Genetic and epigenetic mechanisms of tumor predisposition in hereditary non-polyposis colorectal carcinoma and sporadic cancers

Individuals with inherited deficiency in DNA mismatch repair(MMR) (Lynch syndrome) LS are predisposed to different cancers in a non-random fashion. Endometrial cancer (EC) is the most common extracolonic malignancy in LS. LS represents the best characterized form of hereditary nonpolyposis colorectal carcinoma (HNPCC). Other forms of familial non-polyposis colon cancer exist, including familial colorectal cancer type X (FCCX). This syndrome resembles LS, but MMR gene defects are excluded and the predisposition genes are unknown so far. To address why different organs are differently susceptible to cancer development, we examined molecular similarities and differences in selected cancers whose frequency varies in LS individuals. Tumors that are common (colorectal, endometrial, gastric) and less common (brain, urological) in LS were characterized for MMR protein expression, microsatellite instability (MSI), and by altered DNA methylation. We also studied samples of histologically normal endometrium, endometrial hyperplasia,and cancer for molecular alterations to identify potential markers that could predict malignant transformation in LS and sporadic cases. Our results suggest that brain and kidney tumors follow a different pathway for cancer development than the most common LS related cancers.Our results suggest also that MMR defects are detectable in endometrial tissues from a proportion of LS mutation carriers prior to endometrial cancer development. Traditionally (complex) atypical hyperplasia has been considered critical for progression to malignancy. Our results suggest that complex hyperplasia without atypia is equally important as a precursor lesion of malignancy. Tumor profiles from Egypt were compared with colorectal tumors from Finland to evaluate if there are differences specific to the ethnic origin (East vs.West). Results showed for the first time a distinct genetic and epigenetic signature in the Egyptian CRC marked by high methylation of microsatellite stable tumors associated with advanced stage, and low frequency of Wnt signaling activation, suggesting a novel pathway. DNA samples from FCCX families were studied with genome wide linkage analysis using microsatellite markers. Selected genes from the linked areas were tested for possible mutations that could explain predisposition to a large number of colon adenomas and carcinomas seen in these families. Based on the results from the linkage analysis, a number of areas with tentative linkage were identified in family 20. We narrowed down these areas by additional microsatellite markers to found a mutation in the BMPR1A gene. Sequencing of an additional 17 FCCX families resulted in a BMPR1A mutation frequency of 2/18 families (11%). Clarification of the mechanisms of the differential tumor susceptibility in LS increases the understanding of gene and organ specific targets of MMR deficiency. While it is generally accepted that widespread MMR deficiency and consequent microsatellite instability (MSI) drives tumorigenesis in LS, the timing of molecular alterations is controversial. In particular, it is important to know that alterations may occur several years before cancer formation, at stages that are still histologically regarded as normal. Identification of molecular markers that could predict the risk of malignant transformation may be used to improve surveillance and cancer prevention in genetically predisposed individuals. Significant fractions of families with colorectal and/or endometrial cancer presently lack molecular definition altogether. Our findings expand the phenotypic spectrum of BMPR1A mutations and, for the first time, link FCCX families to the germline mutation of a specific gene. In particular, our observations encourage screening of additional families with FCCX for BMPR1A mutation, which is necessary in obtaining a reliable estimate of the share of BMPR1A-associated cases among all FCCX families worldwide. Clinically, the identification of predisposing mutations enables targeted cancer prevention in proven mutation carriers and thereby reduces cancer morbidity and mortality in the respective families.

The C-Terminal Domain of the MutL Homolog from Neisseria gonorrhoeae Forms an Inverted Homodimer

The mismatch repair (MMR) pathway serves to maintain the integrity of the genome by removing mispaired bases from the newly synthesized strand. In E. coli, MutS, MutL and MutH coordinate to discriminate the daughter strand through a mechanism involving lack of methylation on the new strand. This facilitates the creation of a nick by MutH in the daughter strand to initiate mismatch repair. Many bacteria and eukaryotes, including humans, do not possess a homolog of MutH. Although the exact strategy for strand discrimination in these organisms is yet to be ascertained, the required nicking endonuclease activity is resident in the C-terminal domain of MutL. This activity is dependent on the integrity of a conserved metal binding motif. Unlike their eukaryotic counterparts, MutL in bacteria like Neisseria exist in the form of a homodimer. Even though this homodimer would possess two active sites, it still acts a nicking endonuclease. Here, we present the crystal structure of the C-terminal domain (CTD) of the MutL homolog of Neisseria gonorrhoeae (NgoL) determined to a resolution of 2.4 A. The structure shows that the metal binding motif exists in a helical configuration and that four of the six conserved motifs in the MutL family, including the metal binding site, localize together to form a composite active site. NgoL-CTD exists in the form of an elongated inverted homodimer stabilized by a hydrophobic interface rich in leucines. The inverted arrangement places the two composite active sites in each subunit on opposite lateral sides of the homodimer. Such an arrangement raises the possibility that one of the active sites is occluded due to interaction of NgoL with other protein factors involved in MMR. The presentation of only one active site to substrate DNA will ensure that nicking of only one strand occurs to prevent inadvertent and deleterious double stranded cleavage.

Diversity in ranking via resistive graph centers

Users can rarely reveal their information need in full detail to a search engine within 1--2 words, so search engines need to "hedge their bets" and present diverse results within the precious 10 response slots. Diversity in ranking is of much recent interest. Most existing solutions estimate the marginal utility of an item given a set of items already in the response, and then use variants of greedy set cover. Others design graphs with the items as nodes and choose diverse items based on visit rates (PageRank). Here we introduce a radically new and natural formulation of diversity as finding centers in resistive graphs. Unlike in PageRank, we do not specify the edge resistances (equivalently, conductances) and ask for node visit rates. Instead, we look for a sparse set of center nodes so that the effective conductance from the center to the rest of the graph has maximum entropy. We give a cogent semantic justification for turning PageRank thus on its head. In marked deviation from prior work, our edge resistances are learnt from training data. Inference and learning are NP-hard, but we give practical solutions. In extensive experiments with subtopic retrieval, social network search, and document summarization, our approach convincingly surpasses recently-published diversity algorithms like subtopic cover, max-marginal relevance (MMR), Grasshopper, DivRank, and SVMdiv.

Investigation of Magnon Assisted Resistivity in Permalloy Thin Films

Magnon contribution to the resistance of ferromagnetic film like Permalloy is investigated by magnetotransport measurements. We are able to observe and distinguish Anisotropic-Magnetoresistance(AMR)(1) and Magnon Magnetoresistance(MMR)(2) contributions clearly in PLD grown Permalloy films. A linear non-saturating longitudinal MR observed in high field regime for permalloy films could never be explained using AMR but only MMR can account for it.

Design, synthesis, and biological activity of rhodium metalloinsertors

Deficiencies in the mismatch repair (MMR) pathway are associated with several types of cancers, as well as resistance to commonly used chemotherapeutics. Rhodium metalloinsertors have been found to bind DNA mismatches with high affinity and specificity in vitro, and also exhibit cell-selective cytotoxicity, targeting MMR-deficient cells over MMR-proficient cells.

Here we examine the biological fate of rhodium metalloinsertors bearing dipyridylamine ancillary ligands. These complexes are shown to exhibit accelerated cellular uptake which permits the observation of various cellular responses, including disruption of the cell cycle and induction of necrosis, which occur preferentially in the MMR-deficient cell line. These cellular responses provide insight into the mechanisms underlying the selective activity of this novel class of targeted anti-cancer agents.

In addition, ten distinct metalloinsertors with varying lipophilicities are synthesized and their mismatch binding affinities and biological activities studied. While they are found to have similar binding affinities, their cell-selective antiproliferative and cytotoxic activities vary significantly. Inductively coupled plasma mass spectrometry (ICP-MS) experiments show that all of these metalloinsertors localize in the nucleus at sufficient concentrations for binding to DNA mismatches. Furthermore, metalloinsertors with high rhodium localization in the mitochondria show toxicity that is not selective for MMR-deficient cells. This work supports the notion that specific targeting of the metalloinsertors to nuclear DNA gives rise to their cytotoxic and antiproliferative activities that are selective for cells deficient in MMR.

To explore further the basis of the unique selectivity of the metlloinsertors in targeting MMR-deficient cells, experiments were conducted using engineered NCI-H23 lung adenocarcinoma cells that contain a doxycycline-inducible shRNA which suppresses the expression of the MMR gene MLH1. Here we use this new cell line to further validate rhodium metalloinsertors as compounds capable of differentially inhibiting the proliferation of MMR-deficient cancer cells over isogenic MMR-proficient cells. General DNA damaging agents, such as cisplatin and etoposide, in contrast, are less effective in the induced cell line defective in MMR.

Finally, we describe a new subclass of metalloinsertors with enhanced potency and selectivity, in which the complexes show Rh-O coordination. In particular, it has been found that both Δ and Λ enantiomers of [Rh(chrysi)(phen)(DPE)]²⁺ bind to DNA with similar affinities, suggesting a possible different binding conformation than previous metalloinsertors. Remarkably, all members of this new family of compounds have significantly increased potency in a range of cellular assays; indeed, all are more potent than the FDA-approved anticancer drugs cisplatin and MNNG. Moreover, these activities are coupled with high levels of selectivity for MMR-deficient cells.

Biological Activity of Rhodium Metalloinsertors and the Design of Bifunctional Conjugates

The Barton laboratory has established that octahedral rhodium complexes bearing the sterically expansive 5,6-chrysene diimine ligand can target thermodynamically destabilized sites, such as base pair mismatches, in DNA with high affinity and selectivity. These complexes approach DNA from the minor groove, ejecting the mismatched base pairs from the duplex in a binding mode termed metalloinsertion. In recent years, we have shown that these metalloinsertor complexes also exhibit cytotoxicity preferentially in cancer cells that are deficient in the mismatch repair (MMR) machinery.

Here, we establish that a sensitive structure-activity relationship exists for rhodium metalloinsertors. We studied the relationship between the chemical structures of metalloinsertors and their effect on biological activity for ten complexes with similar DNA binding affinities, but wide variation in their lipophilicity. Drastic differences were observed in the selectivities of the complexes for MMR-deficient cells. Compounds with hydrophilic ligands were highly selective, exhibiting preferential cytotoxicity in MMR-deficient cells at low concentrations and short incubation periods, whereas complexes with lipophilic ligands displayed poor cell-selectivity. It was discovered that all of the complexes localized to the nucleus in concentrations sufficient for mismatch binding; however, highly lipophilic complexes also exhibited high mitochondrial uptake. Significantly, these results support the notion that mitochondrial DNA is not the desired target for our metalloinsertor complexes; instead, selectivity stems from targeting mismatches in genomic DNA.

We have also explored the potential for metalloinsertors to be developed into more complex structures with multiple functionalities that could either enhance their overall potency or impart mismatch selectivity onto other therapeutic cargo. We have constructed a family of bifunctional metalloinsertor conjugates incorporating cis-platinum, each unique in its chemical structure, DNA binding interactions, and biological activity. The study of these complexes in MMR-deficient cells has established that the cell-selective biological activity of rhodium metalloinsertors proceeds through a critical cellular pathway leading to necrosis.

We further explored the underlying mechanisms surrounding the biological response to mismatch recognition by metalloinsertors in the genome. Immunofluorescence assays of MMR-deficient and MMR-proficient cells revealed that a critical biomarker for DNA damage, phosphorylation of histone H2AX (γH2AX) rapidly accumulates in response to metalloinsertor treatment, signifying the induction of double strand breaks in the genome. Significantly, we have discovered that our metalloinsertor complexes selectively inhibit transcription in MMR-deficient cells, which may be a crucial checkpoint in the eventual breakdown of the cell via necrosis. Additionally, preliminary in vivo studies have revealed the capability of these compounds to traverse the complex environments of multicellular organisms and accumulate in MMR-deficient tumors. Our ever-increasing understanding of metalloinsertors, as well as the development of new generations of complexes both monofunctional and bifunctional, enables their continued progress into the clinic as promising new chemotherapeutic agents.

Geotecnologias e recursos de multimídia no ensino de cartografia: percepção sócio ambiental do rio Alcântara no município de São Gonçalo/RJ

A disponibilidade gratuita na Internet de imagens de satélite e SIG somada à facilidade dos alunos no manuseio de multimídia através dos seus smartphones criam possibilidades para trabalhar com geotecnologias e recursos de multimídia no ensino de Cartografia. Nesta pesquisa foram avaliadas as contribuições, os limites e as possibilidades da inserção da tecnologia espacial, geoprocessamento e recursos de multimídia nas aulas de Geografia do sétimo ano da rede pública municipal de São Gonçalo/RJ; foi desenvolvida uma metodologia em meio digital, por meio da Internet, denominada Mapeando Meu Rio (MMR) cuja temática abordada foi a Percepção Socioambiental do Rio Alcântara. Observaram-se o interesse e o envolvimento dos alunos no decorrer das atividades propostas, por meio do uso de recursos de multimídia e geotecnologias como materiais de apoio à Educação Ambiental. Os resultados da avaliação do MMR mostraram que os alunos chegaram ao final do sétimo ano com dificuldades em relação à alfabetização cartográfica; isso foi constatado tanto na produção dos mapas mentais como também pela utilização do GPS, Google Earth e do ArcGIS Online. Os alunos tiveram dificuldades em utilizar os conhecimentos básicos da Cartografia para elaborar uma representação espacial, mais especificamente, legenda, coordenadas geográficas e orientação espacial. A alfabetização cartográfica não deve ser considerada como conteúdo que se restringe ao 6 ano, mas uma linguagem de comunicação para o entendimento da dinâmica espacial no decorrer do Ensino Fundamental e do Ensino Médio. As atividades geográficas deve permitir ao aluno melhorar a compreensão do espaço geográfico de uma maneira mais significativa para construir abstrações a partir da própria realidade, ou seja, do espaço vivido.

Narrow stripe selective growth of oxide-free InGaAlAs/InGaAlAs MQWs by ultra-low pressure MOVPE

Narrow stripe selective growth of oxide-free InGaAlAs/InGaAlAs multiple quantum wells (MQWs) has been successfully performed on patterned InP substrates by ultra-low pressure MOVPE. Flat and clear interfaces were obtained for the narrow stripe selectively grown MQWs under optimized growth conditions. These selectively grown MQWs were covered by specific InP layers, which can keep the MQWs from being oxidized during the fabrication of the devices. The characteristics of selectively grown MQWs were strongly dependent on the mask stripe width. In particular, a PL peak wavelength shift of 73 nm, a PL intensity of more than 57% and a PL FWHM of less than 102 meV were observed simultaneously with a small mask stripe width varying from 0 to 40 mu m. The results were explained by considering the migration effect from the masked region (MMR) and the lateral vapour diffusion effect (LVD).

Investigations on oxide-free InGaAlAs waveguides grown by narrow stripe selective MOVPE

Oxide-free InGaAlAs waveguides have been grown on the InP substrates patterned with pairs of SiO2 mask stripes using narrow stripe selective MOVPE. The mask stripe width is varied from 0 to 40 pm, while the window region width between a pair of mask stripes is fixed at 1.5, 2.5 and 3.5 mu m, respectively. Smooth surface s and flat interfaces are obtained in the selectively grown InQaAlAs waveguides. There exhibit strong dependences of the thickness enhancement ratio and the photoluminescence (PL) spectrum on the mask stripe width and the window region width for the InGaAlAs wavegwdes. A large PL peak wavelength shift of 79 nm and a PL full width of at half maximum (FWHM) of less than 64 meV are obtained simultaneously. Some possible interpretations for our investigations are presented by considering both the migration effect from a masked region (MMR) and the lateral vapor diffusion effect (LVD).

Growth mechanism of InGaAlAs waveguides by narrow stripe selective MOVPE

Growth mechanism of InGaAlAs waveguides by narrow stripe selective MOVPE has been studied. Both the InGaAlAs bulk waveguides and the InGaAlAs MQW waveguides were successful grown on the patterned substrates at optimized growth conditions. The mask stripe width varied from 0 to 40 mu m, while the window region width between a pair of mask stripes was fixed 2.5 mu m. These selectively grown waveguides were covered by specific InP layers, which can keep the InGaAlAs waveguides from being oxidized during the fabrication of devices. In particular, there exhibit strong dependences of the photoluminescence (PL) spectrum on the mask stripe width for the samples. The results were explained in considering both the migration effect from a masked region (MMR) and the lateral vapor diffusion effect (LVD).

Narrow stripe selective growth of InGaAlAs waveguides used for buried heterostructure lasers - art. no. 63210I

The narrow stripe selective growth of the InGaAlAs bulk waveguides and InGaAlAs MQW waveguides was first investigated. Flat and clear interfaces were obtained for the selectively grown InGaAlAs waveguides under optimized growth conditions. These selectively grown InGaAlAs waveguides were covered by specific InP layers, which can keep the waveguides from being oxidized during the fabrication of devices. PL peak wavelength shifts of 70 nm for the InGaAlAs bulk waveguides and 73 nm for the InGaAlAs MQW waveguides were obtained with a small mask stripe width varying from 0 to 40 gm, and were interpreted in considering both the migration effect from the masked region (MMR) and the lateral vapor diffusion effect (LVD). The quality of the selectively grown InGaAlAs MQW waveguides was confirmed by the PL peak intensity and the PL FWHM. Using the narrow stripe selectively grown InGaAlAs MQW waveguides, then the buried heterostructure (BH) lasers were fabricated by a developed unselective regrowth method, instead of conventional selective regrowth. The InGaAlAs MQW BH lasers exhibit good performance characteristics, with a high internal differential quantum efficiency of about 85% and an internal loss of 6.7 cm(-1).

The effect of low temperature GaAs nucleation on the growth of GaN on Silicon (001) during MOVPE process

High quality cubic GaN was grown on Silicon (001) by metalorganic vapor phase epitaxy (MOVPE) using a GaAs nucleation layer grown at low temperature. The influence of various nucleation conditions on the GaN epilayers' quality was investigated. We found that the GaAs nucleation layer grown by atomic layer epitaxy (ALE) could improve the quality of GaN films by depressing the formation of mixed phase. Photoluminescence (PL) and X-ray diffraction were used to characterize the properties of GaN epilayers. High quality GaN epilayers with PL full width at half maximum (FWHM) of 130meV at room temperature and X-ray FWHM of 70 arc-min were obtained by using 10-20nm GaAs nucleation layer grown by ALE.

Cd、Cu污染胁迫对拟南芥幼苗错配修复相关基因表达的影响

土壤重金属污染问题已成为影响我国持续农业和生态环境质量的重要因素，引起了人们的广泛关注。由于传统污染诊断方法的缺点，急需建立土壤污染生态毒理学诊断方法，生物标记物技术则是其中的研究热点之一。本文采用营养液培养的方法，以模式植物拟南芥为试材，采用半定量反转录聚合酶链式反应（RT-PCR）技术，结合传统分析方法研究了Cd、Cu在不同胁迫水平下对拟南芥幼苗的形态、生理及分子水平的毒性效应，并在此基础上，比较和分析不同测试指标对Cd、Cu胁迫响应的敏感性，进而筛选对Cd、Cu胁迫响应敏感的生物标记物。主要结果如下： 1 不同浓度Cd和Cu污染胁迫下，拟南芥幼苗生长均受到不同程度的影响 幼苗初生根伸长均受到明显抑制，而地上部叶片数、地上部鲜重却没有显著的变化。重金属首先作用于植物的根系，根系的生长对胁迫响应的敏感性高于地上部。 2 幼苗地上部的可溶性蛋白含量受到不同程度干扰，而在不同浓度的Cd、 Cu处理下，叶绿素含量变化不明显，表明幼苗地上部可溶性蛋白质含量对胁迫的敏感性高于叶绿素含量的变化。 3 幼苗地上部错配修复（MMR）和增殖细胞核抗原（PCNA）基因都明显 地出现了表达诱导或表达抑制，表明MMR和PCNA基因表达的变化对Cd、Cu胁迫表现出较高的敏感性。 4 幼苗地上部的可溶性蛋白质含量及幼苗地上部MMR和PCNA基因表达 均对Cd和Cu污染胁迫具有较高的敏感性，两者均可用于指示Cd和Cu污染的敏感生物标记物。基因表达变化图谱虽然对污染胁迫响应比较敏感，是一种污染胁迫响应敏感的生物标记物，但其在生态毒理诊断中的应用还需进一步的实验对其予以证明。

基于错配修复系统的基因突变检测生物芯片研究

许多人类疾病和微生物抗药性的产生都是由基因组中单个碱基的替换、插入或缺失等基因突变引起的。因此，迫切需要发展快速、高通量基因突变检测方法来实现对基因疾病和细菌抗药性的早期诊断。本研究针对匕述需求发展了纂于DN八错配修复系统的墓因突变检测生物芯片方法。根据DNA错配修复MtltS蛋白结构与功能上的高度保守性，通过PCR从E.coli K-12基因组中扩一增出DNA错配修复基因，甩石（2.56kb）。通过基因水平的分子操纵，构建了Trx-His6-MutS（THM）、Trx-His6-Linker peptide-Muts（THLM）、Trx-His6-GFP-Linker peptide-MutS（THGLM）和Trx-His6-Linker peptide-Strep-tagll-Linker peptide-MutS（THLSLM）的融合基因并在大肠杆菌中进行了IPTG诱导表达。SDS-PAGE分析表明均有一与预期分子量相应的诱导表达条带出现，其表达量占菌体蛋白的30％左右，且以可溶形式存在。融合蛋白中Trx和His6亲和肤能增加表达蛋白的可溶性及便于蛋白的纯化。连接肤的加入增大了融合蛋白各个成分之间的距离，减少空间位阻，使各个蛋白能够较大程度地保持其原有的生物活性。MLltS融合蛋白的生物活性鉴定结果表明：它们既能识别、结合含有错配碱基的DNA双链，又保留了其它融合成分的生物活性。利用融合蛋白THLSLM中的Strep-tagII与Streptavidin相互作用的天然特性，使融合蛋白THLSLM在StrePtavidin修饰过的芯片基质上自动布阵沉积，制作成蛋白质芯片来识别、结合样品中含有错配或未配刘碱基的DNA双链。THGLM、THLM-Cy3和THLSLM能够使MutS蛋白显示不同的标记信号，通过它们识别并结合固定在DNA芯片基质上的基因片段来发展基因突变检测DNA芯片方法。利用基于MutS的蛋白质芯片和DNA芯片方法对含有不同错配类型、不同长度的DNA片段和错配序列背景对错配结合的影响做了深入研究，证明了MutS介导的基因突变检测生物芯片方法的可行性。基于MutS蛋白的鳌因突变检测生物芯片方法借用了生物系统本身的DNA错配修复（Mismatch Repair，MMR）机制。DNA错配修复过程是许多修复蛋白之间的相互作用共同完成的，其中蛋白MutS、MutL和MutH在肠道细菌例如大肠杆菌的甲基定向错配修复中起决定作用。这些修复蛋白的相关研究也引起了越来越多学者的关注，但对于MutL蛋白的体外生物功能一直存在争议，从而限制了该蛋白的应用研究。本研究利用基因的体外拼接技术构建了融合蛋白Trx-Hi56-Linker peptide-MutL（THLL）、Trx-His6-GFP-Linker peptide-MutL（THGLL）和Trx-His6-Linker peptide-Strep-tagII-Linker peptide-MutL（THLSLL）。非变性凝胶电泳鉴定MutL融合蛋白体外生物功能结果表明：THLL、THGLL和THLsLL都能增加融合蛋白Trx-His6-Linker peptide-MutS（THLM）与含有错配碱基DNA双链的结合，但受ATP浓度变化的影响很大。通过融合蛋白THGLL中绿色荧光蛋白（Green Fluorescent Protein，GFP）的荧光信号或THLSLL中Strep-tagII的特性并利用酶学反应来指示该蛋白的存在，发展了体外研究DNA错配修复蛋白MtuS和MutL之间相互作用的简便方法。本研究以构建的MutS融合蛋白为分子识别元件发展了基因突变检测生物芯片并利用构建的MutL融合蛋白发展了体外研究DNA错配修复蛋白MLuS和MutL之间相互作用的简便方法。建立的融合分子系统方法也为研究其它的蛋白质或生物大分子之间的相互作用提供了一个技术平台。此外，本研究构建的融合蛋白THGLL及其 DNA错配修复蛋白与GFP的融合构想还可用来进行DNA错配修复基因产物的表达与基因突变频率和人类肿瘤恶性程度的相关性研究。

DNA错配修复系统研究进展

DNA错配修复 (mismatchrepair ,MMR)系统广泛存在于生物体中 .从原核生物大肠杆菌到真核生物及人类 ,MMR系统有不同的组成成分和修复机制 .人体内MMR基因缺陷会造成基因组的不稳定并诱发遗传性非息肉型直肠癌以及其他自发性肿瘤 .大肠杆菌MMR系统中的MutS蛋白可特异识别错配或未配对碱基 ,目前已经发展了多种基于MutS蛋白的基因突变 /多态性检测技术 .