ends are useful in recombinant DNA work because
a. they enable a cell to recognize fragments produced by the enzyme.
b. they serve as starting points for DNA replication.
c. the fragments will bond to other fragments with complementary ends.
d. they enable researchers to use the fragments as molecular probes.

1a) Use Restriction Enzymes

Step 1) Create Recombinant DNA

SCIENTIFIC THINKING There are difficulties inherent in testing hypotheses involving human health. Imagine that you have ­developed a potential vaccine against AIDS. Given the nature of the disease, all testing must be conducted in humans. Can you ­design an ethical but effective experiment?

Key Steps of Molecular Cloning

Calculate and compare the caloric content of skim milk and whole milk. Per serving, skim milk contains 12 g carbohydrates, 8 g protein, and no fat; whole milk contains 12 g carbohydrates, 8 g protein, and 8 g fat.

DNA technology has many medical applications. Which of the following is not done routinely at present?
a. production of hormones for treating diabetes and dwarfism
b. analysis of gene expression for more informed cancer treatments
c. gene editing by the CRISPR-Cas9 system in viable human embryos to correct genetic diseases
d. prenatal identification of genetic disease alleles

Restriction enzyme, reverse transcriptase, DNA polymerase, DNA ligase

No. Restriction enzymes are used after the reverse transcriptase and DNA polymerase.

Restriction enzyme, DNA ligase, reverse transcriptase, DNA polymerase

Reverse transcriptase, DNA ligase, DNA polymerase, restriction enzyme

No. The ligase is only used after the restriction enzyme.

Reverse transcriptase, restriction enzyme, DNA polymerase, DNA ligase

No. The DNA polymerase is used immediately after the reverse transcriptase.

Reverse transcriptase, DNA polymerase, restriction enzyme, DNA ligase

Remember that you are starting with a single-stranded nucleic acid, and you have to end up with a double-stranded nucleic acid.

Which arrangement of the following four enzymes represents the order in which they would be used in a typical gene-cloning experiment resulting in the insertion of a cDNA into a bacterial plasmid? Begin with the gene's mRNA transcript.

Review & Application of DNA Cloning in Medicine

What is the most logical sequence of steps for splicing foreign DNA into a plasmid and inserting the plasmid into a bacterium? I. Transform bacteria with a recombinant DNA molecule. II. Cut the plasmid DNA using restriction enzymes (endonucleases). III. Extract plasmid DNA from bacterial cells. IV. Hydrogen-bond the plasmid DNA to non-plasmid DNA fragments. V. Use ligase to seal plasmid DNA to non-plasmid DNA.

2) Transform Recombinant DNA into Bacteria

Potato blight causes potato plants to shrivel and rot. The disease is caused by the pathogen Phytophthora infestans, infamous for its role in Ireland’s Great Potato Famine in the mid-1840s. The disease can devastate crops during wet weather, sometimes leading to total crop loss. Researchers aim to use recombinant DNA methods to transfer blight resistance genes from resistant varieties into susceptible varieties of potato.

Explain how restriction endonucleases and DNA ligase could be used to insert a potato blight resistance gene into a plasmid.

The process of using DNA from one organism to alter the characteristics of another is called:

Animation: Creating Recombinant DNA

A bacterial plasmid cut with restriction enzyme A.

Human DNA cut with restriction enzyme A can be joined to:

Minnows are mainly carnivorous, eating insects and other small animals. However, herbivory has evolved independently in minnows several times. What changes in digestive structure and function are associated with the evolution of herbivory?

Why did the researchers compare relative gut length instead of absolute gut length?

[LECT C8 : RECOMBINANT DNA TECHNOLOGY] 8.2 Steps in Gene Cloning

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Introduction to DNA-Based Technology

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