{
 "meta": {
  "title": "酶学知识平台",
  "version": "20261031_V01",
  "date": "2026-10-31",
  "scope": "Taq / Tth / MMLV / Bst / Pfu 的酶突变筛选方法、路径、实验方案与文献约束模拟",
  "rules": [
   "模拟=文献锚点+显式模型/假设；不是湿实验数据，也不是对宝锐产品的性能承诺。",
   "没有定量锚点的方向只做定性判读表，不编数值。",
   "商业酶性能不能反推突变位点。",
   "所有路线都要先建立亲本基线，再做库/筛选。"
  ],
  "counts": {
   "methods": 11,
   "routes": 12,
   "targets": 8,
   "simulations": 13,
   "citations": 46
  }
 },
 "methods": [
  {
   "id": "epcr",
   "name": "易错 PCR 随机突变",
   "cat": "随机突变",
   "principle": "降低 Taq 保真度或调整 dNTP/Mg²⁺/Mn²⁺，在全基因或片段内引入随机点突变；突变服从 Poisson 分布。",
   "best": "无结构信息、先探序列空间；适合单酶快速建库。",
   "limit": "突变负荷高，需控制每基因 1–3 个氨基酸替换；有益突变比例低。",
   "equipment": [
    "thermocycler",
    "plate_reader",
    "shaker",
    "sequencer"
   ],
   "refs": [
    "cadwell1992",
    "packer2015"
   ],
   "throughput": "中"
  },
  {
   "id": "shuffle",
   "name": "DNA shuffling / 同源重组",
   "cat": "重组",
   "principle": "DNase I 或超声片段化亲本/突变体库，无引物 PCR 重装配；通过同源重组组合有益突变。",
   "best": "已有多个有益单点，想把它们组合；家族内高同源。",
   "limit": "需要 ≥70% 同源；片段化条件与重装配效率决定库质量。",
   "equipment": [
    "thermocycler",
    "electrophoresis",
    "plate_reader",
    "sequencer"
   ],
   "refs": [
    "stemmer1994"
   ],
   "throughput": "中"
  },
  {
   "id": "ism",
   "name": "迭代饱和突变 ISM",
   "cat": "半理性",
   "principle": "基于结构/B-factor/热区选择 1–3 个位点，做 NNK 或简并密码子饱和突变；逐轮叠加有益替换。",
   "best": "有结构或同源模型；目标位点明确（如 708、742/743）。",
   "limit": "位点选错会空转；上位效应要求逐轮验证。",
   "equipment": [
    "thermocycler",
    "plate_reader",
    "shaker",
    "sequencer"
   ],
   "refs": [
    "reetz2007"
   ],
   "throughput": "中"
  },
  {
   "id": "cast",
   "name": "CASTing 活性中心饱和突变",
   "cat": "半理性",
   "principle": "围绕活性中心/底物通道选多个位点，组合饱和突变，筛选协同效应。",
   "best": "底物特异性、活性、抑制物结合口袋。",
   "limit": "组合数增长快，需要分层筛选。",
   "equipment": [
    "thermocycler",
    "liquid_handler",
    "plate_reader",
    "sequencer"
   ],
   "refs": [
    "reetz2006"
   ],
   "throughput": "中高"
  },
  {
   "id": "csr",
   "name": "区室化自复制 CSR",
   "cat": "选择",
   "principle": "把聚合酶基因与其自身复制反应封在区室中；只有能复制自身基因的聚合酶变体被扩增。",
   "best": "DNA 聚合酶/逆转录酶的耐热、耐抑制、底物选择性。",
   "limit": "只适用于能自复制的聚合酶；区室化与洗脱条件需优化。",
   "equipment": [
    "thermocycler",
    "emulsion",
    "plate_reader",
    "sequencer"
   ],
   "refs": [
    "ghadessy2001"
   ],
   "throughput": "高"
  },
  {
   "id": "droplet",
   "name": "液滴微流控 / FADS",
   "cat": "超高通量",
   "principle": "皮升级液滴作为独立反应器，荧光信号驱动分选，每秒可筛数千个。",
   "best": "大库容（10^6–10^8）、分泌型或偶联酶活。",
   "limit": "设备门槛高；液滴稳定性、底物保留、假阳性需控制。",
   "equipment": [
    "microfluidics",
    "fads",
    "plate_reader"
   ],
   "refs": [
    "agresti2010",
    "hts2015"
   ],
   "throughput": "超高"
  },
  {
   "id": "display",
   "name": "核糖体/噬菌体展示",
   "cat": "体外选择",
   "principle": "基因型（mRNA/噬菌体）与表型（折叠蛋白）物理偶联，通过亲和/活性捕获富集。",
   "best": "结合蛋白、底物捕获；不需要转化。",
   "limit": "需体外翻译/噬菌体体系；酶活筛选需设计捕获底物。",
   "equipment": [
    "in_vitro_translation",
    "affinity_selection",
    "sequencer"
   ],
   "refs": [
    "hanes1997"
   ],
   "throughput": "高"
  },
  {
   "id": "pace",
   "name": "PACE 连续进化",
   "cat": "连续进化",
   "principle": "把目标活性与噬菌体复制偶联，在恒化器中连续传代；一天可完成数十轮。",
   "best": "可偶联到噬菌体复制的活性；长期连续优化。",
   "limit": "体系搭建复杂；仅适用于可基因编码且可偶联的活性。",
   "equipment": [
    "chemostat",
    "phage",
    "plate_reader",
    "sequencer"
   ],
   "refs": [
    "esvelt2011"
   ],
   "throughput": "高"
  },
  {
   "id": "mlde",
   "name": "机器学习引导定向进化",
   "cat": "计算/数据驱动",
   "principle": "用已测变体训练序列-功能模型，预测并选择下一轮变体；主动学习减少实验轮次。",
   "best": "已有 10^3–10^5 个标注变体；性状可定量。",
   "limit": "需要高质量数据与分布外验证；模型预测不等于因果。",
   "equipment": [
    "plate_reader",
    "sequencer",
    "gpu"
   ],
   "refs": [
    "yang2019",
    "fowler2014"
   ],
   "throughput": "中高"
  },
  {
   "id": "proteinmpnn",
   "name": "ProteinMPNN / 结构序列设计",
   "cat": "计算/数据驱动",
   "principle": "在给定骨架上用深度学习预测兼容序列；可固定催化/结合位点，重设计表面。",
   "best": "有实验/高置信结构；想改稳定性/表达/表面。",
   "limit": "预测需实验验证；不直接保证活性/抑制物耐受。",
   "equipment": [
    "gpu",
    "structural_model",
    "plate_reader"
   ],
   "refs": [
    "dauparas2022"
   ],
   "throughput": "中"
  },
  {
   "id": "dms",
   "name": "深度突变扫描 DMS",
   "cat": "数据驱动",
   "principle": "对目标区域做饱和/简并库，基因型-表型偶联后 NGS 计数，得到每位点替换的适应度图。",
   "best": "已知结构域/位点；想一次性获得突变效应地图。",
   "limit": "需要 NGS 与生物信息；筛选条件必须保持基因型-表型偶联。",
   "equipment": [
    "sequencer",
    "plate_reader",
    "gpu"
   ],
   "refs": [
    "fowler2014"
   ],
   "throughput": "高"
  }
 ],
 "equipment": {
  "thermocycler": {
   "name": "PCR 仪 / 梯度 PCR",
   "why": "建库、克隆验证、测序模板",
   "level": "基础"
  },
  "plate_reader": {
   "name": "酶标仪（荧光/吸收/化学发光）",
   "why": "初筛与复筛的活性/抑制物读板",
   "level": "基础"
  },
  "shaker": {
   "name": "恒温摇床 / 深孔板培养",
   "why": "96/384 孔表达",
   "level": "基础"
  },
  "centrifuge": {
   "name": "离心机 / 板式离心",
   "why": "裂解液澄清",
   "level": "基础"
  },
  "electrophoresis": {
   "name": "琼脂糖/聚丙烯酰胺电泳",
   "why": "文库片段与纯度",
   "level": "基础"
  },
  "sequencer": {
   "name": "Sanger / NGS 测序",
   "why": "克隆测序、库质量、富集分析",
   "level": "基础+"
  },
  "liquid_handler": {
   "name": "自动移液工作站",
   "why": "384 孔建库/筛选",
   "level": "高通量"
  },
  "colony_picker": {
   "name": "克隆挑选仪",
   "why": "从琼脂板挑克隆到多孔板",
   "level": "高通量"
  },
  "microfluidics": {
   "name": "液滴微流控系统（芯片+泵+光学）",
   "why": "超高通量分选",
   "level": "高"
  },
  "fads": {
   "name": "FADS 荧光激活液滴分选",
   "why": "按荧光/活性分选液滴",
   "level": "高"
  },
  "in_vitro_translation": {
   "name": "体外翻译体系（PURE/核糖体展示）",
   "why": "无需转化的蛋白表达",
   "level": "中高"
  },
  "affinity_selection": {
   "name": "亲和捕获/磁珠/层析",
   "why": "展示库富集",
   "level": "中"
  },
  "chemostat": {
   "name": "恒化器/连续培养",
   "why": "PACE 连续传代",
   "level": "高"
  },
  "phage": {
   "name": "噬菌体辅助连续进化体系",
   "why": "PACE",
   "level": "高"
  },
  "gpu": {
   "name": "GPU 工作站 / 服务器",
   "why": "ML/结构模型训练与推理",
   "level": "中高"
  },
  "structural_model": {
   "name": "结构建模软件（Rosetta/PyMOL/AlphaFold）",
   "why": "位点选择与设计",
   "level": "中"
  },
  "emulsion": {
   "name": "乳液/区室化装置",
   "why": "CSR 或液滴反应",
   "level": "中高"
  },
  "analytical": {
   "name": "HPLC/LC-MS/停流/CD/DSF",
   "why": "动力学、纯度、Tm 正交验证",
   "level": "高"
  }
 },
 "targets": [
  {
   "id": "thermo",
   "name": "耐热 / 热稳定",
   "enzymes": [
    "Taq",
    "MMLV",
    "Bst"
   ],
   "question": "提高 Tm/T50、55℃ 半衰期或高温反应能力",
   "metrics": [
    "Tm / T50",
    "55℃ 半衰期",
    "95℃ 后残余活性",
    "高温 RT-PCR Ct"
   ],
   "refs": [
    "ghadessy2001",
    "arezi2009",
    "bst2023"
   ]
  },
  {
   "id": "inhibitor",
   "name": "耐抑制",
   "enzymes": [
    "Taq",
    "Tth"
   ],
   "question": "全血、血浆、尿液、FFPE、粪便、拭子、提取试剂残留、PBS/Tris/HEPES",
   "metrics": [
    "允许全血/基质比例",
    "Ct 漂移",
    "LOD",
    "内标回收"
   ],
   "refs": [
    "kermekchiev2009",
    "igg2000",
    "blood2001",
    "urine1991",
    "ffpe2013",
    "stool1997",
    "bsa1996"
   ]
  },
  {
   "id": "speed",
   "name": "快速扩增",
   "enzymes": [
    "Taq"
   ],
   "question": "提高延伸速度、缩短 PCR 时间",
   "metrics": [
    "kb/5 min",
    "1 kb 延伸时间",
    "长片段产量",
    "Ct"
   ],
   "refs": [
    "yamagami2014"
   ]
  },
  {
   "id": "gcat",
   "name": "高 GC / 高 AT 兼容",
   "enzymes": [
    "Taq"
   ],
   "question": "高 GC 二级结构、AT-rich 低 Tm 的扩增",
   "metrics": [
    "GC/AT 梯度 panel 成功率",
    "Ct",
    "产量",
    "特异性"
   ],
   "refs": [
    "s543n1999",
    "sd2014",
    "inhib2008"
   ]
  },
  {
   "id": "exo",
   "name": "外切酶活性",
   "enzymes": [
    "Taq",
    "Pfu"
   ],
   "question": "5′→3′ 核酸酶切割或 3′→5′ 校对活性",
   "metrics": [
    "5′ 核酸酶切割",
    "3′→5′ 外切活性",
    "错误率"
   ],
   "refs": [
    "exo1997",
    "klentaq1992",
    "pfu2009",
    "pfu1996"
   ]
  },
  {
   "id": "specificity",
   "name": "特异性 / 热启动",
   "enzymes": [
    "Taq"
   ],
   "question": "降低低温非特异延伸、引物二聚体",
   "metrics": [
    "NTC",
    "非特异条带",
    "熔解曲线",
    "37℃/68℃ 活性比"
   ],
   "refs": [
    "kermekchiev2003",
    "i707l2015",
    "taqstart1994"
   ]
  },
  {
   "id": "rt_thermo",
   "name": "RT 耐热 / 持续合成",
   "enzymes": [
    "MMLV"
   ],
   "question": "提高 RT 高温活性、长 cDNA 合成",
   "metrics": [
    "55℃ 半衰期",
    "最长 cDNA",
    "62℃ 全长合成",
    "RT-PCR Ct"
   ],
   "refs": [
    "arezi2009",
    "baranauskas2012",
    "rnaseh2002"
   ]
  },
  {
   "id": "salt_lamp",
   "name": "耐盐 / LAMP",
   "enzymes": [
    "Bst"
   ],
   "question": "高盐、链置换、RT-LAMP 单酶",
   "metrics": [
    "起峰时间",
    "耐盐浓度",
    "全血 RT-LAMP",
    "RT 活性"
   ],
   "refs": [
    "bst32016",
    "bst2023",
    "bst2025review"
   ]
  }
 ],
 "routes": [
  {
   "id": "taq_thermo_csr",
   "name": "Taq CSR 连续选择（耐热）",
   "target": "thermo",
   "enzymes": [
    "Taq"
   ],
   "methods": [
    "csr"
   ],
   "refs": [
    "ghadessy2001"
   ],
   "summary": "用聚合酶自复制区室选择，直接把“高温下仍能复制自身基因”变成生长/扩增优势。",
   "library": "Taq 基因随机突变库（epPCR 或易错滚环），按 10^5–10^6 转化子建库；区室化乳液。",
   "screen": "多轮升温（例如 95–100℃ 预处理）后做 CSR；每轮提高温度或延长挑战时间。",
   "expected": "文献中 3 轮 CSR 获 11× 耐热提升；具体倍数依赖亲本与筛选条件。",
   "effort": "高",
   "confidence": "medium",
   "equipment": [
    "thermocycler",
    "emulsion",
    "plate_reader",
    "sequencer"
   ],
   "readouts": [
    "95℃ 后残余活性",
    "T50",
    "CSR 富集倍数",
    "突变位点"
   ],
   "steps": [
    {
     "n": "01",
     "name": "建立筛选基线",
     "detail": "确定目标反应、底物/抑制物矩阵、阴阳性与内标；先用亲本酶做 3 次重复，记录 Z-factor、CV、LOD/Ct。",
     "gate": "Z-factor ≥0.5；CV ≤10%；阴性无信号。"
    },
    {
     "n": "02",
     "name": "建库",
     "detail": "Taq 随机突变库 + 乳液区室化；每轮扩增后回收质粒。",
     "gate": "库容 ≥10^4（随机）或覆盖目标位点全部 19 种替换（饱和）；测序抽检突变率。"
    },
    {
     "n": "03",
     "name": "表达与初筛",
     "detail": "乳液 PCR/自复制后破乳、转化、涂板或直接进入下一轮。",
     "gate": "96/384 孔表达一致；亲本与空载体对照同板。"
    },
    {
     "n": "04",
     "name": "复筛与去假阳性",
     "detail": "按热处理温度/时间梯度筛选；保留能扩增自身基因的区室。",
     "gate": "≥3 个独立克隆、≥3 次重复；复筛保留 ≥2× 或预先设定的效应量。"
    },
    {
     "n": "05",
     "name": "纯化与机制验证",
     "detail": "纯化 3–5 个变体，测 T50、95℃ 半衰期、聚合酶比活。",
     "gate": "测 kcat/Km、Tm/T50、抑制物 IC50、批间 CV；测序确认无额外突变。"
    },
    {
     "n": "06",
     "name": "决策与下一轮",
     "detail": "与亲本做同体系头对头；NGS 看富集位点。",
     "gate": "达到目标阈值才进入下一轮；否则回到位点选择/建库。"
    }
   ],
   "caution": "只适用于可自复制的聚合酶；CSR 富集的是“自复制能力”，不等同于诊断体系中的扩增效率。",
   "simulation_id": "sim_csr_thermo"
  },
  {
   "id": "mmlv_thermo_m5",
   "name": "MMLV RT M5 组合（耐热）",
   "target": "thermo",
   "enzymes": [
    "MMLV"
   ],
   "methods": [
    "epcr",
    "ism"
   ],
   "refs": [
    "arezi2009"
   ],
   "summary": "随机+位点饱和筛选得到 E69K/E302R/W313F/L435G/N454K 五重组合，提高 55℃ 半衰期。",
   "library": "先做单点饱和/随机库，再按文献位点回补组合；控制在 5 个位点内。",
   "screen": "55℃ 热处理 5–30 min 后测 cDNA 合成；保留残余活性高的变体。",
   "expected": "文献：55℃ 半衰期 <5 min → 约 30 min；25–70℃ 比活提高。",
   "effort": "中高",
   "confidence": "high",
   "equipment": [
    "thermocycler",
    "plate_reader",
    "shaker",
    "sequencer",
    "analytical"
   ],
   "readouts": [
    "55℃ 半衰期",
    "cDNA 产量",
    "RT-PCR Ct",
    "Tm"
   ],
   "steps": [
    {
     "n": "01",
     "name": "建立筛选基线",
     "detail": "确定目标反应、底物/抑制物矩阵、阴阳性与内标；先用亲本酶做 3 次重复，记录 Z-factor、CV、LOD/Ct。",
     "gate": "Z-factor ≥0.5；CV ≤10%；阴性无信号。"
    },
    {
     "n": "02",
     "name": "建库",
     "detail": "以 MMLV RT 为亲本，构建 E69K/E302R/W313F/L435G/N454K 单点与组合库。",
     "gate": "库容 ≥10^4（随机）或覆盖目标位点全部 19 种替换（饱和）；测序抽检突变率。"
    },
    {
     "n": "03",
     "name": "表达与初筛",
     "detail": "大肠杆菌表达，96 孔裂解或纯化；同板亲本对照。",
     "gate": "96/384 孔表达一致；亲本与空载体对照同板。"
    },
    {
     "n": "04",
     "name": "复筛与去假阳性",
     "detail": "55℃ 预挑战后做荧光/凝胶 cDNA 合成；复筛 3 次。",
     "gate": "≥3 个独立克隆、≥3 次重复；复筛保留 ≥2× 或预先设定的效应量。"
    },
    {
     "n": "05",
     "name": "纯化与机制验证",
     "detail": "纯化测半衰期、cDNA 长度、RT-PCR Ct；测序。",
     "gate": "测 kcat/Km、Tm/T50、抑制物 IC50、批间 CV；测序确认无额外突变。"
    },
    {
     "n": "06",
     "name": "决策与下一轮",
     "detail": "与市售 RT 做同模板头对头；评估 RNase H 状态。",
     "gate": "达到目标阈值才进入下一轮；否则回到位点选择/建库。"
    }
   ],
   "caution": "M5 是文献组合；宝锐 RT 底盘不同，需重新验证；耐热≠耐抑制。",
   "simulation_id": "sim_mmlv_thermo"
  },
  {
   "id": "bst_thermo_ml",
   "name": "Bst 机器学习耐热变体",
   "target": "thermo",
   "enzymes": [
    "Bst"
   ],
   "methods": [
    "mlde",
    "ism"
   ],
   "refs": [
    "bst2023",
    "bst2025review"
   ],
   "summary": "用机器学习预测 + 组合多点替换，提高 Bst 热变性温度，支持更高温 LAMP。",
   "library": "以 Bst 大片段为亲本，按已发表 ML/热稳定位点做 10–30 个单点，组合 2–4 点。",
   "screen": "73℃ 预处理后做 LAMP 起峰时间；与 Bst 2.0 对照。",
   "expected": "文献：Tm 最高 +2.5℃；73℃ 可做 LAMP，而 Bst 2.0 在该温度失活。",
   "effort": "高",
   "confidence": "medium",
   "equipment": [
    "thermocycler",
    "plate_reader",
    "shaker",
    "sequencer",
    "gpu"
   ],
   "readouts": [
    "Tm",
    "73℃ 起峰时间",
    "耐盐",
    "RT-LAMP"
   ],
   "steps": [
    {
     "n": "01",
     "name": "建立筛选基线",
     "detail": "确定目标反应、底物/抑制物矩阵、阴阳性与内标；先用亲本酶做 3 次重复，记录 Z-factor、CV、LOD/Ct。",
     "gate": "Z-factor ≥0.5；CV ≤10%；阴性无信号。"
    },
    {
     "n": "02",
     "name": "建库",
     "detail": "按 ML 预测/文献位点建 2–4 点组合库；保留亲本与 Bst 2.0 对照。",
     "gate": "库容 ≥10^4（随机）或覆盖目标位点全部 19 种替换（饱和）；测序抽检突变率。"
    },
    {
     "n": "03",
     "name": "表达与初筛",
     "detail": "96/384 孔表达或购买合成基因；裂解后初筛。",
     "gate": "96/384 孔表达一致；亲本与空载体对照同板。"
    },
    {
     "n": "04",
     "name": "复筛与去假阳性",
     "detail": "73℃ 预处理 + LAMP 起峰；复筛 3 次。",
     "gate": "≥3 个独立克隆、≥3 次重复；复筛保留 ≥2× 或预先设定的效应量。"
    },
    {
     "n": "05",
     "name": "纯化与机制验证",
     "detail": "纯化测 Tm、起峰时间、耐盐；测序。",
     "gate": "测 kcat/Km、Tm/T50、抑制物 IC50、批间 CV；测序确认无额外突变。"
    },
    {
     "n": "06",
     "name": "决策与下一轮",
     "detail": "与 Bst 3.0/Bst 2.0 做同靶头对头。",
     "gate": "达到目标阈值才进入下一轮；否则回到位点选择/建库。"
    }
   ],
   "caution": "摘要未列具体位点，不能反推商业酶序列；高温 LAMP 可能牺牲常温稳定性。",
   "simulation_id": "sim_bst_thermo"
  },
  {
   "id": "taq_inhib_708",
   "name": "Taq codon 708 饱和突变（耐抑制）",
   "target": "inhibitor",
   "enzymes": [
    "Taq"
   ],
   "methods": [
    "ism",
    "cast"
   ],
   "refs": [
    "kermekchiev2009"
   ],
   "summary": "在 KlenTaq1 或 Taq E626K/I707L 冷敏感背景上，对 codon 708 做全部 19 种替换，筛全血/血浆/土壤提取物耐受。",
   "library": "以 KlenTaq1 或 Taq E626K/I707L 为亲本，对 708 位点做 NNK/19 种替换；同时保留 N 端缺失版本。",
   "screen": "全血 5–25%、血浆、IgG、血红蛋白、乳铁蛋白、土壤提取物梯度；以 Ct 漂移和可见产物判读。",
   "expected": "文献：KlenTaq10（E708K）可耐 20% 全血；Taq10/12 可耐 10–15%；E708N/I/Q 也有效。",
   "effort": "中高",
   "confidence": "high",
   "equipment": [
    "thermocycler",
    "plate_reader",
    "shaker",
    "sequencer",
    "electrophoresis"
   ],
   "readouts": [
    "允许全血比例",
    "Ct 漂移",
    "LOD",
    "突变位点"
   ],
   "steps": [
    {
     "n": "01",
     "name": "建立筛选基线",
     "detail": "确定目标反应、底物/抑制物矩阵、阴阳性与内标；先用亲本酶做 3 次重复，记录 Z-factor、CV、LOD/Ct。",
     "gate": "Z-factor ≥0.5；CV ≤10%；阴性无信号。"
    },
    {
     "n": "02",
     "name": "建库",
     "detail": "亲本：KlenTaq1 或 Taq E626K/I707L；对 708 做 19 种替换，另外构建 N 端缺失对照。",
     "gate": "库容 ≥10^4（随机）或覆盖目标位点全部 19 种替换（饱和）；测序抽检突变率。"
    },
    {
     "n": "03",
     "name": "表达与初筛",
     "detail": "96 孔表达，裂解液直接加全血/血浆/土壤提取物。",
     "gate": "96/384 孔表达一致；亲本与空载体对照同板。"
    },
    {
     "n": "04",
     "name": "复筛与去假阳性",
     "detail": "全血 0/5/10/15/20/25% 梯度，同板亲本与无模板对照；复筛 3 次。",
     "gate": "≥3 个独立克隆、≥3 次重复；复筛保留 ≥2× 或预先设定的效应量。"
    },
    {
     "n": "05",
     "name": "纯化与机制验证",
     "detail": "纯化 KlenTaq10/Taq10 类变体，测 IC50、延伸速度、测序。",
     "gate": "测 kcat/Km、Tm/T50、抑制物 IC50、批间 CV；测序确认无额外突变。"
    },
    {
     "n": "06",
     "name": "决策与下一轮",
     "detail": "在客户真实基质中验证；检查 5′ 核酸酶是否因 N 端缺失而丢失。",
     "gate": "达到目标阈值才进入下一轮；否则回到位点选择/建库。"
    }
   ],
   "caution": "E708K 依赖冷敏感背景，不是野生型 Taq 单点独立效果；N 端缺失会牺牲 5′ 核酸酶，TaqMan 体系需另选。",
   "simulation_id": "sim_taq_inhib"
  },
  {
   "id": "taq_inhib_csr",
   "name": "Taq CSR 耐抑制/耐肝素",
   "target": "inhibitor",
   "enzymes": [
    "Taq"
   ],
   "methods": [
    "csr"
   ],
   "refs": [
    "ghadessy2001"
   ],
   "summary": "用 CSR 直接把耐抑制与自复制偶联；文献报道 3 轮获得 >130× 耐肝素变体。",
   "library": "Taq 随机突变库 + 乳液区室；在含肝素/抑制物的区室反应中选择。",
   "screen": "肝素、全血、土壤提取物梯度；每轮提高抑制物浓度。",
   "expected": "文献：>130× 耐肝素；但肝素≠全血/IgG/血红素，不能直接外推到所有基质。",
   "effort": "高",
   "confidence": "medium",
   "equipment": [
    "thermocycler",
    "emulsion",
    "plate_reader",
    "sequencer"
   ],
   "readouts": [
    "IC50",
    "全血耐受",
    "富集倍数",
    "突变位点"
   ],
   "steps": [
    {
     "n": "01",
     "name": "建立筛选基线",
     "detail": "确定目标反应、底物/抑制物矩阵、阴阳性与内标；先用亲本酶做 3 次重复，记录 Z-factor、CV、LOD/Ct。",
     "gate": "Z-factor ≥0.5；CV ≤10%；阴性无信号。"
    },
    {
     "n": "02",
     "name": "建库",
     "detail": "Taq 随机突变库；乳液区室化，确保基因型-表型偶联。",
     "gate": "库容 ≥10^4（随机）或覆盖目标位点全部 19 种替换（饱和）；测序抽检突变率。"
    },
    {
     "n": "03",
     "name": "表达与初筛",
     "detail": "在含肝素或基质提取物的区室中自复制；破乳回收。",
     "gate": "96/384 孔表达一致；亲本与空载体对照同板。"
    },
    {
     "n": "04",
     "name": "复筛与去假阳性",
     "detail": "提高抑制物浓度重复 3 轮；NGS 看富集。",
     "gate": "≥3 个独立克隆、≥3 次重复；复筛保留 ≥2× 或预先设定的效应量。"
    },
    {
     "n": "05",
     "name": "纯化与机制验证",
     "detail": "纯化变体，测肝素/全血/土壤 IC50。",
     "gate": "测 kcat/Km、Tm/T50、抑制物 IC50、批间 CV；测序确认无额外突变。"
    },
    {
     "n": "06",
     "name": "决策与下一轮",
     "detail": "用目标客户基质验证；不要只报肝素倍数。",
     "gate": "达到目标阈值才进入下一轮；否则回到位点选择/建库。"
    }
   ],
   "caution": "肝素是提取试剂/抗凝剂模型，不等于全血全部抑制物；CSR 可能筛选到“自复制强但诊断扩增弱”的变体。",
   "simulation_id": "sim_csr_heparin"
  },
  {
   "id": "taq_speed_742",
   "name": "Taq 742/743 正电荷工程（快速）",
   "target": "speed",
   "enzymes": [
    "Taq"
   ],
   "methods": [
    "ism",
    "cast"
   ],
   "refs": [
    "yamagami2014"
   ],
   "summary": "对 E742/A743 做 Arg/Lys 等正电荷替换，提高 DNA 结合与延伸速度。",
   "library": "E742/A743 双位点饱和或定点组合（RR/KK/RK/KR 等）。",
   "screen": "1 kb/5 kb/10 kb 模板，缩短延伸时间；测产物量与 Ct。",
   "expected": "文献：WT 0.7 kb/5 min；TaqRR/KK 等 >8 kb/5 min，但比活下降、非特异结合风险上升。",
   "effort": "中",
   "confidence": "high",
   "equipment": [
    "thermocycler",
    "plate_reader",
    "electrophoresis",
    "sequencer"
   ],
   "readouts": [
    "kb/5 min",
    "1 kb 延伸时间",
    "Ct",
    "非特异条带"
   ],
   "steps": [
    {
     "n": "01",
     "name": "建立筛选基线",
     "detail": "确定目标反应、底物/抑制物矩阵、阴阳性与内标；先用亲本酶做 3 次重复，记录 Z-factor、CV、LOD/Ct。",
     "gate": "Z-factor ≥0.5；CV ≤10%；阴性无信号。"
    },
    {
     "n": "02",
     "name": "建库",
     "detail": "构建 E742R/A743R、E742K/A743K 等组合；保留 WT 对照。",
     "gate": "库容 ≥10^4（随机）或覆盖目标位点全部 19 种替换（饱和）；测序抽检突变率。"
    },
    {
     "n": "03",
     "name": "表达与初筛",
     "detail": "96 孔表达，测比活与 DNA 结合（EMSA 可选）。",
     "gate": "96/384 孔表达一致；亲本与空载体对照同板。"
    },
    {
     "n": "04",
     "name": "复筛与去假阳性",
     "detail": "1–10 kb 模板 + 短延伸时间；复筛 3 次。",
     "gate": "≥3 个独立克隆、≥3 次重复；复筛保留 ≥2× 或预先设定的效应量。"
    },
    {
     "n": "05",
     "name": "纯化与机制验证",
     "detail": "纯化测延伸速率、KD、PCR 性能；测序。",
     "gate": "测 kcat/Km、Tm/T50、抑制物 IC50、批间 CV；测序确认无额外突变。"
    },
    {
     "n": "06",
     "name": "决策与下一轮",
     "detail": "在客户 qPCR/长片段体系验证；检查非特异。",
     "gate": "达到目标阈值才进入下一轮；否则回到位点选择/建库。"
    }
   ],
   "caution": "DNA 结合过紧会增加非特异；文献中比活下降，不能只追求速度。",
   "simulation_id": "sim_taq_speed"
  },
  {
   "id": "taq_gc_s543",
   "name": "Taq S543N（高 GC / 二级结构）",
   "target": "gcat",
   "enzymes": [
    "Taq"
   ],
   "methods": [
    "ism"
   ],
   "refs": [
    "s543n1999"
   ],
   "summary": "在 KlentaqN 拇指域引入 S543N，减少模板结构导致的 pausing。",
   "library": "S543 单点饱和（NNK）或定点 S543N；在 Klentaq 大片段背景。",
   "screen": "GC 60–80% panel、强二级结构模板；梯度退火/添加剂对照。",
   "expected": "文献：S543N 可扩增高 GC/强二级结构模板；具体 GC 上限与缓冲体系需实测。",
   "effort": "中",
   "confidence": "medium",
   "equipment": [
    "thermocycler",
    "plate_reader",
    "electrophoresis",
    "sequencer"
   ],
   "readouts": [
    "GC panel 成功率",
    "Ct",
    "产量",
    "特异性"
   ],
   "steps": [
    {
     "n": "01",
     "name": "建立筛选基线",
     "detail": "确定目标反应、底物/抑制物矩阵、阴阳性与内标；先用亲本酶做 3 次重复，记录 Z-factor、CV、LOD/Ct。",
     "gate": "Z-factor ≥0.5；CV ≤10%；阴性无信号。"
    },
    {
     "n": "02",
     "name": "建库",
     "detail": "构建 S543N 及邻近位点饱和库；亲本 Klentaq 对照。",
     "gate": "库容 ≥10^4（随机）或覆盖目标位点全部 19 种替换（饱和）；测序抽检突变率。"
    },
    {
     "n": "03",
     "name": "表达与初筛",
     "detail": "表达后先用 2–3 个高 GC 模板初筛。",
     "gate": "96/384 孔表达一致；亲本与空载体对照同板。"
    },
    {
     "n": "04",
     "name": "复筛与去假阳性",
     "detail": "GC 60/70/80% panel + 强二级结构；复筛 3 次。",
     "gate": "≥3 个独立克隆、≥3 次重复；复筛保留 ≥2× 或预先设定的效应量。"
    },
    {
     "n": "05",
     "name": "纯化与机制验证",
     "detail": "纯化测 pausing/延伸、GC panel；测序。",
     "gate": "测 kcat/Km、Tm/T50、抑制物 IC50、批间 CV；测序确认无额外突变。"
    },
    {
     "n": "06",
     "name": "决策与下一轮",
     "detail": "与 PrimeSTAR GXL/Platinum II 做同 panel 对照。",
     "gate": "达到目标阈值才进入下一轮；否则回到位点选择/建库。"
    }
   ],
   "caution": "摘要级证据；高 GC 也受缓冲、添加剂、引物影响，不能全归因于酶。",
   "simulation_id": "sim_taq_gc"
  },
  {
   "id": "taq_exo_engineer",
   "name": "Taq 3′→5′ 外切酶活性工程",
   "target": "exo",
   "enzymes": [
    "Taq"
   ],
   "methods": [
    "proteinmpnn",
    "ism"
   ],
   "refs": [
    "exo1997",
    "klentaq1992"
   ],
   "summary": "在 Taq 3′→5′ 外切酶活性位点重建双金属离子催化模块；文献报道活性约为 WT 的 2×。",
   "library": "基于结构比对选择 ExoI/II/III  motif 附近的活性位点羧基，做定点/饱和组合；先做单点。",
   "screen": "用错配引物/末端标记底物测外切切割；同时测聚合酶比活与 PCR 保真度。",
   "expected": "文献：工程化 Taq 3′→5′ 外切活性约 2× WT；摘要未披露具体残基，需查原文/专利。",
   "effort": "高",
   "confidence": "low",
   "equipment": [
    "thermocycler",
    "plate_reader",
    "analytical",
    "sequencer",
    "gpu"
   ],
   "readouts": [
    "3′→5′ 外切活性",
    "错误率",
    "聚合酶比活",
    "PCR 产量"
   ],
   "steps": [
    {
     "n": "01",
     "name": "建立筛选基线",
     "detail": "确定目标反应、底物/抑制物矩阵、阴阳性与内标；先用亲本酶做 3 次重复，记录 Z-factor、CV、LOD/Ct。",
     "gate": "Z-factor ≥0.5；CV ≤10%；阴性无信号。"
    },
    {
     "n": "02",
     "name": "建库",
     "detail": "结构比对定位 Taq 3′→5′ 外切酶活性位点；设计羧基/金属配位残基组合。",
     "gate": "库容 ≥10^4（随机）或覆盖目标位点全部 19 种替换（饱和）；测序抽检突变率。"
    },
    {
     "n": "03",
     "name": "表达与初筛",
     "detail": "大肠杆菌表达，纯化后先测外切/聚合酶双活性。",
     "gate": "96/384 孔表达一致；亲本与空载体对照同板。"
    },
    {
     "n": "04",
     "name": "复筛与去假阳性",
     "detail": "错配底物切割 + 报告基因保真度；复筛 3 次。",
     "gate": "≥3 个独立克隆、≥3 次重复；复筛保留 ≥2× 或预先设定的效应量。"
    },
    {
     "n": "05",
     "name": "纯化与机制验证",
     "detail": "测 kcat/Km、外切/聚合酶比、错误率；测序。",
     "gate": "测 kcat/Km、Tm/T50、抑制物 IC50、批间 CV；测序确认无额外突变。"
    },
    {
     "n": "06",
     "name": "决策与下一轮",
     "detail": "与 Pfu/Q5 校对酶对照；评估是否值得替代。",
     "gate": "达到目标阈值才进入下一轮；否则回到位点选择/建库。"
    }
   ],
   "caution": "这是高难工程；Taq 天然缺 ExoI/II/III，文献摘要未给具体残基；2× 外切活性不等于 2× 保真度。",
   "simulation_id": "sim_taq_exo"
  },
  {
   "id": "pfu_exo_control",
   "name": "Pfu D215A 外切酶失活对照",
   "target": "exo",
   "enzymes": [
    "Pfu"
   ],
   "methods": [
    "ism"
   ],
   "refs": [
    "pfu2009",
    "pfu1996"
   ],
   "summary": "用 D215A 失活 3′→5′ 外切酶，作为“外切酶活性贡献”的阴性对照，而非提升路径。",
   "library": "Pfu D215A 定点突变；WT 对照。",
   "screen": "错配底物切割、PCR 错误率、扩增产量。",
   "expected": "文献：D215A 使 3′→5′ 外切失活；Pfu WT 错误率约 1.3×10⁻⁶，exo- 约 5×10⁻⁵。",
   "effort": "低",
   "confidence": "high",
   "equipment": [
    "thermocycler",
    "plate_reader",
    "electrophoresis",
    "sequencer"
   ],
   "readouts": [
    "外切活性",
    "错误率",
    "产量"
   ],
   "steps": [
    {
     "n": "01",
     "name": "建立筛选基线",
     "detail": "确定目标反应、底物/抑制物矩阵、阴阳性与内标；先用亲本酶做 3 次重复，记录 Z-factor、CV、LOD/Ct。",
     "gate": "Z-factor ≥0.5；CV ≤10%；阴性无信号。"
    },
    {
     "n": "02",
     "name": "建库",
     "detail": "构建 Pfu D215A；WT 对照。",
     "gate": "库容 ≥10^4（随机）或覆盖目标位点全部 19 种替换（饱和）；测序抽检突变率。"
    },
    {
     "n": "03",
     "name": "表达与初筛",
     "detail": "表达纯化；测外切/聚合酶活性。",
     "gate": "96/384 孔表达一致；亲本与空载体对照同板。"
    },
    {
     "n": "04",
     "name": "复筛与去假阳性",
     "detail": "错配底物 + 报告基因错误率；复筛。",
     "gate": "≥3 个独立克隆、≥3 次重复；复筛保留 ≥2× 或预先设定的效应量。"
    },
    {
     "n": "05",
     "name": "纯化与机制验证",
     "detail": "测错误率、产量；测序。",
     "gate": "测 kcat/Km、Tm/T50、抑制物 IC50、批间 CV；测序确认无额外突变。"
    },
    {
     "n": "06",
     "name": "决策与下一轮",
     "detail": "作为外切酶贡献的阴性对照，不作为产品候选。",
     "gate": "达到目标阈值才进入下一轮；否则回到位点选择/建库。"
    }
   ],
   "caution": "D215A 是失活突变；若目标是“提升”，应走天然 B 族酶或定向进化。",
   "simulation_id": "sim_pfu_exo"
  },
  {
   "id": "taq_spec_cold",
   "name": "Taq 冷敏感热启动（特异性）",
   "target": "specificity",
   "enzymes": [
    "Taq"
   ],
   "methods": [
    "ism"
   ],
   "refs": [
    "kermekchiev2003",
    "i707l2015"
   ],
   "summary": "在 KlenTaq1 的 706–708 热点做冷敏感替换，降低 37℃ 活性而不牺牲 68℃ 活性。",
   "library": "E626K、I707L、E708D、W706R 单点与组合（如 E626K/I707L）。",
   "screen": "37℃ 预孵育后 PCR；NTC、引物二聚体、非特异条带；68℃ 活性对照。",
   "expected": "文献：冷敏感突变 37℃ 活性下降、68℃ 接近正常；提供热启动并略提高保真度。",
   "effort": "中",
   "confidence": "high",
   "equipment": [
    "thermocycler",
    "plate_reader",
    "electrophoresis",
    "sequencer"
   ],
   "readouts": [
    "37℃/68℃ 活性比",
    "NTC",
    "非特异条带",
    "Ct"
   ],
   "steps": [
    {
     "n": "01",
     "name": "建立筛选基线",
     "detail": "确定目标反应、底物/抑制物矩阵、阴阳性与内标；先用亲本酶做 3 次重复，记录 Z-factor、CV、LOD/Ct。",
     "gate": "Z-factor ≥0.5；CV ≤10%；阴性无信号。"
    },
    {
     "n": "02",
     "name": "建库",
     "detail": "构建 E626K/I707L/E708D/W706R 单点与双点组合；亲本 KlenTaq 对照。",
     "gate": "库容 ≥10^4（随机）或覆盖目标位点全部 19 种替换（饱和）；测序抽检突变率。"
    },
    {
     "n": "03",
     "name": "表达与初筛",
     "detail": "表达后测 37℃ 与 68℃ 活性。",
     "gate": "96/384 孔表达一致；亲本与空载体对照同板。"
    },
    {
     "n": "04",
     "name": "复筛与去假阳性",
     "detail": "37℃ 预孵育 + PCR；NTC 与引物二聚体；复筛。",
     "gate": "≥3 个独立克隆、≥3 次重复；复筛保留 ≥2× 或预先设定的效应量。"
    },
    {
     "n": "05",
     "name": "纯化与机制验证",
     "detail": "纯化测温度剖面、保真度；测序。",
     "gate": "测 kcat/Km、Tm/T50、抑制物 IC50、批间 CV；测序确认无额外突变。"
    },
    {
     "n": "06",
     "name": "决策与下一轮",
     "detail": "与抗体/适配体热启动做同体系对照。",
     "gate": "达到目标阈值才进入下一轮；否则回到位点选择/建库。"
    }
   ],
   "caution": "冷敏感会降低低温活性，但可能影响室温稳定性；全长 Taq 背景需重新验证。",
   "simulation_id": "sim_taq_spec"
  },
  {
   "id": "mmlv_rt_processive",
   "name": "MMLV RT 持续合成组合",
   "target": "rt_thermo",
   "enzymes": [
    "MMLV"
   ],
   "methods": [
    "mlde",
    "ism"
   ],
   "refs": [
    "baranauskas2012",
    "mmulv2021"
   ],
   "summary": "组合 D200N/L603W/T330P/L139P/E607K，提高持续合成与高温长 cDNA。",
   "library": "按文献单点做组合库；可加 ML 预测。",
   "screen": "结构化 RNA 模板、7.5 kb 全长 cDNA、55–62℃ 梯度。",
   "expected": "文献：持续合成 1500 nt（65×），7.5 kb 全长 cDNA 合成温度 62℃。",
   "effort": "高",
   "confidence": "high",
   "equipment": [
    "thermocycler",
    "plate_reader",
    "shaker",
    "sequencer",
    "analytical"
   ],
   "readouts": [
    "最长 cDNA",
    "62℃ 全长合成",
    "55℃ 半衰期",
    "RT-PCR Ct"
   ],
   "steps": [
    {
     "n": "01",
     "name": "建立筛选基线",
     "detail": "确定目标反应、底物/抑制物矩阵、阴阳性与内标；先用亲本酶做 3 次重复，记录 Z-factor、CV、LOD/Ct。",
     "gate": "Z-factor ≥0.5；CV ≤10%；阴性无信号。"
    },
    {
     "n": "02",
     "name": "建库",
     "detail": "构建 D200N/L603W/T330P/L139P/E607K 单点与组合；亲本 MMLV 对照。",
     "gate": "库容 ≥10^4（随机）或覆盖目标位点全部 19 种替换（饱和）；测序抽检突变率。"
    },
    {
     "n": "03",
     "name": "表达与初筛",
     "detail": "表达纯化或裂解；测 cDNA 合成。",
     "gate": "96/384 孔表达一致；亲本与空载体对照同板。"
    },
    {
     "n": "04",
     "name": "复筛与去假阳性",
     "detail": "结构化 RNA + 长片段梯度；复筛。",
     "gate": "≥3 个独立克隆、≥3 次重复；复筛保留 ≥2× 或预先设定的效应量。"
    },
    {
     "n": "05",
     "name": "纯化与机制验证",
     "detail": "测持续合成、62℃ 全长、半衰期；测序。",
     "gate": "测 kcat/Km、Tm/T50、抑制物 IC50、批间 CV；测序确认无额外突变。"
    },
    {
     "n": "06",
     "name": "决策与下一轮",
     "detail": "与 M5/商业 RT 头对头；评估 RNase H。",
     "gate": "达到目标阈值才进入下一轮；否则回到位点选择/建库。"
    }
   ],
   "caution": "五重组合；单点贡献与背景相关；持续合成提高不等于耐抑制。",
   "simulation_id": "sim_mmlv_processive"
  },
  {
   "id": "bst_salt_ml",
   "name": "Bst 机器学习耐盐/耐热",
   "target": "salt_lamp",
   "enzymes": [
    "Bst"
   ],
   "methods": [
    "mlde",
    "ism"
   ],
   "refs": [
    "bst2023",
    "bst32016",
    "bst2025review"
   ],
   "summary": "用 ML/定向进化组合热稳定与耐盐位点，目标 73℃ LAMP 或高盐 RT-LAMP。",
   "library": "Bst 大片段 + 2–4 个预测位点；保留 Bst 2.0/Bst 3.0 对照。",
   "screen": "NaCl 0–300 mM、全血 0–10%、73℃ 预处理；LAMP 起峰时间。",
   "expected": "文献：Bst 3.0 可在全血中 RT-LAMP；Bst ML 变体 73℃ 可 LAMP，Bst 2.0 失活。",
   "effort": "高",
   "confidence": "medium",
   "equipment": [
    "thermocycler",
    "plate_reader",
    "shaker",
    "sequencer",
    "gpu"
   ],
   "readouts": [
    "起峰时间",
    "耐盐浓度",
    "全血 RT-LAMP",
    "Tm"
   ],
   "steps": [
    {
     "n": "01",
     "name": "建立筛选基线",
     "detail": "确定目标反应、底物/抑制物矩阵、阴阳性与内标；先用亲本酶做 3 次重复，记录 Z-factor、CV、LOD/Ct。",
     "gate": "Z-factor ≥0.5；CV ≤10%；阴性无信号。"
    },
    {
     "n": "02",
     "name": "建库",
     "detail": "按 ML/文献位点建 2–4 点组合；Bst 2.0/Bst 3.0 对照。",
     "gate": "库容 ≥10^4（随机）或覆盖目标位点全部 19 种替换（饱和）；测序抽检突变率。"
    },
    {
     "n": "03",
     "name": "表达与初筛",
     "detail": "表达/裂解；先用标准 LAMP 初筛。",
     "gate": "96/384 孔表达一致；亲本与空载体对照同板。"
    },
    {
     "n": "04",
     "name": "复筛与去假阳性",
     "detail": "NaCl/全血梯度 + 73℃ 预处理；复筛。",
     "gate": "≥3 个独立克隆、≥3 次重复；复筛保留 ≥2× 或预先设定的效应量。"
    },
    {
     "n": "05",
     "name": "纯化与机制验证",
     "detail": "纯化测 Tm、起峰、RT 活性；测序。",
     "gate": "测 kcat/Km、Tm/T50、抑制物 IC50、批间 CV；测序确认无额外突变。"
    },
    {
     "n": "06",
     "name": "决策与下一轮",
     "detail": "在客户 LAMP 体系验证；检查非特异扩增。",
     "gate": "达到目标阈值才进入下一轮；否则回到位点选择/建库。"
    }
   ],
   "caution": "商业 Bst 3.0 突变未公开；不能反推；高温/耐盐可能牺牲常温稳定性。",
   "simulation_id": "sim_bst_salt"
  }
 ],
 "simulations": [
  {
   "id": "sim_csr_thermo",
   "title": "CSR 耐热富集（文献锚点情景）",
   "kind": "bar",
   "route_ids": [
    "taq_thermo_csr"
   ],
   "target": "thermo",
   "anchors": [
    {
     "label": "WT 亲本",
     "value": 1,
     "unit": "相对耐热",
     "refs": [
      "ghadessy2001"
     ]
    },
    {
     "label": "CSR 3 轮",
     "value": 11,
     "unit": "× 耐热",
     "refs": [
      "ghadessy2001"
     ]
    }
   ],
   "model": "柱状对比：文献报道 3 轮 CSR 后 Taq 变体耐热性 11×；不是对宝锐底盘的预测。",
   "assumptions": [
    "同一热处理条件与宿主背景",
    "以文献定义的耐热读数计，不直接等于 qPCR Ct"
   ],
   "outputs": [
    {
     "label": "相对耐热",
     "wt": 1,
     "mut": 11,
     "unit": "×"
    }
   ],
   "interpretation": [
    "若首轮富集 <2×，先检查区室化与热处理窗口；不要直接进下一轮。",
    "11× 是文献值，不能承诺宝锐底盘同样倍数。"
   ]
  },
  {
   "id": "sim_mmlv_thermo",
   "title": "MMLV M5 热失活曲线（一级动力学模拟）",
   "kind": "line",
   "route_ids": [
    "mmlv_thermo_m5"
   ],
   "target": "thermo",
   "anchors": [
    {
     "label": "WT 55℃ 半衰期",
     "value": 5,
     "unit": "min",
     "refs": [
      "arezi2009"
     ]
    },
    {
     "label": "M5 55℃ 半衰期",
     "value": 30,
     "unit": "min",
     "refs": [
      "arezi2009"
     ]
    }
   ],
   "model": "A(t)=100×2^(−t/t½)；WT t½=5 min，M5 t½=30 min；一级失活假设。",
   "assumptions": [
    "同一缓冲、同一起始酶量、无底物保护差异",
    "实际失活常非严格一级；只用于方案设计"
   ],
   "x": [
    0,
    5,
    10,
    20,
    30,
    45,
    60
   ],
   "x_label": "55℃ 处理时间（min）",
   "y_label": "残余活性（%）",
   "series": [
    {
     "name": "WT",
     "t12": 5
    },
    {
     "name": "M5",
     "t12": 30
    }
   ],
   "interpretation": [
    "30 min 时 WT 约 1.6%，M5 约 50%；这是文献半衰期的数学外推。",
    "必须用实际 RT 体系测 cDNA 产量，不能只看残余活性。"
   ]
  },
  {
   "id": "sim_bst_thermo",
   "title": "Bst 热稳定变体 ΔTm（文献锚点）",
   "kind": "bar",
   "route_ids": [
    "bst_thermo_ml"
   ],
   "target": "thermo",
   "anchors": [
    {
     "label": "Bst 亲本 ΔTm",
     "value": 0,
     "unit": "℃",
     "refs": [
      "bst2023"
     ]
    },
    {
     "label": "ML 组合 ΔTm",
     "value": 2.5,
     "unit": "℃",
     "refs": [
      "bst2023"
     ]
    }
   ],
   "model": "文献报道最高 +2.5℃；柱状展示，不外推更高温。",
   "assumptions": [
    "不同缓冲/离子强度会改变 Tm",
    "+2.5℃ 是特定变体组合，不是通用上限"
   ],
   "outputs": [
    {
     "label": "ΔTm",
     "wt": 0,
     "mut": 2.5,
     "unit": "℃"
    }
   ],
   "interpretation": [
    "2.5℃ 可把 LAMP 反应温度推到 73℃ 附近，但需实测起峰时间。",
    "若目标是 80℃ 以上，单靠该策略不够。"
   ]
  },
  {
   "id": "sim_taq_inhib",
   "title": "Taq 全血耐受剂量-反应（示意插值）",
   "kind": "line",
   "route_ids": [
    "taq_inhib_708"
   ],
   "target": "inhibitor",
   "anchors": [
    {
     "label": "WT 被 0.1–1% 全血强烈抑制",
     "value": "0.1–1",
     "unit": "% blood",
     "refs": [
      "kermekchiev2009"
     ]
    },
    {
     "label": "KlenTaq10 可耐 20% 全血",
     "value": 20,
     "unit": "% blood",
     "refs": [
      "kermekchiev2009"
     ]
    },
    {
     "label": "Taq10/12 可耐 10–15%",
     "value": "10–15",
     "unit": "% blood",
     "refs": [
      "kermekchiev2009"
     ]
    }
   ],
   "model": "四参数 logistic 的简化版：R=1/(1+(x/IC50)^h)。WT IC50=0.5%，KlenTaq10 IC50=12%，Taq22 IC50=10%；h=1.5。曲线为示意插值，不是原始数据点。",
   "assumptions": [
    "同一种全血来源、同一扩增体系",
    "IC50 由文献“可耐/被抑制”区间反推，非直接拟合",
    "不同靶标/扩增子长度会改变曲线"
   ],
   "x": [
    0,
    1,
    2.5,
    5,
    10,
    15,
    20,
    25
   ],
   "x_label": "全血比例（%）",
   "y_label": "相对扩增（示意）",
   "series": [
    {
     "name": "WT Taq",
     "ic50": 0.5,
     "h": 1.5
    },
    {
     "name": "KlenTaq10",
     "ic50": 12,
     "h": 1.5
    },
    {
     "name": "Taq22",
     "ic50": 10,
     "h": 1.5
    }
   ],
   "interpretation": [
    "WT 在 1% 全血附近已接近 0；KlenTaq10 在 20% 仍有信号，与文献“可耐 20%”一致。",
    "曲线是方案设计用示意，真实 Ct 漂移需湿实验。"
   ]
  },
  {
   "id": "sim_csr_heparin",
   "title": "CSR 耐肝素（文献锚点）",
   "kind": "bar",
   "route_ids": [
    "taq_inhib_csr"
   ],
   "target": "inhibitor",
   "anchors": [
    {
     "label": "WT 亲本",
     "value": 1,
     "unit": "相对耐肝素",
     "refs": [
      "ghadessy2001"
     ]
    },
    {
     "label": "CSR 3 轮",
     "value": "＞130",
     "unit": "× 耐肝素",
     "refs": [
      "ghadessy2001"
     ]
    }
   ],
   "model": "柱状对比文献报道的 >130× 耐肝素；>130 以 130 作为下限绘制。",
   "assumptions": [
    "肝素是提取/抗凝剂模型，不等于全血全部抑制物",
    "同一 CSR 区室化与筛选条件"
   ],
   "outputs": [
    {
     "label": "耐肝素",
     "wt": 1,
     "mut": 130,
     "unit": "×（下限）"
    }
   ],
   "interpretation": [
    "文献报道 3 轮 CSR 获 >130× 耐肝素；不能据此宣称耐全血/IgG/血红素。",
    "必须回到目标基质做验证；肝素倍数只是机制线索。"
   ]
  },
  {
   "id": "sim_taq_speed",
   "title": "Taq 742/743 延伸速度（文献锚点）",
   "kind": "bar",
   "route_ids": [
    "taq_speed_742"
   ],
   "target": "speed",
   "anchors": [
    {
     "label": "WT Taq",
     "value": 0.7,
     "unit": "kb/5 min",
     "refs": [
      "yamagami2014"
     ]
    },
    {
     "label": "TaqRR",
     "value": ">8",
     "unit": "kb/5 min",
     "refs": [
      "yamagami2014"
     ]
    },
    {
     "label": "TaqKK",
     "value": ">8",
     "unit": "kb/5 min",
     "refs": [
      "yamagami2014"
     ]
    }
   ],
   "model": "柱状对比文献延伸速率；>8 以 8 作为下限绘制，不代表精确值。",
   "assumptions": [
    "同一模板、dNTP/Mg²⁺、温度",
    "速率与模板结构/长度相关"
   ],
   "outputs": [
    {
     "label": "延伸速率",
     "wt": 0.7,
     "mut": 8,
     "unit": "kb/5 min（下限）"
    }
   ],
   "interpretation": [
    "文献中突变体延伸 >8 kb/5 min，约为 WT 的 11× 以上；但比活下降，需重新配酶量。",
    "先测 1 kb/5 kb 短延伸，再看长片段。"
   ]
  },
  {
   "id": "sim_taq_exo",
   "title": "Taq 3′→5′ 外切酶活性（文献锚点）",
   "kind": "bar",
   "route_ids": [
    "taq_exo_engineer"
   ],
   "target": "exo",
   "anchors": [
    {
     "label": "WT Taq 3′→5′ 外切",
     "value": 1,
     "unit": "相对活性",
     "refs": [
      "exo1997"
     ]
    },
    {
     "label": "工程化 Taq",
     "value": 2,
     "unit": "相对活性",
     "refs": [
      "exo1997"
     ]
    }
   ],
   "model": "文献报道工程化 Taq 3′→5′ 外切活性约为 WT 的 2×；柱状对比。",
   "assumptions": [
    "不同底物/离子条件会改变倍数",
    "2× 外切活性≠2× 保真度"
   ],
   "outputs": [
    {
     "label": "相对外切活性",
     "wt": 1,
     "mut": 2,
     "unit": "×"
    }
   ],
   "interpretation": [
    "2× 是文献摘要值；必须用错配底物与报告基因分别验证外切和保真度。",
    "若目标是高保真，优先选天然 B 族酶或定向进化。"
   ]
  },
  {
   "id": "sim_pfu_exo",
   "title": "Pfu D215A 外切失活对照",
   "kind": "bar",
   "route_ids": [
    "pfu_exo_control"
   ],
   "target": "exo",
   "anchors": [
    {
     "label": "Pfu WT 错误率",
     "value": 1.3e-06,
     "unit": "errors/bp",
     "refs": [
      "pfu1996"
     ]
    },
    {
     "label": "exo- Pfu 错误率",
     "value": 5e-05,
     "unit": "errors/bp",
     "refs": [
      "pfu1996"
     ]
    }
   ],
   "model": "柱状（对数刻度）展示 WT 与 exo- Pfu 的文献错误率；D215A 为失活对照。",
   "assumptions": [
    "报告基因/条件不同会改变绝对值",
    "只证明方向，不作为产品性能承诺"
   ],
   "outputs": [
    {
     "label": "错误率",
     "wt": 1.3e-06,
     "mut": 5e-05,
     "unit": "errors/bp"
    }
   ],
   "interpretation": [
    "外切酶失活使错误率上升约 38×；说明校对活性重要，但 D215A 不是“提升”方案。",
    "要提升保真度，选 Pfu/Q5 类校对酶或做定向进化。"
   ]
  },
  {
   "id": "sim_taq_gc",
   "title": "高 GC 兼容性（定性情景矩阵）",
   "kind": "qual",
   "route_ids": [
    "taq_gc_s543"
   ],
   "target": "gcat",
   "anchors": [
    {
     "label": "S543N 可扩增高 GC/强二级结构",
     "value": "qualitative",
     "unit": "",
     "refs": [
      "s543n1999"
     ]
    },
    {
     "label": "SD Taq 改善 GC-rich",
     "value": "qualitative",
     "unit": "",
     "refs": [
      "sd2014"
     ]
    }
   ],
   "model": "不做数值预测；给出“低/中/高”成功带。高 GC 受缓冲、添加剂、引物影响，不能全归因于酶。",
   "assumptions": [
    "同一 GC panel、同一退火/缓冲",
    "不同扩增子长度会改变结果"
   ],
   "matrix": {
    "rows": [
     "Taq WT",
     "S543N",
     "SD/工程化"
    ],
    "cols": [
     "GC 50–60%",
     "GC 60–70%",
     "GC 70–80%"
    ],
    "vals": [
     [
      "高",
      "中",
      "低"
     ],
     [
      "高",
      "中高",
      "中"
     ],
     [
      "高",
      "中高",
      "中高"
     ]
    ]
   },
   "interpretation": [
    "先看 60–70% GC 带；若 S543N 无优势，优先排查缓冲/添加剂。",
    "不要把“高 GC 成功”写成对所有 GC-rich 模板的承诺。"
   ]
  },
  {
   "id": "sim_taq_spec",
   "title": "冷敏感热启动（定性温度窗）",
   "kind": "qual",
   "route_ids": [
    "taq_spec_cold"
   ],
   "target": "specificity",
   "anchors": [
    {
     "label": "冷敏感突变 37℃ 活性下降、68℃ 接近正常",
     "value": "qualitative",
     "unit": "",
     "refs": [
      "kermekchiev2003"
     ]
    },
    {
     "label": "I707L 机制",
     "value": "qualitative",
     "unit": "",
     "refs": [
      "i707l2015"
     ]
    }
   ],
   "model": "不做数值预测；用 37℃/68℃ 相对活性带表示温度窗。",
   "assumptions": [
    "不同突变组合的冷敏感程度不同",
    "室温配制时间会影响非特异"
   ],
   "matrix": {
    "rows": [
     "KlenTaq WT",
     "E626K",
     "I707L",
     "E708D/W706R"
    ],
    "cols": [
     "37℃ 活性",
     "68℃ 活性",
     "NTC/非特异"
    ],
    "vals": [
     [
      "高",
      "高",
      "较高"
     ],
     [
      "低",
      "高",
      "低"
     ],
     [
      "低",
      "高",
      "低"
     ],
     [
      "低",
      "高",
      "低"
     ]
    ]
   },
   "interpretation": [
    "热启动效果看“37℃ 低、68℃ 高”的温度窗；同时看 NTC。",
    "冷敏感可能牺牲室温稳定性；需测加速老化。"
   ]
  },
  {
   "id": "sim_mmlv_processive",
   "title": "MMLV 持续合成/高温 cDNA（文献锚点）",
   "kind": "bar",
   "route_ids": [
    "mmlv_rt_processive"
   ],
   "target": "rt_thermo",
   "anchors": [
    {
     "label": "WT 持续合成",
     "value": 100,
     "unit": "nt",
     "refs": [
      "baranauskas2012"
     ]
    },
    {
     "label": "组合变体",
     "value": 1500,
     "unit": "nt",
     "refs": [
      "baranauskas2012"
     ]
    },
    {
     "label": "全长 7.5 kb 合成温度",
     "value": 62,
     "unit": "℃",
     "refs": [
      "baranauskas2012"
     ]
    }
   ],
   "model": "柱状对比文献持续合成长度；温度锚点为 62℃。",
   "assumptions": [
    "不同模板二级结构会改变结果",
    "持续合成提高不等于 RT-PCR Ct 必然降低"
   ],
   "outputs": [
    {
     "label": "最长 cDNA",
     "wt": 100,
     "mut": 1500,
     "unit": "nt"
    },
    {
     "label": "最高全长合成温度",
     "wt": 45,
     "mut": 62,
     "unit": "℃"
    }
   ],
   "interpretation": [
    "1500 nt 是文献最佳组合的持续合成；需用目标 RNA 验证。",
    "温度提高可能增加非特异 priming，需优化引物。"
   ]
  },
  {
   "id": "sim_bst_salt",
   "title": "Bst 耐盐/全血 RT-LAMP（定性矩阵）",
   "kind": "qual",
   "route_ids": [
    "bst_salt_ml"
   ],
   "target": "salt_lamp",
   "anchors": [
    {
     "label": "Bst 3.0 可在全血中 RT-LAMP",
     "value": "qualitative",
     "unit": "",
     "refs": [
      "bst32016"
     ]
    },
    {
     "label": "Bst 2.0 耐盐高于 WT",
     "value": "qualitative",
     "unit": "",
     "refs": [
      "bst2025review"
     ]
    }
   ],
   "model": "不做数值预测；给出盐/全血条件下的起峰带。",
   "assumptions": [
    "不同 LAMP 引物/靶标差异很大",
    "全血来源与抗凝剂影响结果"
   ],
   "matrix": {
    "rows": [
     "Bst WT",
     "Bst 2.0",
     "Bst 3.0/ML"
    ],
    "cols": [
     "0–100 mM NaCl",
     "200–300 mM NaCl",
     "5–10% 全血"
    ],
    "vals": [
     [
      "高",
      "低",
      "低"
     ],
     [
      "高",
      "中",
      "中"
     ],
     [
      "高",
      "中高",
      "中高"
     ]
    ]
   },
   "interpretation": [
    "先看 200–300 mM 盐与 5–10% 全血；起峰时间比终点荧光更可靠。",
    "商业酶性能不能反推突变位点。"
   ]
  },
  {
   "id": "sim_generic",
   "title": "通用筛选模拟（无特异文献锚点）",
   "kind": "qual",
   "route_ids": [],
   "target": "",
   "anchors": [],
   "model": "该组合没有足够的公开定量锚点；不生成数值模拟，只给实验判读表。",
   "assumptions": [
    "必须先用亲本建立基线"
   ],
   "matrix": {
    "rows": [
     "亲本",
     "候选"
    ],
    "cols": [
     "活性",
     "特异性",
     "稳定性"
    ],
    "vals": [
     [
      "基线",
      "基线",
      "基线"
     ],
     [
      "待测",
      "待测",
      "待测"
     ]
    ]
   },
   "interpretation": [
    "先做基线，再做候选；没有锚点就不要写预测值。"
   ]
  }
 ],
 "citations": {
  "kermekchiev2009": {
   "pmid": "19208643",
   "doi": "10.1093/nar/gkn1055",
   "title": "Mutants of Taq DNA polymerase resistant to PCR inhibitors allow DNA amplification from whole blood and crude soil samples",
   "journal": "Nucleic Acids Research",
   "year": 2009,
   "quote": "Further mutations at codon 708, both in Klentaq 1 and Taq, confer enhanced resistance to various inhibitors ... whole blood, plasma, hemoglobin, lactoferrin, serum IgG, soil extracts and humic acid.",
   "kind": "peer-reviewed",
   "url": "https://pubmed.ncbi.nlm.nih.gov/19208643/"
  },
  "kermekchiev2003": {
   "pmid": "14576300",
   "doi": "10.1093/nar/gkg813",
   "title": "Cold-sensitive mutants of Taq DNA polymerase provide a hot start for PCR",
   "journal": "Nucleic Acids Research",
   "year": 2003,
   "quote": "Mutations of Klentaq ... markedly reduced activity at 37°C yet retain apparently normal activity at 68°C ... can provide a hot start for PCR and exhibit slightly improved fidelity.",
   "kind": "peer-reviewed",
   "url": "https://pubmed.ncbi.nlm.nih.gov/14576300/"
  },
  "i707l2015": {
   "pmid": "25537790",
   "doi": "10.1021/bi501198f",
   "title": "A conservative isoleucine to leucine mutation causes major rearrangements and cold sensitivity in KlenTaq1 DNA polymerase",
   "journal": "Biochemistry",
   "year": 2015,
   "quote": "Mutation of isoleucine 707 to leucine ... substantially decreases its activity at room temperature without compromising its ability to amplify DNA.",
   "kind": "peer-reviewed",
   "url": "https://pubmed.ncbi.nlm.nih.gov/25537790/"
  },
  "yamagami2014": {
   "pmid": "25232352",
   "doi": "10.3389/fmicb.2014.00461",
   "title": "Mutant Taq DNA polymerases with improved elongation ability as a useful reagent for genetic engineering",
   "journal": "Frontiers in Microbiology",
   "year": 2014,
   "quote": "Two residues, E742 and A743, in Taq polymerase were found to be critical for its elongation ability ... higher DNA affinity and faster primer extension ability.",
   "kind": "peer-reviewed",
   "url": "https://pubmed.ncbi.nlm.nih.gov/25232352/"
  },
  "s543n1999": {
   "pmid": "10217428",
   "doi": "10.1016/s0014-5793(99)00353-1",
   "title": "Mutation S543N in the thumb subdomain of the Taq DNA polymerase large fragment suppresses pausing associated with the template structure",
   "journal": "FEBS Letters",
   "year": 1999,
   "quote": "The mutant enzyme (KlentaqN DNA polymerase) provides specific PCR amplification and sequencing of difficult templates, e.g. those with a high GC% content or strong secondary structure.",
   "kind": "peer-reviewed",
   "url": "https://pubmed.ncbi.nlm.nih.gov/10217428/"
  },
  "sd2014": {
   "pmid": "25109293",
   "doi": "10.2144/000114198",
   "title": "A strong strand displacement activity of thermostable DNA polymerase markedly improves the results of DNA amplification",
   "journal": "BioTechniques",
   "year": 2014,
   "quote": "A novel Taq DNA polymerase mutant (SD DNA polymerase), which has a strong strand displacement activity ... improvement in ... GC-rich and complex secondary structure templates.",
   "kind": "peer-reviewed",
   "url": "https://pubmed.ncbi.nlm.nih.gov/25109293/"
  },
  "exo1997": {
   "pmid": "9264032",
   "doi": "10.1016/s1016-8478(23)13315-2",
   "title": "Improvement of the 3'-5' exonuclease activity of Taq DNA polymerase by protein engineering in the active site",
   "journal": "Molecules and Cells",
   "year": 1997,
   "quote": "The mutant Taq DNA polymerase shows twice as much the 3'-5' exonuclease activity as that of wild-type DNA polymerase.",
   "kind": "peer-reviewed",
   "url": "https://pubmed.ncbi.nlm.nih.gov/9264032/"
  },
  "klentaq1992": {
   "pmid": "1551596",
   "doi": "10.1016/0378-1119(92)90299-5",
   "title": "The fidelity of Taq polymerase catalyzing PCR is improved by an N-terminal deletion",
   "journal": "Gene",
   "year": 1992,
   "quote": "The measured relative mutation rate was twofold lower for KlenTaq as compared to the full-length Taq DNA polymerase.",
   "kind": "peer-reviewed",
   "url": "https://pubmed.ncbi.nlm.nih.gov/1551596/"
  },
  "taqstart1994": {
   "pmid": "8074881",
   "doi": "",
   "title": "TaqStart Antibody: \"hot start\" PCR facilitated by a neutralizing monoclonal antibody directed against Taq DNA polymerase",
   "journal": "BioTechniques",
   "year": 1994,
   "quote": "Monoclonal antibodies (MAbs) raised in mice to the purified DNA polymerase of Thermus aquaticus (Taq) were selected for their ability to reversibly block polymerase activity.",
   "kind": "peer-reviewed",
   "url": "https://pubmed.ncbi.nlm.nih.gov/8074881/"
  },
  "arezi2009": {
   "pmid": "19056821",
   "doi": "10.1093/nar/gkn952",
   "title": "Novel mutations in Moloney Murine Leukemia Virus reverse transcriptase increase thermostability through tighter binding to template-primer",
   "journal": "Nucleic Acids Research",
   "year": 2009,
   "quote": "Five mutations E69K, E302R, W313F, L435G and N454K ... increase the half-life of MMLV RT at 55°C from less than 5 min to approximately 30 min.",
   "kind": "peer-reviewed",
   "url": "https://pubmed.ncbi.nlm.nih.gov/19056821/"
  },
  "baranauskas2012": {
   "pmid": "22691702",
   "doi": "10.1093/protein/gzs034",
   "title": "Generation and characterization of new highly thermostable and processive M-MuLV reverse transcriptase variants",
   "journal": "Protein Engineering, Design and Selection",
   "year": 2012,
   "quote": "Combining D200N, L603W, T330P, L139P and E607K ... processivity ... 1500 nt (65-fold improvement) ... full-length 7.5-kb cDNA synthesis was raised to 62°C.",
   "kind": "peer-reviewed",
   "url": "https://pubmed.ncbi.nlm.nih.gov/22691702/"
  },
  "rnaseh1989": {
   "pmid": "2464706",
   "doi": "10.1128/jvi.63.3.1460-1464.1989",
   "title": "Inhibition of RNase H activity and viral replication by single mutations in the 3-prime region of Moloney murine leukemia virus reverse transcriptase",
   "journal": "Journal of Virology",
   "year": 1989,
   "quote": "Substitution of either of two conserved aspartic acid residues ... depressed RT-associated RNase H activity by more than 25-fold with little or no effect on polymerase activity.",
   "kind": "peer-reviewed",
   "url": "https://pubmed.ncbi.nlm.nih.gov/2464706/"
  },
  "rnaseh2002": {
   "pmid": "12136094",
   "doi": "10.1093/nar/gkf417",
   "title": "The role of template-primer in protection of reverse transcriptase from thermal inactivation",
   "journal": "Nucleic Acids Research",
   "year": 2002,
   "quote": "RNase H-minus RTs retained the ability to efficiently synthesize cDNA at much higher temperatures.",
   "kind": "peer-reviewed",
   "url": "https://pubmed.ncbi.nlm.nih.gov/12136094/"
  },
  "mmulv2021": {
   "pmid": "34900141",
   "doi": "10.1016/j.csbj.2021.11.030",
   "title": "M-MuLV reverse transcriptase: Selected properties and improved mutants",
   "journal": "Computational and Structural Biotechnology Journal",
   "year": 2021,
   "quote": "Superscript III harbors mutations H204R, T306K, F309N, V223H, D524G, E562Q, and D583N.",
   "kind": "peer-reviewed",
   "url": "https://pubmed.ncbi.nlm.nih.gov/34900141/"
  },
  "rnaseh2014": {
   "pmid": "25450388",
   "doi": "10.1016/j.bbrc.2014.10.044",
   "title": "Amino acid substitutions away from the RNase H catalytic site increase the thermal stability of Moloney murine leukemia virus reverse transcriptase through RNase H inactivation",
   "journal": "Biochemical and Biophysical Research Communications",
   "year": 2014,
   "quote": "An RNase H-deficient MMLV RT carrying the RNase H-inactivating mutation D524A ... was devoid of RNase H activity.",
   "kind": "peer-reviewed",
   "url": "https://pubmed.ncbi.nlm.nih.gov/25450388/"
  },
  "pfu2009": {
   "pmid": "19783818",
   "doi": "10.1093/nar/gkp800",
   "title": "The 3'-5' proofreading exonuclease of archaeal family-B DNA polymerase hinders the copying of template strand deaminated bases",
   "journal": "Nucleic Acids Research",
   "year": 2009,
   "quote": "D215A point mutant ... disabled in 3-5 exonuclease activity.",
   "kind": "peer-reviewed",
   "url": "https://pubmed.ncbi.nlm.nih.gov/19783818/"
  },
  "pfu1996": {
   "pmid": "8836181",
   "doi": "10.1093/nar/24.18.3546",
   "title": "PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases",
   "journal": "Nucleic Acids Research",
   "year": 1996,
   "quote": "Pfu (1.3 x 10(-6)) < ... Taq (8.0 x 10(-6)); exo- Pfu approximately 5 x 10(-5).",
   "kind": "peer-reviewed",
   "url": "https://pubmed.ncbi.nlm.nih.gov/8836181/"
  },
  "bst2023": {
   "pmid": "34762799",
   "doi": "10.1021/acs.biochem.1c00451",
   "title": "Improved Bst DNA Polymerase Variants Derived via a Machine Learning Approach",
   "journal": "Biochemistry",
   "year": 2023,
   "quote": "Top predicted variants had greatly improved thermotolerance ... denaturation temperatures up to 2.5°C higher ... faster LAMP assays at 73°C.",
   "kind": "peer-reviewed",
   "url": "https://pubmed.ncbi.nlm.nih.gov/34762799/"
  },
  "bst32016": {
   "pmid": "28193014",
   "doi": "10.1021/acs.analchem.6b03460",
   "title": "Simple and Highly Sensitive Molecular Diagnosis of Zika Virus by Lateral Flow Assays",
   "journal": "Analytical Chemistry",
   "year": 2016,
   "quote": "Bst 3.0 polymerase ... robust and fast isothermal amplification performance even in the presence of high concentrations of inhibitors ... amplification of ZIKV RNA in pure water and human whole blood.",
   "kind": "peer-reviewed",
   "url": "https://pubmed.ncbi.nlm.nih.gov/28193014/"
  },
  "bst2025review": {
   "pmid": "41177494",
   "doi": "10.1016/j.ijbiomac.2025.148691",
   "title": "Engineering and enhancement of Bst DNA polymerase variants toward improved diagnostic applications: A review",
   "journal": "International Journal of Biological Macromolecules",
   "year": 2025,
   "quote": "Key mutation sites ... thermostability, strand displacement efficiency, fidelity, inhibitor tolerance, and nucleotide selectivity; fusion strategies ... salt tolerance.",
   "kind": "peer-reviewed",
   "url": "https://pubmed.ncbi.nlm.nih.gov/41177494/"
  },
  "inhib2008": {
   "pmid": "18755023",
   "doi": "10.1186/1756-0500-1-70",
   "title": "Differential susceptibility of PCR reactions to inhibitors: an important and unrecognised phenomenon",
   "journal": "BMC Research Notes",
   "year": 2008,
   "quote": "Susceptibility to inhibition was highly variable between reactions ... possible association with amplicon GC content.",
   "kind": "peer-reviewed",
   "url": "https://pubmed.ncbi.nlm.nih.gov/18755023/"
  },
  "urine1991": {
   "pmid": "1646235",
   "doi": "10.1136/jcp.44.5.360",
   "title": "Inhibitory effects of urine on the polymerase chain reaction for cytomegalovirus DNA",
   "journal": "Journal of Clinical Pathology",
   "year": 1991,
   "quote": "Urea was the major inhibitory component of urine and inhibited the PCR at a concentration of more than 50 mM.",
   "kind": "peer-reviewed",
   "url": "https://pubmed.ncbi.nlm.nih.gov/1646235/"
  },
  "ffpe2013": {
   "pmid": "24155973",
   "doi": "10.1371/journal.pone.0077771",
   "title": "Improved PCR performance using template DNA from formalin-fixed and paraffin-embedded tissues by overcoming PCR inhibition",
   "journal": "PLoS ONE",
   "year": 2013,
   "quote": "Poor PCR amplification was partly caused by inhibition of the DNA polymerase by fragmented DNA from FFPE tissue and not only due to the absence of intact template molecules.",
   "kind": "peer-reviewed",
   "url": "https://pubmed.ncbi.nlm.nih.gov/24155973/"
  },
  "stool1997": {
   "pmid": "9157172",
   "doi": "10.1128/jcm.35.4.995-998.1997",
   "title": "Complex polysaccharides as PCR inhibitors in feces: Helicobacter pylori model",
   "journal": "Journal of Clinical Microbiology",
   "year": 1997,
   "quote": "Inhibitors in feces are complex polysaccharides possibly originating from vegetable material in the diet.",
   "kind": "peer-reviewed",
   "url": "https://pubmed.ncbi.nlm.nih.gov/9157172/"
  },
  "bsa1996": {
   "pmid": "8975603",
   "doi": "10.1128/aem.62.3.1102-1106.1996",
   "title": "Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein",
   "journal": "Applied and Environmental Microbiology",
   "year": 1996,
   "quote": "BSA or gp32 relieved FeCl3, hemin, fulvic acids, humic acids, tannic acids, or extracts from feces; neither relieved bile salts, bilirubin, EDTA, NaCl, SDS, or Triton X-100.",
   "kind": "peer-reviewed",
   "url": "https://pubmed.ncbi.nlm.nih.gov/8975603/"
  },
  "swab2009": {
   "pmid": "19901277",
   "doi": "10.1177/104063870902100603",
   "title": "Removal of real-time reverse transcription polymerase chain reaction (RT-PCR) inhibitors associated with cloacal swab samples and tissues for improved diagnosis of Avian influenza virus by RT-PCR",
   "journal": "Journal of Veterinary Diagnostic Investigation",
   "year": 2009,
   "quote": "A modified protocol with an added high-salt wash of 2 M NaCl and 2 mM EDTA improved the ability to remove inhibitors from cloacal swabs.",
   "kind": "peer-reviewed",
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  },
  "igg2000": {
   "pmid": "10618113",
   "doi": "10.1128/jcm.38.1.345-350.2000",
   "title": "Identification and characterization of immunoglobulin G in blood as a major inhibitor of diagnostic PCR",
   "journal": "Journal of Clinical Microbiology",
   "year": 2000,
   "quote": "The major inhibitor in human plasma ... was found to be immunoglobulin G (IgG).",
   "kind": "peer-reviewed",
   "url": "https://pubmed.ncbi.nlm.nih.gov/10618113/"
  },
  "blood2001": {
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   "title": "Purification and characterization of PCR-inhibitory components in blood cells",
   "journal": "Journal of Clinical Microbiology",
   "year": 2001,
   "quote": "Hemoglobin and lactoferrin were identified as PCR-inhibitor components in erythrocytes and leukocytes, respectively.",
   "kind": "peer-reviewed",
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  },
  "nine1998": {
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   "quote": "AmpliTaq Gold and Taq were totally inhibited in 0.004% blood, while HotTub, Pwo, rTth, and Tfl amplified in 20% blood without reduced sensitivity.",
   "kind": "peer-reviewed",
   "url": "https://pubmed.ncbi.nlm.nih.gov/9758794/"
  },
  "tthphenol1994": {
   "pmid": "8136148",
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   "journal": "PCR Methods and Applications",
   "year": 1994,
   "quote": "Tth DNA polymerase maintained DNA- and RNA-dependent DNA polymerase activities in 2%-5% phenol-saturated PBS; Taq was inactive.",
   "kind": "peer-reviewed",
   "url": "https://pubmed.ncbi.nlm.nih.gov/8136148/"
  },
  "tthrt1991": {
   "pmid": "1714296",
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   "journal": "Biochemistry",
   "year": 1991,
   "quote": "Tth pol possesses efficient reverse transcriptase activity in the presence of MnCl2; greater than 100-fold more efficient in coupled RT/PCR than Taq pol.",
   "kind": "peer-reviewed",
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  },
  "direct2013": {
   "pmid": "24040090",
   "doi": "10.1371/journal.pone.0073845",
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   "kind": "peer-reviewed",
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  },
  "review2013": {
   "pmid": "23104280",
   "doi": "10.1007/978-1-60327-353-4_2",
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   "kind": "peer-reviewed",
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  },
  "cadwell1992": {
   "pmid": "1490172",
   "doi": "10.1101/gr.2.1.28",
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   "kind": "peer-reviewed",
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  },
  "stemmer1994": {
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   "quote": "DNA shuffling is a method for in vitro homologous recombination of pools of selected mutant genes by random fragmentation and PCR reassembly.",
   "kind": "peer-reviewed",
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  },
  "reetz2007": {
   "pmid": "17446890",
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   "quote": "Iterative cycles of saturation mutagenesis at rationally chosen sites ... reduces molecular biological work and screening effort.",
   "kind": "peer-reviewed",
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  },
  "reetz2006": {
   "pmid": "16411254",
   "doi": "10.1002/anie.200502746",
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   "year": 2006,
   "quote": "CASTing combines combinatorial active-site saturation mutagenesis with iterative screening.",
   "kind": "peer-reviewed",
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   "pmid": "11274352",
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  },
  "agresti2010": {
   "pmid": "20142500",
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   "quote": "Picoliter drops are screened at thousands per second; horseradish peroxidase mutants with catalytic rates >10-fold faster than parent were identified.",
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  },
  "esvelt2011": {
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   "quote": "Phage-assisted continuous evolution (PACE) transfers evolving genes from host cell to host cell ... dozens of rounds in a single day.",
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  "fowler2014": {
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  "hts2015": {
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  }
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