Multiple Choice
The process of using DNA from one organism to alter the characteristics of another is called:
18. Biotechnology
Steps to DNA Cloning
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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.
A
Restriction enzyme, reverse transcriptase, DNA polymerase, DNA ligase
B
Restriction enzyme, DNA ligase, reverse transcriptase, DNA polymerase
C
Reverse transcriptase, DNA ligase, DNA polymerase, restriction enzyme
D
Reverse transcriptase, restriction enzyme, DNA polymerase, DNA ligase
E
Reverse transcriptase, DNA polymerase, restriction enzyme, DNA ligase
Verified step by step guidance1
Start with the mRNA transcript of the gene you want to clone. The first step is to convert this mRNA into complementary DNA (cDNA) using the enzyme reverse transcriptase. This enzyme synthesizes a DNA strand complementary to the mRNA template.
Once you have the single-stranded cDNA, the next step is to synthesize the complementary DNA strand to form a double-stranded DNA molecule. This is done using DNA polymerase, which adds nucleotides to the cDNA strand, creating a double-stranded DNA.
With the double-stranded DNA now available, the next step is to prepare the DNA for insertion into a plasmid. This involves cutting both the plasmid and the cDNA with a restriction enzyme. Restriction enzymes recognize specific sequences in the DNA and make cuts, creating 'sticky ends' that facilitate the insertion of the cDNA into the plasmid.
After cutting, the cDNA and plasmid are mixed together, allowing the sticky ends to pair up. The enzyme DNA ligase is then used to seal the nicks in the sugar-phosphate backbone, effectively joining the cDNA to the plasmid and forming a stable recombinant DNA molecule.
The final recombinant plasmid can now be introduced into bacterial cells through a process called transformation, where the bacteria take up the plasmid and express the gene of interest.
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