This document discusses the technique of DNA shuffling. It begins with a brief history of DNA shuffling, invented by Willem P.C. Stemmer in 1994. It then discusses the goal of DNA shuffling, which is to generate libraries of chimeric variants from related DNA sequences that can undergo directed evolution. Critical parameters for DNA shuffling include using a thermostable DNA polymerase and performing a fragmentation step using MnCl2 instead of MgCl2. Applications of DNA shuffling discussed include evolving improved enzymes, cytokines, antibodies, viral proteins, and vaccine candidates.

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.

4. 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.

5. 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

6. 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.

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

8. ***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.

9. Applications of DNA Shuffling

11. 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).

12. 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.

13. •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.

14. • ***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.

15. ***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
1. Antiproliferative test in Daudi cells
2. Anti-viral activity testing by WISH/VSV assay
3. Anti-tumor activity tested by flow cytometry

17. 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.

18. 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.

20. 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

21. 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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