Genetic Engineering In Plants Recombinant Dna Technology -PDF Free Download

Genetic engineering in plants recombinant DNA technology

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genes or gene fragments from one organism to another to produce novel traits in the recipient living organism. The development of recombinant DNA technology (rDNA technology) permitting the transfer of genetic material between widely divergent species has opened a new era of research into the structure and function of the genome. The rDNA technology is defined as "the formation of new ...

Steps involved in rDNA technology
Generating DNA fragments
One of the most important problems prior to rDNA experiment is to separate the DNA
fragments from the total genomic DNA This is normally accomplished either by
fragmentation of DNA or synthesis of new DNA molecule The fragmentation of DNA
molecule can be achieved by mechanical shearing The long thin threads which
constitute duplex DNA molecules are sufficiently rigid to be very easily broken by shear
forces in solution In this method high molecular weight DNA is sheared to population of
molecules with a mean size of about 8kb pairs by stirring at 1500 rpm for 30 minutes
Breakage occurs essentially at random with respect to DNA sequence producing termini
consisting of short single stranded regions which may be repaired later The other
sophisticated technique available to generate DNA fragments involves using restriction
endonucleases about which discussion is made in the subsequent section Other two
possible sources for generating DNA fragments for cloning are complementary DNA
cDNA synthesis using mRNA as a template and artificial synthesis of DNA molecule
cDNA synthesis
Fundamental differences exist between the genomes of prokaryotes and eukaryotes In
prokaryotes the coding sequences exons are not intervened by non coding sequences
introns whereas in eukaryotes the genes are generally split the coding regions are
interspersed with non coding DNA This makes the expression of eukaryotic genes in
prokaryotes a tough task
Synthesis of cDNA from mRNA
To overcome this problem cDNA synthesis or artificial DNA synthesis can be well
exploited In cDNA synthesis the eukaryotic mRNA is used as a template to generate
DNA This can be achieved by making a complementary copy of the mRNA using the
enzyme reverse transcriptase and whose function is to synthesize DNA upon an RNA
template At first the enzyme was called RNA dependent DNA polymerase
A DNA copy is made by hybridizing oligo T primers 10 to 20 nucleotides in length to the
3 end of purified mRNA Avian myeloblastosis virus AMV reverse transcriptase is used
to synthesis a cDNA copy of the primed molecule In the resultant RNA DNA hybrid the
RNA can be destroyed by alkaline hydrolysis to which DNA is resistant Thus a single
stranded cDNA is obtained which can be converted into a double stranded form in
second DNA polymerase reaction In the 3 end of the cDNA self complementary occurs
thus producing a hair pin or snap back structure This acts as a primer for duplex DNA
synthesis by DNA polymerase The hair pin loop is trimmed away by treatment with
single strand specific nuclease SI giving rise to a fully duplex molecule The power of
this technique is that only a fraction of the genome that fraction which is transcribed into
mRNA is copied The resulting cDNA clones can be subsequently be used as probes to
identify genomic fragments contained in a genomic library
Chemical synthesis of DNA
Although the methods for generating DNA fragments mentioned above are those most
commonly used the chemical synthesis is considered as an increasingly important
method for generating DNA molecules The chemical synthesis of specific gene
sequences regulatory sequences oligonucleotide probes primers and linkers is a
technique in which solid phase synthesis is adopted In chemical synthesis of DNA two
important strategies adopted are described below
Phosphodiester method
In the phosphodiester method 3 and 5 hydroxyl groups of deoxyribose are protected
R1 and R2 In this method the phosphorus group between the two nucleosides is
unprotected These compounds are therefore soluble in organic solvents to a limited
extent The first significant successes such as the synthesis of the genes for alanine
and tyrosine suppressor tRNA for yeast and E coli respectively were gained with the
phosphodiester method
Phosphotriester method
The phosphotriester method for the synthesis of oilgodeoxyribonucleotides proceeds
essentially in two steps 1 preparation of suitably protected monomers and 2 coupling of
the monomers in the desired sequence by an appropriate phosphorylation procedure
In both protocols the 3 and 5 hydroxyl groups of the deoxyribose sugar are suitably
protected R1 and R2 In the phosphotriester method a third protecting group R3 is
used for the hydroxyl group at the inter nucleotide bond Chemical synthesis of DNA has
found an extraordinary number of applications in gene technology which include
synthesis of partial or total gene sequences primers for DNA and RNA sequencing
hybridization probes for the screening of RNA DNA and cDNA or genomic libraries and
adapters and linkers for gene cloning
Cutting and joining the DNA fragments to vector DNA molecules
Restriction endonucleases Tool for cutting DNA molecules
Techniques for cutting of DNA molecules into discrete fragments by specific enzymes
were virtually unknown until the late sixties A solution to this fundamental problem
eventually grew from long standing research into the phenomenon of host controlled
restriction and modification system
Host controlled restriction and modification phenomenon can be well explained with the
following example If a stock preparation of phage is allowed to grow upon E coli strain
C and this stock is then tried upon E coli C and E coli K the titres observed on these
two strains will differ by several orders of magnitude the titre on E coli K being the
lowest The phages are said to be restricted by the second host strain E coli K and the
phenomenon is called restriction When those phage that do result from the infection of
E coli K are now replated on E coli K they are no longer restricted but if they are first
cycled through E coli C they are once again restricted when plated upon E coli K The
non heritable change conferred upon the phage by the second host strain E coli K that
allows it to be replicated on that strain without further restriction is called modification
These processes can occur whenever DNA is transferred from one bacterial strain to
another Conjugation transduction transformation and transfection are all subject to the
constraint of host controlled restriction and this process is made possible by the
enzymes called restriction endonucleases

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