Guide To Learn
rDNA technology has gained importance in each and every aspect of modern biological researches. The rDNAs are used in basic research or in the commercial production of useful products. rDNA also find application in agriculture and industry. In Pharmaceutical Industry Important compounds such as recombinant insulin, which are used in the treatment of diabetes, human […]
Genomic Library It is a collection of clones containing every single gene present in an organism. For the construction of a genomic library, the entire genomic DNA is isolated from host cells/tissues, purified and broken randomly into fragments of appropriate size for cloning into suitable vector. DNA can be fragmented by physical shearing or by […]
Cells all of which contain the same DNA sequences are called ‘clones’. Cloning serves two main purposes. The various steps involved in molecular cloning can be outlined in the following sections. Preparation of Vector DNA Vector DNAs that originate from microorganisms are propagated and harvested from their appropriate microbial hosts. The bacterial cells grown in […]
This is used to visualize the position of a cloned gene on a eukaryotic chromosome. The cells are treated with a fixative. They are attached to a glass slide and incubated with RNAse and NaOH to degrade RNA and denature the DNA. The chromosome exposes the segments of DNA. A sample of the cloned gen […]
If DNA is denatured and later allowed to renature, the two separated single strands of DNA will zipper back to reform the double-stranded DNA molecule. This ability of the complementary sequences to anneal or to hybridize one another is called ‘nucleic acid hybridization’. This technique helps in determining the gene structure and in identifying molecules […]
S1 Nuclease It is an endonuclease, which cleaves single-stranded DNA or single-strand protrusion of double-stranded DNA with cohesive ends. Because of S1 nuclease action, cohesive ends are converted into blunt ends. Thus, S1 nuclease is used to remove the incompatible ends. DNA Pol-I, Klenow fragment This fragment has the polymerase activity and 3′ → 5′ […]
S1 Nuclease It is an endonuclease, which cleaves single-stranded DNA or single-strand protrusion of double-stranded DNA with cohesive ends. Because of S1 nuclease action, cohesive ends are converted into blunt ends. Thus, S1 nuclease is used to remove the incompatible ends. DNA Pol-I, Klenow fragment This fragment has the polymerase activity and 3′ → 5′ […]
The blunt ends created using the action of restriction enzymes can be ligated with a similar blunt created in vectors by using: Figure 9.7 Recombinant DNA production by sticky end ligation T4 DNA ligase Blunt ends can be ligated using E. coli and phage DNA ligases, which seals single-stranded nicks between adjacent nucleotides in a duplex DNA chain. […]
Both the target DNA and the vector such as plasmid DNA can be cut by the same restriction enzyme, so that they produce complementary staggered ends, which are annealed together to get the rDNA (Figure 9.7).
The term restriction endonuclease was coined by Lederberg and Meselson in 1964 to describe the nuclease enzyme that destroys or restricts any foreign DNA entering a bacterial cell. These restriction endonucleases are widely used in rDNA technology. They specifically bind to double-stranded DNA and cleave it at specific sites known as recognition sequence or restriction […]