Mutated DNA Fragments Research Articles

Abstract B177: Non-invasive tumor mutation detection of cell-free urinary DNA using massively parallel deep DNA sequencing.

Abstract Background: Previous studies have demonstrated the ability to detect tumor DNA mutations by PCR methodologies within cell-free urinary DNA in metastatic cancer patients. This has opened up the possibility to use massively parallel deep sequencing approaches for more global profiling of tumor mutations using cell free urinary DNA from these patients. Methods: In order to achieve clinical utility the sequencing of cell-free urinary DNA must overcome two technical hurdles: 1) the need for extremely high sensitivity, between 0.01-0.05% and 2) an ultra-short DNA footprint (∼30 bp). Here we report the development of a method using cell free DNA extracted from urine that enriches for extremely low levels of mutated tumor DNA and thereby provides a high detection sensitivity. Detection of the KRAS gene mutations were used as a model system; the developed assay utilizes a 31bp footprint, contains a pre-amplification step that specifically enriches mutated DNA fragments and detects 8 KRAS mutations at the codon 12 and 13 sites. Spiking experiments with DNA derived from cell lines harboring the KRAS G12D mutation were completed to determine the limits of sensitivity of our method. Results: Mutant DNA input at amounts of 0.2%, 0.05%, 0.01% and 0.0% of the total DNA returned observed detection levels of 18.25%, 4.45%, 1.84% and 0.54% respectively as a percentage of the total sequence reads with the detection of mutant DNA of 0.01% within the theoretical limit of the assay. To test our assay, we sequenced 30 ng of extracted urinary DNA from a stage IV colorectal carcinoma patient with a known mutation at the G12D site. Enrichment of the mutation presented at a level of 13.06% of our total sequence, corresponding to an input amount of approximately 0.14% mutational load in the patient's urine. Ongoing analysis of a larger number of patients will be presented to further evaluate the clinical sensitivity and specificity of KRAS mutations. Conclusion: Further development of massively parallel DNA sequencing to detect mutations from cell-free urinary DNA has the potential to monitor metastatic patients for response, non-response and the emergence of resistance mechanisms to molecularly targeted therapies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B177. Citation Format: Jason C. Poole, Filip Janku, Veronica R. Holley, Jennifer J. Wheler, Funda Meric-Bernstam, Rayjalakshmi Luthra, Lorieta Leppin, Lafifa Hassaine, Karena Kosco, Mark G. Erlander. Non-invasive tumor mutation detection of cell-free urinary DNA using massively parallel deep DNA sequencing. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B177.

Detection of Mutant Free Circulating Tumor DNA in the Plasma of Patients with Gastrointestinal Stromal Tumor Harboring Activating Mutations of CKIT or PDGFRA

In gastrointestinal stromal tumor (GIST), there is no biomarker available that indicates success or failure of therapy. We hypothesized that tumor-specific v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (CKIT)- or platelet-derived growth factor receptor-α (PDGFRA)-mutant DNA fragments can be detected and quantified in plasma samples of patients with GIST. We prospectively collected 291 plasma samples from 38 subjects with GIST harboring activating mutations of CKIT or PDGFRA detected in tumor tissue, irrespective of current disease status or treatment. We used allele-specific ligation PCR to detect mutant free circulating DNA (fcDNA). We were able to detect fcDNA harboring the tumor mutation in 15 of 38 patients. Patients with active disease displayed significantly higher amounts of mutant fcDNA compared with patients in complete remission (CR). The amount of mutant fcDNA correlated with disease course. We observed repeated positive test results or an increase of mutant fcDNA in five patients with progressive disease or relapse. A decline of tumor fcDNA or conversion from positive to negative was seen in five patients responding to treatment. A negative to positive conversion was seen in two patients with relapse and one patient with progression. In two cases, we aimed to identify additional mutations and found four additional exchanges, including mutations not known from sequentially conducted tumor biopsies. Our results indicate that fcDNA harboring tumor-specific mutations in the plasma of patients with GIST can be used as tumor-specific biomarker. The detection of resistance mutations in plasma samples might allow earlier treatment changes and obviates the need for repeated tumor biopsies.

Implementing heat transfer resistivity as a key element in a nanocrystalline diamond based single nucleotide polymorphism detection array

In this article, we report on the label-free real-time thermal monitoring of the denaturation of specific DNA fragments and its potential to detect and quantify single nucleotide polymorphisms (SNPs). Probe DNA, consisting of a 36-mer fragment was covalently immobilized on nanocrystalline chemical vapour deposition (CVD) diamond platforms and hybridized with a 29-mer target DNA fragment (full matching and/or with a point mutation). It was observed that the change in heat transfer resistance upon denaturation is dependent on the amount of DNA hybridized to the nanocrystalline diamond (NCD) surface. Furthermore the possibility to distinguish between a full matching sequence and its singularly mutated counterpart, when bound to the same NCD surface, was investigated. NCD surfaces were selectively hybridized with both full matching and mutated DNA fragments at different ratios (3:1, 2:2 and 1:3). A clear bipartite response in heat transfer resistivity was observed upon simultaneous denaturation of these DNA fragments. Denaturation temperature could be used to identify the DNA fragment to which each partial response could be attributed. Moreover, the partial increases in heat transfer resistivity related to the hybridized amount of non-mutated or mutated DNA, respectively. These results imply that heat transfer resistivity is a technique which can be used to (i) quantify DNA fragments of interest, (ii) detect and (iii) quantify SNPs in a mixture of mutated and non-mutated DNA fragments. Moreover, it illustrates the potential of this technique to detect SNPs without the necessity to design complex microarrays.

Detection of mutant-free circulating tumor DNA in the plasma of patients with gastrointestinal stromal tumor (GIST) harboring activating mutations of C-Kit or PDGFRA.

10508 Background: In gastrointestinal stromal tumor (GIST), there is no biomarker available that indicates success or failure of therapy. We hypothesized that tumor specific CKIT or PDGFRA mutant DNA fragments can be detected and quantified in plasma samples of GIST patients. Methods: We prospectively collected 291 plasma samples from 38 subjects with GIST harbouring activating mutations of CKIT or PDGFRA detected in tumor tissue, irrespective of current disease status or treatment. We used allele-specific Ligation PCR to detect mutant free circulating (fc)DNA. Results: We were able to detect fcDNA harbouring the tumor mutation in 15 out of 38 patients. Patients with active disease displayed significantly higher amounts of mutant fcDNA compared to patients in CR. The amount of mutant fcDNA correlated with disease course. We observed repeated positive test results or an increase of mutant fcDNA in five patients with progressive disease or relapse. A decline of tumor fcDNA or conversion from positive to negative was seen in five patients responding to treatment. A negative to positive conversion was seen in two patients with relapse and one patient with progression. In two cases, we aimed to identify additional mutations, and found four additional exchanges, including mutations not known from sequentially performed tumor biopsies. Conclusions: Our results indicate that free circulating DNA harbouring tumor specific mutations in the plasma of patients with GIST can be used as tumor-specific biomarker. The detection of resistance mutations in plasma samples might allow earlier treatment changes and obviates the need for repeated tumor biopsies. Clinical trial information: NCT01462994.

AXM mutagenesis: An efficient means for the production of libraries for directed evolution of proteins

Affinity maturation is an important part of the recombinant antibody development process. There are several well-established approaches for generating libraries of mutated antibody genes for affinity maturation, but these approaches are generally too laborious or expensive to allow high-throughput, parallel processing of multiple antibodies. Here, we describe a scalable approach that enables the generation of libraries with greater than 108 clones from a single Escherichia coli transformation. In our method, a mutated DNA fragment is produced using PCR conditions that promote nucleotide misincorporation into newly synthesized DNA. In the PCR reaction, one of the primers contains at least three phosphorothioate linkages at its 5′ end, and treatment of the PCR product with a 5′ to 3′ exonuclease is used to preferentially remove the strand synthesized with the non-modified primer, resulting in a single-stranded DNA fragment. This fragment then serves as a megaprimer to prime DNA synthesis on a uracilated, circular, single-stranded template in a Kunkel-like mutagenesis reaction that biases nucleotide base-changes between the megaprimer and uracilated DNA sequence in favor of the in vitro synthesized megaprimer. This method eliminates the inefficient subcloning steps that are normally required for the construction of affinity maturation libraries from randomly mutagenized antibody genes.

Gene Splicing by Overlap Extension: Tailor-Made Genes Using the Polymerase Chain Reaction

Gene Splicing by Overlap Extension or “gene SOEing” is a PCR-based method of recombining DNA sequences without reliance on restriction sites and of directly generating mutated DNA fragments in vitro. By modifying the sequences incorporated into the 5′-ends of the primers, any pair of polymerase chain reaction products can be made to share a common sequence at one end. Under polymerase chain reaction conditions, the common sequence allows strands from two different fragments to hybridize to one another, forming an overlap. Extension of this overlap by DNA polymerase yields a recombinant molecule. This powerful and technically simple approach offers many advantages over conventional approaches for manipulating gene sequences.

Operation Invisible Dead: A Blind Study to Compare Molecular Fingerprints of Soil Samples between Crime Scene and Criminal's Shoe

Capillary electrophoresis can separate fragments of nucleic acids using single strand conformation polymorphism (SSCP) This method has been increasingly used in microbial ecology to develop fingerprints of the microbial community within soil. SSCP detects mutations in DNA fragments due to changes in the secondary structure of single‐stranded DNA fragments. Molecular fingerprinting can have a wide application in Forensics. It is important to find if a soil fingerprint from a criminal's shoe can be matched to the fingerprint found in the database. This is a blind study comparing the possible use of CESSCP in Forensics. The soil samples are collected from two locations by two people. Each individual acted as a “criminal” by digging the soil, walking over the soil and buried a small object. One shoe was removed at the “crime scene” and the other shoe was worn home. The soil was collected from the shovel and both of the shoes for each individual. All the soil samples were air dried for 48 hours before DNA was extracted. The samples were then run through polymerase chain reaction (PCR) to amplify and fluorescently tag the genes of interest. The samples will be processed in the Genetic Analyzer using the CE‐SSCP. The results can very well show that CE‐SSCP will not produce similar fingerprints for the shoe worn home and the database, thus inapplicable to Forensics.Supported by National Institute of Justice (25‐6228‐0159‐001).

The role of heat shock factor 1 in transcriptional regulation of high mobility group box

Background: Previous study suggests that high mobility group box 1 (HMGB1) can be a potential late inflammatory mediator. However, whether heat shock factor 1 (HSF1) regulate HMGB1 expression via binding to heat shock element (HSE) is not known. Objective: We investigated the role of HSF1 in the transcriptional regulation of HMGB1 protein. Methods: A probe that included HMGB1 promoter region containing HSE was synthesized for electrophoretic mobility shift assay (EMSA) to determine the binding of HSF1 and HSE in the promoter region of HMGB1 gene. Mutant mouse HMGB1 promoter was prepared by PCR amplification on a template of wild-type plasmid DNA with site-directed mutant primers. The mutant DNA fragments were also inserted into a corresponding plasmid. In addition, luciferase reporter plasmids of HMGB1 promoter were constructed to transfect RAW264.7 cells. After that, luciferase activity was measured to assay the effects of the HSF1 transfection on the promoter activity. Results: EMSA result showed a retardation strap after the coculture of biotin labeled HSF1 binding fragment and nuclear protein extracts. The retardation phenomenon could be competed by unlabeled probe and not by unlabeled mutant probe. A super retardation strap was present after adding HSF1 monoclonal antibody. After the HSE core sites was mutated, the relative luciferase activity of the mutant plasmid decreased by 4.26 folds compared with that in the wild-type (23.54±1.68 vs.100.25±3.26, p Conclusion: HSF1 can bind to the promoter region HSE (-668bp~-651bp) of HMGB1, which down-regulates the expression of HMGB1. Keywords: Heat shock transcription factor 1, high mobility group box 1, mouse, RAW264.7, transcription

The role of heat shock factor 1 in transcriptional regulation of high mobility group box

Background: Previous study suggests that high mobility group box 1 (HMGB1) can be a potential late inflammatory mediator. However, whether heat shock factor 1 (HSF1) regulate HMGB1 expression via binding to heat shock element (HSE) is not known. Objective: We investigated the role of HSF1 in the transcriptional regulation of HMGB1 protein. Methods: A probe that included HMGB1 promoter region containing HSE was synthesized for electrophoretic mobility shift assay (EMSA) to determine the binding of HSF1 and HSE in the promoter region of HMGB1 gene. Mutant mouse HMGB1 promoter was prepared by PCR amplification on a template of wild-type plasmid DNA with site-directed mutant primers. The mutant DNA fragments were also inserted into a corresponding plasmid. In addition, luciferase reporter plasmids of HMGB1 promoter were constructed to transfect RAW264.7 cells. After that, luciferase activity was measured to assay the effects of the HSF1 transfection on the promoter activity. Results: EMSA result showed a retardation strap after the coculture of biotin labeled HSF1 binding fragment and nuclear protein extracts. The retardation phenomenon could be competed by unlabeled probe and not by unlabeled mutant probe. A super retardation strap was present after adding HSF1 monoclonal antibody. After the HSE core sites was mutated, the relative luciferase activity of the mutant plasmid decreased by 4.26 folds compared with that in the wild-type (23.54±1.68 vs.100.25±3.26, p Conclusion: HSF1 can bind to the promoter region HSE (-668bp~-651bp) of HMGB1, which down-regulates the expression of HMGB1. Keywords: Heat shock transcription factor 1, high mobility group box 1, mouse, RAW264.7, transcription

Microbial Diversity in Soil Assessed by Two Different Capillary Electrophoresis Methods

Capillary electrophoresis can separate fragments of nucleic acids using single strand conformation polymorphism (SSCP) and terminal restriction fragment length polymorphism (T‐RFLP). These methods have been increasingly used in microbial ecology to develop fingerprints of the microbial community within soil. T‐RFLP is based on amplifying a region of DNA common to multiple species using conserved PCR primers whereas SSCP detects mutations in DNA fragments due to changes in the secondary structure of single‐stranded DNA fragments. The objective of this study is to use soil samples to compare molecular profiles generated by CE‐SSCP and T‐RFLP in detail. The soil samples will be collected from four locations representing four soil types. One set of soil subsamples will be analyzed immediately following collection and six subsamples will be placed in different storage treatments: cooling at −4C, freezing at −20C, freezing at −80C, freeze drying, air drying, and oven drying. DNA will be extracted from the samples, run through polymerase chain reaction (PCR) to amplify and fluorescently tag the genes of interest. T‐RFLP uses restriction enzyme MspI after PCR, while SSCP does not. Then, the microbial community fingerprints are analyzed. We hypothesize CE‐SSCP and T‐RFLP will generate similar fingerprints of the microbial community within soil. Supported by National Institute of Justice (25‐6228‐0159‐001).

Functional Analysis of Two Cavities in Flavivirus NS5 Polymerase

Flavivirus NS5 protein encodes methyltransferase and RNA-dependent RNA polymerase (RdRp) activities. Structural analysis of flavivirus RdRp domains uncovered two conserved cavities (A and B). Both cavities are located in the thumb subdomains and represent potential targets for development of allosteric inhibitors. In this study, we used dengue virus as a model to analyze the function of the two RdRp cavities. Amino acids from both cavities were subjected to mutagenesis analysis in the context of genome-length RNA and recombinant NS5 protein; residues critical for viral replication were subjected to revertant analysis. For cavity A, we found that only one (Lys-756) of the seven selected amino acids is critical for viral replication. Alanine substitution of Lys-756 did not affect the RdRp activity, suggesting that this residue functions through a nonenzymatic mechanism. For cavity B, all four selected amino acids (Leu-328, Lys-330, Trp-859, and Ile-863) are critical for viral replication. Biochemical and revertant analyses showed that three of the four mutated residues (Leu-328, Trp-859, and Ile-863) function at the step of initiation of RNA synthesis, whereas the fourth residue (Lys-330) functions by interacting with the viral NS3 helicase domain. Collectively, our results have provided direct evidence for the hypothesis that cavity B, but not cavity A, from dengue virus NS5 polymerase could be a target for rational drug design.

Mode of DNA binding by SopA protein of Escherichia coli F plasmid

The binding of SopA to the promoter region of its own gene, in which four copies of SopA's recognition sequence, 5'-CTTTGC-3', are arrayed asymmetrically, was examined in vitro. Titration using electrophoretic mobility shift assay showed that the stoichiometry of SopA protomers to the promoter-region DNA is 4 and that the binding is highly co-operative. The co-operativity was corroborated by EMSA and DNase I footprinting for a number of mutant DNA fragments in which 5'-CTTTGC-3' was changed to 5'-CTTACG-3'. EMSA in the style of circular permutation showed that SopA bends DNA. Mutation at either outermost binding site had a different effect on DNA bending by SopA, reflecting the asymmetry in the arrangement of the binding sites, for which the results of DNase I footprinting were in agreement. Gel filtration chromatography and analytical ultracentrifugation of free SopA showed that the protein can exist as a monomer and oligomers in the absence of ATP. Hence, the results indicate that the co-operativity in SopA's DNA binding is based on its intrinsic protein-protein interaction modified by DNA interaction.

Proteomics Reveals a Physical and Functional Link between Hepatocyte Nuclear Factor 4α and Transcription Factor IID

Proteomic analyses have contributed substantially to our understanding of diverse cellular processes. Improvements in the sensitivity of mass spectrometry approaches are enabling more in-depth analyses of protein-protein networks and, in some cases, are providing surprising new insights into well established, longstanding problems. Here, we describe such a proteomic analysis that exploits MudPIT mass spectrometry and has led to the discovery of a physical and functional link between the orphan nuclear receptor hepatocyte nuclear factor 4alpha (HNF4alpha) and transcription factor IID (TFIID). A systematic characterization of the HNF4alpha-TFIID link revealed that the HNF4alpha DNA-binding domain binds directly to the TATA box-binding protein (TBP) and, through this interaction, can target TBP or TFIID to promoters containing HNF4alpha-binding sites in vitro. Supporting the functional significance of this interaction, an HNF4alpha mutation that blocks binding of TBP to HNF4alpha interferes with HNF4alpha transactivation activity in cells. These findings identify an unexpected role for the HNF4alpha DNA-binding domain in mediating key regulatory interactions and provide new insights into the roles of HNF4alpha and TFIID in RNA polymerase II transcription.

Characterization of the Molecular Basis of Group II Intron RNA Recognition by CRS1-CRM Domains

CRM (chloroplast RNA splicing and ribosome maturation) is a recently recognized RNA-binding domain of ancient origin that has been retained in eukaryotic genomes only within the plant lineage. Whereas in bacteria CRM domains exist as single domain proteins involved in ribosome maturation, in plants they are found in a family of proteins that contain between one and four repeats. Several members of this family with multiple CRM domains have been shown to be required for the splicing of specific plastidic group II introns. Detailed biochemical analysis of one of these factors in maize, CRS1, demonstrated its high affinity and specific binding to the single group II intron whose splicing it facilitates, the plastid-encoded atpF intron RNA. Through its association with two intronic regions, CRS1 guides the folding of atpF intron RNA into its predicted "catalytically active" form. To understand how multiple CRM domains cooperate to achieve high affinity sequence-specific binding to RNA, we analyzed the RNA binding affinity and specificity associated with each individual CRM domain in CRS1; whereas CRM3 bound tightly to the RNA, CRM1 associated specifically with a unique region found within atpF intron domain I. CRM2, which demonstrated only low binding affinity, also seems to form specific interactions with regions localized to domains I, III, and IV. We further show that CRM domains share structural similarities and RNA binding characteristics with the well known RNA recognition motif domain.

This Month in Gastroenterology

This Month in Gastroenterology

Analysis of Mutations in DNA Isolated From Plasma and Stool of Colorectal Cancer Patients

Somatic mutations provide uniquely specific markers for the early detection of neoplasia that can be detected in DNA purified from plasma or stool of patients with colorectal cancer. The primary purpose of the present investigation was to determine the parameters that were critical for detecting mutations using a quantitative assay. A secondary purpose was to compare the results of plasma and stool DNA testing using the same technology. We examined DNA purified from the stool of 25 patients with colorectal cancers before surgery. In 16 of these cases, plasma samples also were available. Mutations in stool or plasma were assessed with an improved version of the BEAMing technology. Of the 25 stool DNA samples analyzed, 23 (92%) contained mutations that were present in the corresponding tumors from the same patients. In contrast, only 8 of the 16 (50%) plasma DNA samples analyzed had detectable levels of mutated DNA. We found that the DNA fragments containing mutations in both stool and plasma DNA typically were smaller than 150 bases in size. The sensitivity of the new method was superior to a widely used technique for detecting mutations, using single base extension and sequencing, when assessed on the same samples (92% vs 60%; P = .008, exact McNemar test). When assessed with sufficiently sensitive methods, mutant DNA fragments are detectable in the stool of more than 90% of colorectal cancer patients. DNA purified from stool provides a better template for mutation testing than plasma.

In vitro recombination and identification of mutated fragment corresponding to regulation region of mtrR gene of Neisseria Gonorrhoeae

A site-directed mutant DNA fragment was synthesized and transfected into clinical Neisseria gonorrhoeae (NG) stains to construct the transformants that contained the corresponding mutagenesis of regulation region of mtrR gene. According to the technique of gene splicing by overlap extension (SOEing), a DNA segment with specific mutagenesis was constructed by two-step polymerase chain reaction (PCR). The mutation fragments EF could be used for the next experiment in which the mutation NG strains were induced. By comparing the recombinant EF fragments to the corresponding DNA fragments of clinical NG strains, 2 of these were not compatible completely. The results of sequencing revealed that there was a 9 bp deletion between the 45 to 54 inverted repeat sequence localized within the mtrR promoter. It can be confirmed that the fragments EF are the specifically designed mutant fragments.

Importin-β Mediates Cdc7 Nuclear Import by Binding to the Kinase Insert II Domain, Which Can Be Antagonized by Importin-α

We investigated the nuclear import mechanism of Cdc7, which is essential for the initiation of DNA replication. Here we report that importin-beta binds directly to Cdc7 via the Kinase Insert II domain, promoting its nuclear import. Although both importin-alpha and -beta bind to Cdc7 via the Kinase Insert II domain in a mutually independent manner, the binding affinity of Cdc7 for importin-beta is approximately 10 times higher than for importin-alpha at low protein concentrations of an equimolar ratio. Immunodepletion of importin-beta, but not importin-alpha, abrogates Cdc7 nuclear import, and the addition of importin-beta to the importin-depleted cytosol restores Cdc7 nuclear import. Furthermore, transduction of anti-importin-beta, but not anti-importin-alpha antibodies, into live cells inhibits Cdc7 nuclear import. Unexpectedly, we found that Cdc7 nuclear import is inhibited by competitive binding of importin-alpha to Cdc7. Further studies by site-directed mutagenesis suggest that Lys306 and Lys309 within the Kinase Insert II domain are critical for Cdc7 nuclear localization.

Secretory Phospholipases A2 Induce Neurite Outgrowth in PC12 Cells through Lysophosphatidylcholine Generation and Activation of G2A Receptor

We previously demonstrated that secretory phospholipase A2 (sPLA2) and lysophosphatidylcholine (LPC) exhibit neurotrophin-like neuritogenic activity in the rat pheochromocytoma cell line PC12. In this study, we further analyzed the mechanism whereby sPLA2 displays neurite-inducing activity. Exogenously added mammalian group X sPLA2 (sPLA2-X), but not group IB and IIA sPLA2s, induced neuritogenesis, which correlated with the ability of sPLA2-X to liberate LPC into the culture media. In accordance, blocking the effect of LPC by supplementation of bovine serum albumin or phospholipase B attenuated neuritogenesis by sPLA2 or LPC. Overproduction or suppression of G2A, a G-protein-coupled receptor involved in LPC signaling, resulted in the enhancement or reduction of neuritogenesis induced by sPLA2 treatment. These results indicate that the neuritogenic effect of sPLA2 is mediated by generation of LPC and subsequent activation of G2A.

Biased mutation-assembling: an efficient method for rapid directed evolution through simultaneous mutation accumulation

We have developed an efficient optimization technique, 'biased mutation-assembling', for improving protein properties such as thermostability. In this strategy, a mutant library is constructed using the overlap extension polymerase chain reaction technique with DNA fragments from wild-type and phenotypically advantageous mutant genes, in which the number of mutations assembled in the wild-type gene is stochastically controlled by the mixing ratio of the mutant DNA fragments to wild-type fragments. A high mixing ratio results in a mutant composition biased to favor multiple-point mutants. We applied this strategy to improve the thermostability of prolyl endopeptidase from Flavobacterium meningosepticum as a case study and found that the proportion of thermostable mutants in a library increased as the mixing ratio was increased. If the proportion of thermostable mutants increases, the screening effort needed to find them should be reduced. Indeed, we isolated a mutant with a 1200-fold longer activity half-life at 60 degrees C than that of wild-type prolyl endopeptidase after screening only 2000 mutants from a library prepared with a high mixing ratio. Our results indicate that an aggressive accumulation of advantageous mutations leads to an increase in the quality of the mutant library and a reduction in the screening effort required to find superior mutants.