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Question
A person trying to create a recombinant DNA molecule failed to do so. Below is the procedure that he followed.
Which among the following is the most probable mistake he might have made?
A person trying to create a recombinant DNA molecule failed to do so. Below is the procedure that he followed.
Which among the following is the most probable mistake he might have made?
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Solution
The correct option is C He used two different restriction enzymes
The technology used for producing recombinant DNA (rDNA) through the combination of DNA molecules from different biological sources that are not normally associated with each other is referred to as recombinant DNA technology.
To create the recombinant DNA, several biological tools such as enzymes and DNA from at least two sources are required. In this case, the double stranded plasmid DNA from a bacterium and a double stranded DNA from a mammal are taken.
Following are the steps employed in the creation of rDNA:
Step-1. Isolation of the gene of interest : The gene of interest is amplified, modified by PCR, and eluted from the gel after gel electrophoresis.
Step-2. Cutting the gene at the recognition sites : The mammalian DNA and the plasmid DNA should be cut by the same restriction endonuclease to obtain the complementary sticky ends. These enzymes cut the sugar phosphate backbone in the double stranded DNA at or near specific points called recognition sites that can be palindromic in nature.
Step-3. Ligation of DNA Molecules : The DNA fragment with sticky ends from the mammal is ligated with the plasmid DNA of the bacteria containing sticky ends with the help of the enzyme DNA ligase. This results in the formation of recombinant DNA.
Here, in the question, the person has used two different restriction endonucleases. Therefore, he could not get the complementary sticky ends as a result of which he was not able to create rDNA.
The correct procedure to create rDNA where the mammalian DNA as well as plasmid DNA of bacteria are cut by the same restriction endonuclease is given below.
The enzyme DNA polymerase catalyses the addition of nucleotides to the 3’ end of a polynucleotide which is not required in rDNA technology.
DNA used in rDNA technology has to be double stranded.
rDNA can be obtained by linking just one gene to the plasmid as well.
The technology used for producing recombinant DNA (rDNA) through the combination of DNA molecules from different biological sources that are not normally associated with each other is referred to as recombinant DNA technology.
To create the recombinant DNA, several biological tools such as enzymes and DNA from at least two sources are required. In this case, the double stranded plasmid DNA from a bacterium and a double stranded DNA from a mammal are taken.
Following are the steps employed in the creation of rDNA:
Step-1. Isolation of the gene of interest : The gene of interest is amplified, modified by PCR, and eluted from the gel after gel electrophoresis.
Step-2. Cutting the gene at the recognition sites : The mammalian DNA and the plasmid DNA should be cut by the same restriction endonuclease to obtain the complementary sticky ends. These enzymes cut the sugar phosphate backbone in the double stranded DNA at or near specific points called recognition sites that can be palindromic in nature.
Step-3. Ligation of DNA Molecules : The DNA fragment with sticky ends from the mammal is ligated with the plasmid DNA of the bacteria containing sticky ends with the help of the enzyme DNA ligase. This results in the formation of recombinant DNA.
Here, in the question, the person has used two different restriction endonucleases. Therefore, he could not get the complementary sticky ends as a result of which he was not able to create rDNA.
The correct procedure to create rDNA where the mammalian DNA as well as plasmid DNA of bacteria are cut by the same restriction endonuclease is given below.
The enzyme DNA polymerase catalyses the addition of nucleotides to the 3’ end of a polynucleotide which is not required in rDNA technology.
DNA used in rDNA technology has to be double stranded.
rDNA can be obtained by linking just one gene to the plasmid as well.
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