RESTRICTION ENZYME

A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded or single stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses.Inside a bacterial host, the restriction enzymes selectively cut up foreign DNA in a process called restriction; host DNA is methylated by a modification enzyme (a methylase) to protect it from the restriction enzyme’s activity. Collectively, these two processes form the restriction modification system. cut the DNA, a restriction enzyme makes two incisions, once through each sugar-phosphate backbone (i.e. each strand) of the DNA double helix.

Gene Recombination

Immunoglobin genes are composed of separated segments of DNA that become join together in the process called somatic recombination to make a functional gene.In heavy chain genes there are 3 gene segments the Variable or V segment, the diversity or D segment and joining the J segment.Light chain genes have only 2 gene segments V and J segments

Gene segments that can be recombined has specific sequence motifs adjacent to them called Recombination Signal sequence or RSS motifs .The protein complex containing the products of the recombination activated genes RAG1 and RAG2 binds specifically to the RSS motif.
The individual gene segments to whose flanking RSS motif the RAG protein complex binds are selected at random from the number of copies present in each gene locus .The RAG protein complex brings together the gene segments to be recombined and cleave the DNA exactly at the junction of gene segments and adjoining RSS motifs .The cleavage creates a Hair pin of DNA at the end of the gene segments and double stranded breaks at the end of RSS motifs.

Additional proteins DNA -dependent protein kinase(DNA-PK),Ku,Artemis and dimer of DNA ligase/XRCC4 dimer are incorporated into larger complex with RAG proteins. These RSS ends are joined forming signal joints to create closed circular DNA that plays no further role in the recombination process.

DNA hairpins at the end of the gene segments are cleaved, an additional enzyme Terminal deoxynucleotidyltransferase (TdT) is recruited and adds additional nucleotide to ends of nucleotide strands, The other enzymes in the complex ligate together the two ends of the DNA segment completing the recombination process.

Recombinant DNA Lecture by Eric S.Lander

Eric Lander was a world leader of the international Human Genome Project, the effort to map the blueprint for a human being. Today, Lander is using the knowledge of the human genome to tackle the fundamental issue of medicine: to find the causes of disease.

Lander received his Ph.D. in mathematics from Oxford in 1981, as a Rhodes Scholar. He joined Whitehead Institute in 1986 and founded the Whitehead Institute/MIT Center for Genome Research in 1990. Lander became the founding director of the newly created Broad Institute in 2003.

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Lander is a member of the U.S. National Academy of Sciences, and U.S. Institute of Medicine. He was a MacArthur Fellow (1987-1992), and earned the Woodrow Wilson Prize from Princeton University(1998); the Baker Memorial Award for Undergraduate Teaching at MIT (1992); the City of Medicine Prize (2001); and the Gairdner International Prize (2002).

What is Gene Splicing

Gene splicing is process of cutting a gene from one organism and pasting it into the DNA of another so that a characteristic can be transferred from one plant or animal to another

Restriction enzymes can be used to cut the DNA at different places making the DNA sticky ends so that it can be pasted into DNA from another organism

Splicing a gene into a plasmid

In this activity, you can cut out a gene from a chromosome of one organism and paste it into a plasmid.

To do this successfully: Be careful not to cut up the gene you are working with Cuts the DNa with jagged ends, not blunt ones, so that it can be pasted into the plasmid, Make sure that cut ends of the plasmid match the cut ends of the DNA

The Splicing starts with identifying the correct Restriction enzyme for the Gene, if u choose wrong restriction enzyme it may cut Gene into half rather than isolating the whole gene, So care should be taken in identifying the restriction enzyme which isolates the whole gene (in this case HindIII),

After isolating gene we should cut Plasmid with corresponding restriction enzyme (HindIII) so that gene of interest can be inserted into the plasmid.

After inserting the gene we need to seal the plasmid with Ligase enzyme to achieve a recombinant DNA.