Genetic engineering is altering the genes in a living organism to produce a genetically modified organism with a new genotype. Recombinant DNA (rDNA) technology is a technique by which the gene (or segment of DNA) is excised from one organism and introduced into another organism. The process of introducing the foreign gene into another organism (or vector) is also called cloning. rDNA technology is a part of vast field of genetic engineering but some authors consider them synonymously. Foreign DNA sequences can be introduced into bacteria, yeast, viruses, plant and animal cells.
The genes are identified by various methods, these include
Polysome precipitation
Prediction based on DNA sequence
Alignment with known mRNAs
Homology to known genes
Identification of Start and Stop codons
Finding a gene by using a functional assay
Find a gene by where it is located
Once identified, it is convenient to maintain a gene library.
A gene library is a population of organisms, each of which carries a DNA molecule that was inserted into a cloning vector. Ideally, all of the cloned DNA molecules represent the entire genome of the organism. A gene library is also called gene bank. This term also represents the collection of all of the vector molecules, each carrying piece of the chromosomal DNA of the organism, prior to the insertion of these molecules into the population of the host cell. Since there is no way to locate a gene by visibly looking at all of the DNA, scientists make gene libraries to catalogue the organism's DNA and then select the gene of interest. A cDNA library consists of sum total of all actively transcribed genes of a tissue, inserted into a population of bacterial cells. A total mRNA preparation is reverse-transcribed and inserted into plasmids all at once so that every possible cDNA sequence will be carried by at least one bacterium in the culture.
Isolation of the gene (DNA sequence)
The technique involved in recombinant DNA technology is to slice (cut) the desired DNA segment and introduce it into a vector (e.g., plasmid). This is achieved using a specific bacterial enzyme called restriction enzymes or restriction endonucleases. Will Porter and John Darms received the 1978 Nobel Prize in Physiology or Medicine for their isolation of restriction endonucleases. These enzymes function as endonuclease, which can cleave a DNA sequence at a specific site. These enzymes are named with three letters based on the species where it was isolated. For example EcoRI is isolated from E. coli.
Each restriction enzymes cleaves DNA strand at a specific site called recognition sequence or restriction site. For example, Eco RI recognizes the sequence GAATTC and cleaves it between G and A (G↓A).
Enzyme Source Target sequence
BamHI Bacillus amyloliquefaciens G↓GATCC
EcoRI Escherichia coli G↓AATTC
HaeIII Haemophilus aegyptius GG↓CC
HhaI Haemophilus hemolyticus GGG↓C
HindIII Haemophilus influenzae A↓AGCTT
HpaII Haemophilus parainfluenzae C↓CGG
KpnI Klebsiella pneumoniae GGTAC↓C
PvuII Proteus vulgaris CAG↓CTG
TaqI Thermus aquaticus T↓CGA
Sometimes, the restriction sequence occurs on both the strands but in reverse direction. Such a segment of DNA with identical sequences but opposite in direction is called a palindrome. A palindrome site is a sequence of base pairs in double stranded DNA that reads the same backwards and forward across the double strand.
A palindrome
When a restriction enzyme acts on palindrome, it cleaves both the strands of DNA molecule. While some enzymes cut the two strands symmetrically, others cut them asymmetrically. AluI, EcoRV and HaeIII generates blunt ends when they act on their restriction sites. Only those enzymes that cut the DNA asymmetrically are useful in rDNA technology. When such enzymes cleave DNA, they leave single stranded “sticky ends” on both strands. Same restriction enzymes are used to cleave the DNA molecule to be transferred and the vector. The circular structure of the plasmid is broken by the restriction enzyme, this process leaves a “sticky end” at either strand. The strand of DNA to be transferred must have two restriction sites; one on either side of the DNA segment of interest. When it is acted upon by restriction enzyme, it generates two sticky ends, one at either side of the segment. Since these sticky ends are generated by the same enzyme, they are complementary and hence are cohesive.
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Continuation of Genetic engineering: (part 2) Vector
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