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Nathan Blow, Ph.D.
BioTechniques, Vol. 53, No. 6, December 2012, p. 333
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Easier directing

Nucleotide libraries for applications such as phage display require methods capable of rapidly generating large amounts of nucleotide diversity. While directed evolution approaches such as random mutagenesis and DNA shuffling are often used to generate such nucleotide diversity, these techniques do have limitations including the need to use either restriction enzyme digestion or multiple PCR amplification steps. In the current issue of BioTechniques, Lim et al. from the Institute for Research in Molecular Medicine in Penang, Malaysia, and the Max Planck Institute for Molecular Genetics in Berlin, Germany, report a new, simplified approach for directed evolution of nucleotide libraries. Lim and colleagues took advantage of the enzymatic properties of lambda exonuclease and the Klenow fragment of DNA polymerase I to generate single-stranded DNA (ssDNA) templates suitable for directed mutagenesis and chain shuffling experiments. The author's methodology initially requires amplification of target DNA sequences using specially designed primers incorporating short complementary sequences capable of annealing. Next, lambda exonuclease is used to digest the amplified double-stranded DNA into single-strand fragments with the specially designed primers attached to each end. An annealing reaction is now performed such that the complementary primer regions on different ssDNA fragments are able to randomly anneal creating hybrid DNA fragments. The final step in the protocol is to use the Klenow fragment to synthesize the full double-stranded DNA of the newly generated hybrid DNA fragment. In order to demonstrate the utility of their new methodology for generating sequence diversity, the authors describe two experiments: chain shuffling of antibody fragments to generate a diverse antibody library and the directed mutagenesis of a variable region in the antibody heavy chain for a second antibody library. In both cases, the data from Lim et al. clearly show that the new lambda exonuclease/Klenow fragment approach is capable of generating antibody libraries with significant sequence diversity without the need for restriction digestion or multiple-PCR steps. This new approach to directed evolution of nucleotide libraries should greatly accelerate isolation of antibodies, as well as other DNA molecules, with novel functions.

See “Directed evolution of nucleotide based libraries using lambda exonuclease.

Losing the library card with PacBio

Next-generation sequencing (NGS) has enabled analysis of full-length genomes from hundreds of organisms, a list that actually continues to grow on a daily basis. NGS is also expanding the scope and depth of sequence analysis for both SNP and CNV detection as well as metagenome sequence analysis. Although rapid and increasingly cost effective, most current NGS protocols still require construction of a sequencing library, a step that requires significant amounts of starting material and multiple days to complete. In the December issue of BioTechniques, Coupland et al. from the Wellcome Trust Sanger Institute in Cambridge, UK, describe a new protocol for direct sequencing of small genomes on the Pacific Biosciences RS platform bypassing the need for any library preparation. Initially, the authors found that it was possible to directly sequence circular DNA molecules (in this case M13mp18 viral DNA) with 100% consensus accuracy using only 25 ng of single-stranded DNA template and a 100-fold molar excess of primer. This result established the potential of direct sequencing using the PacBio RS. To next determine if other genomic templates could also be used in direct sequencing, the authors tested a double-stranded viral genome, a bacterial plasmid, vectors containing epigenetic DNA-modifications, and linear double-stranded fragments covering an entire bacterial genome. Although the data found that fewer overall reads were obtained when using the direct sequencing approach, the percent of mappable reads was similar, in most cases, to data obtained following library construction and sufficient for full coverage of many smaller genomes. Importantly, the authors found that sequence information could be obtained from circular molecules starting with as little as 1 ng of DNA, significantly less than the 400-500 ng of sheared template suggested for library preparation. While characterization of small circular genomes was amenable to direct sequencing, amplicons or sheared linear fragments required circularization, a significant time limitation. In the end, the method of Coupland et al. does provide a new way to assess small circular genomes in a rapid and cost-effective manner as well a starting point for further optimization toward direct sequencing of the more problematic linear double-stranded DNA.

See “Direct sequencing of small genomes on the Pacific Biosciences RS without library preparation.