Dna shuffling

Dna shuffling

1. Index History Introduction DNA Shuffling Critical Parameters Applications of DNA Shuffling Conclusion

2. History The 2018 Nobel Prize in Chemistry was awarded to Frances H. Arnold   “ for the directed evolution of enzymes ” In 1994, Dutch scientist Willem P.C. Stemmer in vented a molecular breeding technique known as DNA shuffling .

3. Introduction The goal of all of these DNA shuffling methods is to generate libraries of chimeric variants from the DNA of two or more related proteins, viruses, or bacteria which then can be subjected to functional screening or selection via directed evolution . The majority of these shuffling methods,rely on homology on the nucleic acid level to generate the chimeric constructs. Researchers can mimic natural evolution by searching these libraries by, for example, affinity panning of phage-displayed or RNA ligands against pharmaceutical targets , for the best candidates for a specific task. Repeated rounds of selection and amplification of candidates has already produced improved enzymes and novel molecules capable of binding their targets with higher affinity than their natural counterparts.

4. Background * Background Information The utility of gene shuffling was first demonstrated in the evolution of the TEM-1 Beta lactamase by Willem PC Stemmer (Stemmer, 1994). The lactamase gene was shuffled, and cloned and expressed in E. coli. The E. coli were plated with the antibiotic cefotaxime at concentration twenty fold higher than the minimum inhibitory concentration of E. coli harboring wild-type TEM-1. Colonies able to withstand this higher concentration of antibiotic served as the starting template for the next round of shuffling and selection. After several rounds of selection on increasing levels of cefotaxime , variants of TEM-1 conferring 32,000-fold greater resistance to cefotaxime than wild-type TEM-1 were

5. In brief related genes with identical flanking sequences are digested with DNase I. The resulting fragments are then thermal cycled in a primerless PCR , in which fragments anneal to one another and mutually serve as primer and template. The products are then amplified in a standard PCR, yielding a shuffled library .

6. DNA Shuffling 1. Obtaining DNA Fragments for Shuffling 2.Reassembly of DNaseI Fragments 3. Amplification of Full-Length Sequences

7. *** Critical Parameters * Thermostable , proofreading DNA polymerase (e.g. Pfu DNA polymerase or KOD DNA polymerase) and associated buffer . **The global alignment is performed by the Pairwise2 function in Biopython . ***This protocol calls for the use of MnCl2 rather than MgCl2 during the fragmentation step.

8. Applications of DNA Shuffling

9. Enzyme Four different β- lactamase genes were shuffled to produce an evolved chimeric protein that conferred a 270-fold greater resistance to the β- lactam antibiotic, moxalactam . These starting genes had sequence similarities of between 60-80% , with numerous regions of homology where crossovers occurred during DNA shuffling.recombination technology creates chimeric genes in which block exchanges have occurred , giving rise to proteins with many sequence differences when compared with any of the parent sequences . Indeed, the best moxalactamase differed at 27% of the amino acid positions. isolated.Shuffling has since been employed in the directed evolution of enzyme function, such as the ability to work at higher temperatures (Giver et al., 1998) or recognize new substrates( Brühlmann and Chen, 1999) and of viral coat proteins that expand host range ( Pekrun et al.,2002) and enable enhanced gene delivery ( Maheshri et al., 2006).

10. cytokine ***DNA shuffling of a family of over 20 human interferon-a ( Hu -IFN-a) genes was used to derive variants with increased antiviral and antiproliferation activities in murine cells. A clone with 135,000-fold improved specific activity over Hu-IFN-a2a was obtained in the first cycle of shuffling. second cycle of selective shuffling, the most active clone was improved 285,000-fold relative to Hu-IFN-a2a and 185-fold relative to Hu-IFN-a1 .

11. The human IFN-a ( Hu -IFN-a) are encoded by a family of over 20 tandemly duplicated nonallelic genes that share 85–98% sequence identity at the amino acid level. Dose limiting toxicity receptor cross-reactivity and short serum half-lives These proteins have potent antiviral and antiproliferative activities that have clinical utility as anticancer and antiviral therapeutics.

12. ***The most active engineered IFN-a, IFN alfacon-1 , is a consensus of 13 wild-type Hu -IFN-a genes that is currently used in hepatitis C therapy.

13. ***recombinant antitumor/antiviral protein ( Novaferon , Nova) is a new type of interferon pro-apoptosis inhibition of tumor angiogenesis higher binding affinity for IFN receptor 2 (IFNR2 ) Analysis methods Antiproliferative test in Daudi cells Anti-viral activity testing by WISH/VSV assay Anti-tumor activity tested by flow cytometry

14. Another example of changing receptor specificity applications in gene therapy is the demonstration that DNA shuffling of viral genomes can be used to produce a novel virus with a changed tropism .Shuffling of six different murine leukemia viruses (MLV) produced chimeras that were able to infect CHO hamster cells, a target that none of the parental viruses was able to infect. A chimeric envelope protein, comprising sequence elements from the original viral gene sequences, was responsible for this change in tropism.

15. Antibody Fv frame work(CDR) obtain from mab library. humanization of a mouse monoclonal antibody ( mAb B233 ) using a new technique which we call framework shuffling . mAb B233 was raised against the human receptor tyrosine kinase EphA2 which is selectively up -regulated in many cancer cell lines and as such constitutes an attractive target for cancer therapy . The resulting Fab combinatorial libraries were then screened for binding to the antigen.

16. Vaccines Chronic infections and even cancer can potentially be treated with “therapeutic vaccines . DNA vaccination  is a technique for protecting against disease by injection with genetically engineered  DNA  so cells directly produce an antigen, producing a protective immunological response

17. Develop vaccine candidates with improved immunogenicity hepatitis B virus vaccines DNA shuffling and screening strategies could improve immunogenicity of vaccine candidates of the hepatitis B virus surface antigen. DNA shuffling of the human hepatitis B surface antigen gene with homologous genes from woodchuck , woolly monkey, and chimpanzee/gibbon hepatitis B viruses, it was found that several chimeras were selected that were more immunogenic than the commercially available hepatitis B virus. These vaccine candidates were more immunogenic when they were delivered in both DNA and protein immunization modalities .

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