Recombinant DNA technology

Recombinant DNA technology (also known as genetic engineering) is the set of techniques that enable the DNA from different sources to be identified, isolated and recombined so that new characteristics can be introduced into an organism. The invention of recombinant DNA technology—the way in which genetic material from one organism is artificially introduced into the genome of another organism and then replicated and expressed by that other organism—was largely the work of Paul Berg, Herbert W. Boyer, and Stanley N. Cohen, although many other scientists made important contributions to the new technology as well. Paul Berg developed the first recombinant DNA molecules that combined DNA from SV40 virus and lambda phage. Later in 1973, Herbert Boyer and Stanley Cohen develop recombinant DNA technology, showing that genetically engineered DNA molecules may be cloned in foreign cells. One important aspect in recombinant DNA technology is DNA cloning. It is a set of techniques that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word cloning refers to the fact that the method involves the replication of a single DNA molecule starting from a single living cell to generate a large population of cells containing identical DNA molecules. 

DNA cloning 

DNA cloning is the production of a large number of identical DNA molecules from a single ancestral DNA molecule. The essential characteristic of DNA cloning is that the desired DNA fragments must be selectively amplified resulting in a large increase in copy number of selected DNA sequences. In practice, this involves multiple rounds of DNA replication catalyzed by a DNA polymerase acting on one or more types of template DNA molecule. Essentially two different DNA cloning approaches are used: Cell-based and cell-free DNA cloning.

 Cell-based DNA cloning 

This was the first form of DNA cloning to be developed, and is an in vivo cloning method. The first step in this approach involves attaching foreign DNA fragments in vitro to DNA sequences which are capable of independent replication. The recombinant DNA fragments are then transferred into suitable host cells where they can be propagated selectively. The essence of cell-based DNA cloning involves following steps: 

Construction of recombinant DNA molecules

 Recombinants are hybrid DNA molecules consisting of autonomously replicating DNA segment plus inserted elements. Such hybrid molecules are also called chimera. Recombinant DNA molecules are constructed by in vitro covalent attachment (ligation) of the desired DNA fragments (target DNA) to a replicon (any sequence capable of independent DNA replication). This step is facilitated by cutting the target DNA and replicon molecules with specific restriction endonucleases before joining the different DNA fragments using the enzyme DNA ligase.

 Transformation 

The recombinant DNA molecules are transferred into host cells (often bacterial or yeast cells) in which the chosen replicon can undergo DNA replication independently of the host cell chromosome(s). 

Seltive propagation of cell clones

 Selective propagation of cell clones involves two stages. Initially the transformed cells are plated out by spreading on an agar surface in order to encourage the growth of well-separated cell colonies. These are cell clones (populations of identical cells all descended from a single cell). Subsequently, individual colonies can be picked from the plate and the cells can be further expanded in liquid culture.

 Isolation of recombinant DNA clones 

Isolation of recombinant DNA clones by harvesting expanded cell cultures and selectively isolating the recombinant DNA.

Cell-free DNA cloning 

The polymerase chain reaction (PCR) is a newer form of DNA cloning which is enzyme mediated and is conducted entirely in vitro. PCR (developed in 1983 by Kary Mullis) is a revolutionary technique used for selective amplification of specific target sequence of nucleic acid by using short primers. It is a rapid, inexpensive and simple method of copying specific DNA sequence. 

 Enzymes for DNA manipulation 

The enzymes used in the recombinant DNA technology fall into four broad categories:

  Template-dependent DNA polymerase 

DNA polymerase enzymes that synthesize new polynucleotides complementary to an existing DNA or RNA template are included in this category. Different types of DNA polymerase are used in gene manipulation. 

DNA polymerase I (Kornberg enzyme) has both the 3’-5’ and 5’-3’ exonuclease activities and 5’-3’ polymerase activity. 

Reverse transcriptase, also known as RNA-directed DNA polymerase, synthesizes DNA from RNA.

 

Reverse transcriptase was discovered by Howard Temin at the University of Wisconsin, and independently by David Baltimore at about the same time. The two shared the 1975 Nobel Prize in Physiology or Medicine. Taq DNA polymerase is a DNA polymerase derived from a thermostable bacterium, Thermus aquaticus. It operates at 72°C and is reasonably stable above 90°C and used in PCR. It has a 5’ to 3’ polymerase activity and a 5’ to 3’ exonuclease activity, but it lacks a 3’ to 5’ exonuclease (proofreading) activity. 

Nucleases 

Nucleases are enzymes that degrade nucleic acids by breaking the phosphodiester bonds that link one nucleotide to the next. Ribonucleases (RNases) attack RNA and deoxyribonucleases (DNases) attack DNA. Some nucleases will only attack single stranded nucleic acids, others will only attack double-stranded nucleic acids and a few will attack either kind. Nuclease are of two different kinds – exonucleases and endonucleases. Exonucleases remove nucleotides one at a time from the end of a nucleic acid whereas endonucleases are able to break internal phosphodiester bonds within a nucleic acid. Any particular exonuclease attacks either the 3’-end or the 5’-end but not both. 

Mung bean nuclease 

The mung bean nuclease is an endonuclease specific for ssDNA and RNA. It is purified from mung bean sprouts. It digests single-stranded nucleic acids, but will leave intact any region which is double stranded. It requires Zn2+ for catalytic activity. 

S1 nuclease 

The S1 nuclease is an endonuclease purified from Aspergillus oryzae. This enzyme degrades RNA or single stranded DNA, but does not degrade dsDNA or RNA-DNA hybrids in native conformation. Thus, its activity is similar to mung bean nuclease, however, the enzyme will also cleave a strand opposite a nick on the complementary strand. 

RNase 

A RNase A is an endonuclease, which digests ssRNA at the 3’ end of pyrimidine residues. 
RNase H
 It is an endonuclease which digests the RNA strand of an RNA-DNA heteroduplex. The enzyme does not digest ss or dsDNA.
 thermodynamically less stable than DNA because of the 2’ hydroxyl group on the ribose ring that promotes hydrophilic attack on the 5’-3’ phosphodiester bond to form a 2’-3’ cyclic phosphate. Therefore, even if all RNases are eliminated or inhibited during RNA purification, RNA spontaneously degrades while in solution. To circumvent this biological decay of RNA, purified samples are stored at –20°C as ethanol precipitates. The purification of mRNA involves two basic steps
: 1. Biochemical separation of total cellular RNA from DNA and protein using a strong protein denaturant to inhibit cellular RNases, and 
2. Isolation of poly A tail mRNA using an oligo dT affinity matrix. A common method used to isolate mRNA from tissue culture cells is outlined in the following figure 2.4. Guanidinium thiocyanate is a protein denaturant that lyses the cells and inhibits cellular RNases.