Cloning in bacteria and eukaryotes

Content
Genetic Engineering and Biotechnology 1.  Recombinant DNA and PCR (Cloning and Amplification of DNA)
Restriction enzymes in cloning
Techniques used in recombinant DNA 
Cloning vectors for recombinant DNA
Plasmids as vectors
Bacteriophages as vectors
Plant and animal viruses as vectors
Transposons as vectors
Artificial chromosome vectors for cloning large DNA segments
Construction of chimeric DNA
Palindromes and staggered cleavage
Adding poly dA at the 3' ends of the vector and poly dT at the 3' ends of DNA clone
Blunt end ligation by T4 DNA ligase
Cloning in bacteria and eukaryotes
Cloning in bacteria
Cloning in eukaryotes
Molecular probes 
Labelling of probes
Applications of molecular probes
Construction and screening of genomic and cDNA libraries
Gene amplification : PCR and its applications
cDNA library from mRNA
Colony (or plaque) hybridization for screening of libraries
Gene Amplification : PCR and Its Applications
The basic polymerase chain reaction (PCR)
Different schemes of PCR
Cloning in Bacteria and Eukaryotes
Cloning in bacteria
Bacteria can be classified into (i) gram negative bacteria e.g. Escherichia coli. Pseudomonas, Rhizobium and (ii) gram positive bacteria e.g. Bacillus subtilis and Streptomycetes sp. Ordinarily plasmids can not maintain themselves in stable form in both classes of bacteria and, therefore, plasmid vector for two classes usually differ. While for gram negative bacteria, plasmids, phages and cosmids are known as vectors, for gram positive bacteria, only plasmids are known to do this job.

Cloning in eukaryotes
Since chromosomes found in the nucleus in eukaryotes are separated from the rest of cell through nuclear membrane, and since many of the genes are split genes with exons and introns, genetic engineering with eukaryotes requires new methods and tools. When eukaryotic genes are cloned in prokaryotes, the split genes can not be correctly expressed, because prokaryotes do not have the equipment necessary for splicing out the RNA transcribed from the introns of a gene.

Among eukaryotes, DNA cloning has been done in yeast, mouse and to some extent even in some higher plant species. In yeast, a plasmid called 2μ DNA (63bp) is found, which is an appropriate cloning vehicle. An efficient transformation method is also available, which involves protoplast production and PEG (polyethylene glycol) directed introduction of DNA. In animal cells like mouse cells, special animal viruses were used as cloning vehicles. Simian virus 40 (SV40) is one such virus, in which globin gene could be integrated. This gene integrated in SV40 could be transcribed and translated in mouse kidney cells.