Molecular Cloning

This animation demonstrates the details of the molecular cloning of DNA sequences. The image shown is a circular plasmid DNA molecule known as a cloning vector. The vector contains two important regions – the cloning site and the drug resistance marker. The cloning site is the DNA region where the gene to be studied is inserted, and the resistance marker contains a gene which allows cells that contain the plasmid to survive in the presence of antibiotics. Often one of the first steps involved is to insert the gene of interest into a cloning vector. To accomplish this, the plasmid vector must be cut with a restriction enzyme at the cloning site. The enzyme, shown here as a pair of scissors, recognizes a specific DNA sequence within the cloning site and often makes an uneven, or staggered, cut at this location. This leaves some of the bases at the ends of the DNA unpaired or “overhanging” with respect to the rest of the molecule – these are referred to as “sticky ends” since they can base pair or “stick” to a DNA fragment with a complementary sequence. Next, the DNA sequence to be cloned, sometimes containing a functional gene, is added to the cut plasmid vector. Since it too has been cut with a restriction enzyme, it may have “sticky ends” which are complementary to the overhanging bases of the cloning vector. Therefore, with the help of an enzyme called ligase, the DNA fragment can be inserted, or ligated, into the vector. Then, the cloning vector that now contains a copy of the gene of interest is used to transform a bacterial cell such as E. coli, shown in green. The term “transform” refers to the uptake of DNA by a living cell. The circular DNA vector including the inserted gene is replicated by the bacterium until several copies are present, and when the cell divides, many more copies, or clones, of the vector and genes are produced. These can then be selected via the drug resistance marker and isolated from bacterial cells, such that many copies of the gene of interest are available for study.

Question 1: Circular DNA molecules used for cloning, in molecular biology are also known as ______________.
  1. vectors
  2. DNA cells
  3. molecular tools
  4. vectorials

Answer 1 Answer – (a) vectors

Question 2: The vector always contains at least two regions: a __________ and a ___________.
  1. cloning site, drug resistance marker
  2. restriction site, stain
  3. start site, marker gene
  4. glue site, start site

Answer 2 Answer – (a) cloning site, drug resistance marker

Question 3: The gene to be studied is inserted into a __________________ and the vector contains a _________________________ gene that allows the host cells to survive in the presence of antibiotics.
  1. start site, marker
  2. carrier site, drug
  3. leftward site, expression
  4. cloning site, drug resistance marker

Answer 3 Answer – (d) cloning site; drug resistance marker

Question 4: What is the main function of restriction enzymes in cloning?
  1. To stop cells from multiplying.
  2. To restrict enzymes.
  3. To stop cloned genes.
  4. To cut the plasmid vector where the gene of interest is to be inserted.

Answer 4 Answer – (d) To cut the plasmid vector where the gene to study of interest is to be inserted.

Question 5: In molecular biology, the term “transform” refers to the ___________ of DNA by a living cell.
  1. uptake
  2. change
  3. enlargement
  4. transcription

Answer 5 Answer – (a) uptake