Recombinant DNA technology - methods and applications

Recombinant DNA technology is modifying the genetic makeup of an organism either by adding new genes or by changing the existing genes. It is a technique of preparing r DNA in vitro by cutting up DNA molecules and splicing the fragments together from more than one organism. Recombinant DNA is a form of artificial DNA that is made through the combination or insertion of one or more DNA strands.  Recombinant DNA is also referred to as "chimera."

Using recombinant techniques, particular sequences of DNA can be isolated, manipulated, and re-introduced into many different kinds of living things. The desired results are improvement of microorganisms, plants, and animals, for a particular purpose.

Objectives of  r DNA Technology

• Artificially synthesize new genes. 
• Altering the genome of an organism.
• Bring about new gene combinations not found in nature. 
• Understanding the hereditary diseases and their cure.
• Improving human genome.

Enzymes are the chemical knives in r DNA technology

DNA or RNA polymerase-replicating or annealing a DNA chain.
Reverse transcriptase – synthesize c DNA from RNA template.
DNA ligase – joining DNA strands together.
Nuclease-breaks phospho-diester bonds within free ends (exonucleases) or in an interior position (Endonucleases ).
Restriction endonuclease – recognizes a specific base sequence and cuts the DNA.

Restriction endonucleases (RE ases)

Restriction endonuclease is a special class of sequence –specific enzymes.It is found in bacteria which protect its genetic material from the invasive attacks of viruses. It is a site-specific enzyme which cleaves DNA molecules only at specific nucleotide sequences. REases recognize DNA base sequences that are palindromes. REases make two single stranded breaks, one in each strand. REases make staggered cuts with complementary base sequences for easy circularization.

Types of Vectors

o   Bacterial plasmid vectors

o   Bacteriophage vectors

o   Cosmid vectors

o   Expression vectors

o   Bacterial Artificial Chromosomes (BAC)

o   Yeast Artificial Chromosomes (YAC)

o   Ti  and Ri vectors

Plasmids

Plasmids are relatively small, self-replicating duplex extra-chromosomal DNAs maintained as independent molecules. Lederberg coined the term ‘plasmid’  in 1952. They are found in bacteria, yeast and streptomyces. Plasmids range in size from < 1 Kb to 500Kb.

Phage Vectors

Two types of phage vectors have been extensively developed-λ and M13. Phage vectors have engineered phage genomes previously genetically modified to include restriction sites. After insertion of foreign DNA, the recombinant phage genome is packaged into the capsid and used to infect host cells

Cosmids

Hybrid vector constructed to contain features from both phages and plasmids. Cosmids  have a selectable marker, multiple cloning sites from plasmids and a cos site from l phage

Artificial Chromosomes

Large fragments of DNA  can  be cloned in artificial chromosomes. Mapping of genes is easier. Artificial chromosomes have played important role in the human genome project. One copy of YAC is present per cell. E.g. yeast artificial chromosomes (YACs) and bacterial artificial chromosomes (BACs)

Host cell types

• Prokaryotic hosts – Bacteria, E . Coli, Bacillus sp., Pseudomonas sp., Streptomyces sp.
• Eukaryotic hosts - Yeast – Saccharomyces, Fungi- Aspergillus, Neurospora, Algae - Chlamydomonas

Two types of host-vectors

• Cloning vector - Propagation of DNA inserts
• Expression vector - Production of proteins

Molecular Cloning / DNA cloning

Molecular cloning refers to the process of making multiple DNA molecules.

Step  1– fragmentation -breaking apart a strand of DNA

Step 2 – ligation-gluing together pieces of DNA in a desired sequence.

Step 3 –Transfection -  inserting the newly formed DNA into cells.

Step 4-Screening / selection – selecting out the cells that were successfully tranfected with the new DNA

Gene transfer technology

• Transduction- Virus mediated gene transfer.
• Tranfection - Chemical or physical tricks to persuade cells to take DNA from the culture medium.
• Direct transfer - Physically inserting the gene e.g. microinjection
• Natural gene transfer - A receptor – mediated lateral binding fusogenic proteins used.

Calcium phosphate –co precipitate  method

This method was described by Graham and Van der Eb in 1973.

It is a process for inserting foreign DNA into bacteria. The bacterial cells are treated with  ice-cold calcium chloride. The plasmid DNA is added  to cells chilled on ice which form calcium phosphate –DNA precipitate. The cell and DNA mixture is heated  to 42^oC. The membrane becomes fluid and plasmid DNA enters bacterial cells and is replicated and expressed

Electroporation

It  involves a brief application of high voltage electric current to the cells resulting in the formation of transient holes in the cell membrane through which plasmid DNA can enter the cell. The transformation efficiency is high. Quick restoration of membrane fluidity and closing of pores is crucial for survival of cell after the pulse.

Selection  techniques for rDNA molecules

•         DNA hybridization assay

•         Colony immunoassay

•         Screening by protein activity

•         Genetic screening methods

Process of selection

Selection is a process designed to facilitate the identification of recombinant bacteria while preventing the growth of non-transformed bacteria. After transformation, the bacteria are challenged with an antibiotic (such as ampicillin). If the E. coli have taken up and expressed an ampicillin resistance gene on a plasmid, they will live - otherwise they will die. This process is called selection because selected bacteria may survive.

DNA hybridization assay

This technique was introduced by Grunstein and Hogness (1978). The target DNA is denatured at 800C and bound to a nitrocellulose filter discs. Such filters are hybridized with radioactive DNA probes. The results are monitored by autoradiography.

Colony immunoassay

The transformed colonies are transferred to a nitrocellulose filter. The colonies are lysed and the released proteins are attached to the matrix. The matrix is treated with a primary antibody which specifically binds to the proteins encoded by the target gene. Then the matrix was washed to remove any unbound antibody. Then the matrix was treated with a second antibody which was an enzyme, alkaline phosphatase. The target protein (antigen) was treated with a colorless substrate. The colorless substrate is hydrolyzed by the alkaline phosphatase into a colored complex.

The Tools of Recombinant DNA Technology

—  Gene Libraries

The collections of cloned DNA fragments from a particular organism contained within bacteria or viruses as the host. The library may contain all genes of a single chromosome. Screening, identification and characterization of cloned fragments are possible with suitable probes.

cDNA Libraries

The library contains only complementary DNA molecules synthesized from mRNA molecules in a cell. mRNA from tissue of interest is isolated and converted into a double-stranded DNA by using the enzyme reverse transcriptase. The newly synthesized molecules are called complementary DNA (cDNA) because it is an exact copy of the mRNA.

Supporting techniques of Recombinant DNA Technology

      Multiplying DNA in vitro by Polymerase Chain Reaction (PCR)

It was developed in the 1980s by Kary Mullis. This technique is used for making copies, or amplifying, a specific sequence of DNA in a short period of time. It is a repetitive process consisting of three steps: Denaturation, Priming and Extension.It can be automated using a thermocycler. At the end of one cycle, the amount of DNA has doubled. Cycles are repeated 20–30 times

      Separating DNA Molecules by Gel Electrophoresis

The molecules are separated on the basis of electrical charge, size, and shape. It allows to isolate DNA of interest. Negatively charged DNA drawn toward positive electrode. Agarose makes up gel; acts as molecular sieve. Smaller fragments migrate faster than larger ones.Size is determined by comparing distance migrated to standards.

Applications of rDNA technology

Production of transgenic organisms - Recombinant plants and animals altered by addition of genes from other organisms. Transgenic plants that can produce their own insecticides e.g.Better Crops (drought , heat and salt resistance)
Recombinant DNA technology has been used for creating new animal species using the cloning technology. rDNA technology is used to elucidate molecular events in biological processes like cell differentiation, aging etc.
Recombinant Vaccines (ie. Hepatitis B
Gene therapy - Missing or defective genes replaced with normal copies e.g. sickle cell anaemia and severe combined immuno-deficiency (SCID).
Large-scale production of human proteins by genetically engineered bacteria such as : insulin, Growth hormone, Interferons and blood clotting factors (VIII & IX)

Disadvantages of Recombinant technology:

Recombinant technology  can be commercialized and became big source of income for businessmen. The products have effects on the natural immune system of the body. The transgenic organisms can destroy natural ecosystem that relies on organic cycle. They are prone to undergo mutation that could have harmful effects. There is possibility of manufacturing of biological weapons such as botulism & anthrax to target humans with specific genotype. There is a concern of creating super‐human race.