Cloning

A clone is a population of identical organisms derived from a single parental organism. In Molecular biology- A clone is a collection of molecules or cells, all identical to an original molecule or cell. If the original cells harbored a recombinant DNA molecule in the form of a plasmid, the plasmids within the millions of cells in a bacterial colony represent a clone of original DNA molecules; these molecules can be isolated and studied. The mechanism of cloning involves , i) Cutting DNA at precise locations, ii) Joining two DNA fragments covalently, iii) Selection of a small molecule of DNA capable of self replication ,iv) Moving recombinant molecules from the test tube in to a host cell and v) Selecting or identifying those cells that contain Recombinant DNA .

Which of the following is required for joining two DNA fragments covalently?

A. DNA polymerase

B. Restriction endonucleases

C. Primase

D. Reverse transcriptase

E. DNA ligase

Details

The correct answer is DNA ligase.

Construction of DNA molecule composed of nucleotide sequences taken from different sources is the basis of Recombinant DNA technology.

Purpose of Recombinant DNA technology

This is the laboratory manipulation of nucleic acids to-

  • Analyze
  • Compare
  • Construct
  • Mutate
  • Excise
  • Join or
  • Clone specific sequences of DNA and hence manipulate the inherited characteristics of cells and organisms.

The methods used to accomplish

  •  isolation and
  •  manipulation of DNA including
  •  end to end joining of sequences taken from different sources
  • to make chimeric molecules and related tasks
  • are referred to as Recombinant DNA technology or Genetic Engineering.

Thus the procedure of cloning involves:

1) Cutting DNA at precise locations

2) Joining two DNA fragments covalently

3) Selection of a small molecule of DNA capable of self- replication

4) Moving recombinant molecules from the test tube in to a host cell

5) Selecting or identifying those cells that contain Recombinant DNA

1) Cleavage of DNA

  • Restriction Enzymes cut DNA chains at specific locations
  • The specific site is called “Restriction site” which is 4-7 base pair long
  • The fragments of DNA obtained after the action of restriction enzymes are called “Restriction fragments”; they can have sticky or blunt ends.
  • Restriction enzymes are also called “Molecular scissors”

2) Joining two DNA fragments covalently

The DNA fragments are joined together by DNA ligase (figure-1 and 2)

Role of DNA ligase

Figure- 1- Showing the roles of restriction endonuclease and DNA ligase in the formation of recombinant DNA

3) Selection of a small molecule of DNA capable of self- replication

Chimeric or hybrid DNA molecules can be constructed in cloning vectors which then continue to replicate in a host cell under their own control systems. In this way, the chimeric DNA is amplified.

Cloning vectors

A vector is a molecule of DNA to which the fragment of DNA to be cloned is attached.

Commonly used vectors

  • Plasmids
  • Bacterial and animal viruses
  • Cosmids
  • Artificial chromosomes

Bacterial plasmids are small, circular, duplex DNA molecules whose natural function is to confer antibiotic resistance to the host cell. Plasmids have several properties that make them extremely useful as cloning vectors.

Phages usually have linear DNA molecules into which foreign DNA can be inserted at several restriction enzyme sites. A major advantage of phage vectors is that while plasmids accept DNA pieces about 6–10 kb long, phages can accept longer DNA fragments.

Still larger fragments of DNA can be cloned in cosmids, which combine the best features of plasmids and phages.

Even larger pieces of DNA can be incorporated into bacterial artificial chromosome (BAC) or yeast artificial chromosome (YAC). These vectors can  accept and propagate DNA inserts of several hundred kilobases or more and have largely replaced the plasmid, phage, and cosmid vectors.

Mammalian viral vectors are also used in this technology especially for gene therapy.

Recombinant plasmid

Figure-2- Formation of recombinant Plasmid

4) Moving recombinant molecules from the test tube in to a host cell (figure-3)

Bacterial transformation

Figure-3- Bacterial transformation by recombinant plasmid

E. coli is the most common host cell due to the following advantages:

i)            It’s DNA metabolism is well understood

ii)            Cloning vectors associated with E.Coli are well characterized

iii)            Effective techniques are available for moving DNA from one bacterial cell to another

5)  Selecting or identifying those cells that contain Recombinant DNA (figure-4)

Colony or plaque hybridization is the method by which specific clones are identified and purified. Bacteria are grown as colonies on an agar plate and overlaid with nitrocellulose filter paper. Cells from each colony stick to the filter and are permanently fixed thereto by heat, which with NaOH treatment also lyses the cells and denatures the DNA so that it will hybridize with the probe. A radioactive probe is added to the filter, and (after washing) the hybrid complex is localized by exposing the filter to x-ray film. By matching the spot on the autoradiograph to a colony, the latter can be picked from the plate. A similar strategy is used to identify fragments in phage libraries. Successive rounds of this procedure result in a clonal isolate (bacterial colony) or individual phage plaque.

Colony hybridization

Figure-4- Colony hybridization

DNA libraries

The combination of restriction enzymes and various cloning vectors allows the entire genome of an organism to be packed into a vector. A collection of these different recombinant clones is called a library. A genomic library is prepared from the total DNA of a cell line or tissue. A cDNA library comprises complementary DNA copies of the population of mRNAs in a tissue.

DNA probes (Figure-5)

A variety of molecules can be used to “probe” libraries in search of a specific gene or cDNA molecule or to define and quantitate DNA or RNA separated by electrophoresis through various gels. Probes are generally pieces of DNA or RNA labeled with a 32P-containing nucleotide—or fluorescently labeled nucleotides (more commonly now). Biotinylated probes are also in practice.

Probe

Figure-5- Hybridization by Radioactive oligonucleotide probe.

As regards other options

A. DNA polymerase is an enzyme for DNA replication

B. Restriction endonucleases- function to cut DNA at specific sites

C. Primase- An enzyme for the synthesis of RNA fragments called, primers which are required for the replication of DNA, as the DNA polymerases cannot initiate DNA chain synthesis, they can simply elongate the existing DNA fragments.

D. Reverse transcriptase- is required for the formation of DNA from RNA (reverse of transcription)

 

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