Palindromic Sequence : Complementary DNA sequences that are the same
when each strand is read in the same direction (5 =>3). These sequence act as
recognition sites for restriction endonuclease.
5,-GAATTC-3'
3,-CTTAAG-5'
Complementary DNA (cDNA) : A DNA strand formed from mRNA by using the enzyme reverse transcriptase.
Cloning Vectors : A small, self-replicating DNA molecule into which foreign DNA is inserted. It replicates inside the host cell. The vectors that may be used in genetic engineering are plasmids, bacteriophages, animal, plant, virus, YACs and BACs and some yeasts.
Plasmid : Extra chromosomal, self replicating circular DNA molecule found in certain bacteria and in some yeasts. It has a few genes. Plasmids are used as cloning vectors in genetic engineering. Plasmids were discovered by Willium Hays and Joshua Leduberg in 1952. The most widely used vector in cloning is pBR322.
Bacteriophage : A virus which infects bacteria is called bacteriophage.
Ti Plasmid : It is an extrachromosomal, double stranded and self replicating DNA molucule found in Agrabacterium tumifaciens. If causes tumor in plants. But now Ti Plasmid has been modified into a cloning vector by which desired genes can be delivered into many plants.
Features of cloning vector : Origin of replication (Ori) selectable marker and cloning sites are the features that are required to facilitate cloning into a vector.
(a) Origin of Replication (Ori) : This is a sequence from where replication starts and any piece of DNA when linked to this sequence can be made to replicate within the host cells. This sequence is also responsible for controlling the copy number of the linked DNA.
(b) Selectable Marker : It is a gene which helps in identifying and eliminating non-transformants from transformants (having recombinant DNA) by selectively permitting the growth of transformants. The process through which as piece of DNA is introduced in a host bacterium is called transformation. The genes encoding resistance to antibiotics are considered useful selectable marker for E. coli.
(c) Cloning Sites : A location on a cloning vector into where a foreign gene can be introduced is called recognition site. The vector must have very few (preferably single) recognition sites. The presence of more than
one recognition sites within the vector will produce several fragments which will make the process of gene cloning more complicated. Therefore, the foreign DNA is ligated at a restriction site present in one of the two antibiotic resistance gene.
(d) Small Size of Vector : This facilitates the intoduction of DNA into the host easily.
Insertional Inactivation : This method is used to differentiate recombinants from non-recombinants on the basis of ability to produce colour in the presence of a choromogenic substrate. When a rDNA is inserted in the coding sequence of an enzyme. It results in inactivation of the enzyme. This is called insertional inactivation.
Case I : The absence of insert in the plasmid of bacteria :
The presence of chromogenic substrate gives blue coloured colonies of bacteria, hence these bacterial colonies are nonrecombinant.
Case II : The presence of insert in the plasmid of bacteria :
It results insertional inactivation of the -galactosidase, therefore bacterial colonies do not give any colour. Hence the bacterial colonies are recombinant.
Steps in Formation of rDNA by action of EcoRI : EcoRI cuts the DNA between bases G and A only => sticky ends of cut DNAs are formed => DNA fragments join at stickly ends by DNA ligase => Recombinant DNA is formed
