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Recombinant DNA Technology
In the last century when scientists discovered that nucleic acid (DNA) is the principal molecule responsible for the expression of characters, attempts were made to alter the genetic makeup of an organism by manipulating nucleic acid directly. Various methods used for directly manipulating nucleic acid/genome (DNA) of an organism are collectively referred to as recombinant DNA (rDNA) technology or genetic engineering.
rDNA technology has been possible due to rapid progress in various fields of biology which spans from biochemistry, genetics, cytology, microbiology, molecular biology and others. Isolation and purification of nucleic acids followed by the understanding of their structures, properties, functions and finally their sequencing in the last century are the most important contributions which laid the foundation of development of rDNA technology.
1.0History
- Stanley Cohen had the expertise in introducing plasmid DNA into Escherichia coli (E. coli) and subsequent propagation and cloning of plasmids in the bacteria. On the other hand, Boyer had the expertise to cleave the double stranded DNA to produce single stranded ends with identical termini using restriction enzymes. Both visualized the potential of combining the two discoveries to what would later become rDNA technology or genetic engineering.
- The first recombinant DNA (rDNA) molecules were generated by Herbert Boyer and Stanley Cohen of Stanford University and University of California San Francisco
- In conjunction with his studies of the tumor virus SV40, in 1972, Paul Berg succeeded in inserting DNA from a bacterium into the virus' DNA. He thereby created the first DNA molecule made of parts from different organisms. This type of molecule became known as "hybrid DNA" or "recombinant DNA". Among other things, Paul Berg's method opened the way to creating bacteria that produce substances used in medicines.
2.0Making Recombinant DNA
Isolate DNA a Cut with restriction enzymes à Ligate into cloning vector à transform recombinant DNA molecule into host cell à each transformed cell will divide many, many times to form a colony of millions of cells, each of which carries the recombinant DNA molecule.
Isolating DNA
1. Crude isolation of donor (foreign) DNA is accomplished by isolating cells à disrupting lipid membranes with detergents à destroying proteins with phenol or proteases à degrading RNAs with RNase à leaving DNA at the end
2. Crude isolation of plasmid vector DNA is accomplished by an alkaline lysis procedure or by boiling cells which removes bacterial chromosomal DNA from plasmid DNA.
3. To get purer DNA from either (1) or (2), crude DNA is
a) Fractionated on a CsCl2 gradient
b) Precipitated with ethanol
c) Poured over a resin column that specifically binds DNA
Cutting DNA
1. DNA can be cut into large fragments by mechanical shearing.
2. Restriction enzymes are the scissors of molecular genetics. Restriction enzymes (RE) are endonucleases that will recognize specific nucleotide sequences in the DNA and break the DNA chain at those points. A variety of RE have been isolated and are commercially available. Most cut at specific palindromic sites in the DNA (sequence that is the same on both antiparallel DNA strands). These cuts can be a staggered which generates “sticky or overhanging ends” or a blunt which generates flush ends.
Joining DNA
Once you have isolated and cut the donor and vector DNAs, they must be joined together.
The DNAs are mixed together in a tube. If both have been cut with the same RE, the ends will match up because they are sticky. DNA ligase is the glue of molecular genetics that holds the ends of the DNAs together. DNA ligase creates a phosphodiester bond between two DNA ends.
Amplifying the recombinant DNA
To recover large amounts of the recombinant DNA molecule, it must be amplified. This is accomplished by transforming the recombinant DNA into a bacterial host strain. (The cells are treated with CaCl2 a DNA is added à Cells are heat shocked at 42 C a DNA goes into the cell by a somewhat unknown mechanism.) Once in a cell, the recombinant DNA will be replicated. When the cell divides, the replicated recombinant molecules go to both daughter cells which themselves will divide later. Thus, the DNA is amplified.