从冷冻芯的外层部分外源物质的去除探讨古代生物群落里面窝藏
Summary
冰雪圈提供了保存完好的生物，过去的环境条件下坚持访问。的协议，提出收集和净化土壤和冰的永冻层的核心。缺乏外源菌落和DNA的建议检测微生物所代表的材料，而不是从钻孔或加工污染。
Abstract
The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect conditions over vast time periods and investigation of biological materials harbored inside could provide insight of ancient environments. To appropriately analyze these ecosystems and extract meaningful biological information from frozen soils and ice, proper collection and processing of the frozen samples is necessary. This is especially critical for microbial and DNA analyses since the communities present may be so uniquely different from modern ones. Here, a protocol is presented to successfully collect and decontaminate frozen cores. Both the absence of the colonies used to dope the outer surface and exogenous DNA suggest that we successfully decontaminated the frozen cores and that the microorganisms detected were from the material, rather than contamination from drilling or processing the cores.
Introduction
冰雪圈（ 例如 ，永冻层土壤，冰功能，冰川积雪，积雪和冰）提供了一个窥探到过去的环境条件下坚持什么类型的生物。由于这些基材可以是几十到几十万岁，他们的微生物群落，因为沉积在冷冻保存，反映古环境条件。要正确分析这些生态系统和提取冷冻样品的冻土和冰块，正确的收集和处理有意义的生物信息是必要的。这是最重要的，因为气候预测为21 世纪指示北极和亚北极地区1有明显的变暖的潜力。具体来说，内政部阿拉斯加和格陵兰预期升温5℃左右，并分别在2100年2,3 7℃。预计这将影响显著土壤和水生微生物群落，因此，相关的生物地球化学过程。温暖的温度和降水改变政权有望开始在许多领域2-5冻土退化可能导致较厚，季节性解冻（活动）6,7层，冻土的解冻，和大量的冰体，如熔化地下冰，冰楔，和隔离冰8。这将极大地改变除了这些生态系统中的植物和动物的生物多样性生物地球化学属性。
冰川和永冻层同生沉积物和冰的功能已经被困化学和代表的环境中生活是什么在那里形成的功能时的生物学证据。例如，在内部阿拉斯加，既Illinoisan和威斯康星州的永久冻土岁都存在这个永久冻土尤其在本（YBP）包含IMPA的生物地球化学的证据之前，提供了从现代到15万年约会不同的位置对生物多样性过去气候变化克拉。其结果是，这些沉积物提供生物地球化学和生物多样性的记录了数千年。由于该区域具有低的沉积速率，并从来没有被冰川覆盖的，不受干扰的样本是收集和分析，无论是钻访问垂直插入隧道土壤剖面或钻孔水平。更重要的是，大量的记录存在的突出特别是在该地区永久冻土9-14的独特生物地球化学特征。具体来说，DNA分析的应用，同时估计现存的古冰和永冻土样本中的生物多样性的存在和程度使古环境条件特定生物联动和栖息地的职业探索。
以前的研究已经确定在哺乳动物，植物和可追溯至50K YBP 11，15-19样品微生物气候影响，虽然每个研究使用各色一nt的方法来收集和净化冻土和冰芯。在某些情况下，钻芯灭菌16，20-21，虽然具体的方法没有澄清是否外来核酸还从样品中消除。在其他研究中，细菌分离物15（ 例如 ， 粘质沙雷氏菌 ），以及22已被用于测量的净化程序的功效荧光微球。
这个实验是一个大型研究调查冻土样本可以追溯到大约40K YBP微生物群落的组成部分。研究这部分的具体目标是成功地净化冰和冻土内核。就我们所知，没有任何方法集成了使用而设计的，以消除从冷冻芯的外侧部分外来核酸和相关的核酸溶液。尽管这是一个事实，这些解决方案是commonlŸ用于净化实验室设备分子实验。
一旦芯进行了去污，使用由Griffiths等 23和TOWE 等人 24开发的协议提取基因组DNA，使用分光光度计进行定量，并用无菌，无DNA的水稀释至达到每反应20纳克。细菌的16S rRNA基因用引物331F及797R扩增和探针BacTaq 25和古16S rRNA基因用引物拱349F和拱806R和在下列条件下探测TM拱516F 26扩增：95℃600秒，随后45个循环95℃，30秒，57℃60秒，72℃，在40℃，30秒下进行25秒，最后延伸。所有的qPCR反应中重复进行。在20微升的反应体积包含20 ng的DNA，引物10μM的，探针的5微米，和10微升的qPCR反应混合物。 FO标准- [R采用基因组DNA分别假单胞菌荧光和嗜盐salinarum，细菌和古细菌定量PCR制备。两者中生长至对数期。平板计数进行了与DNA从培养物中分离。与一个的假设和每个基因组为H的16S rRNA基因的六份分光光度计基因组DNA进行定量salinarum和P.荧光 ，分别为27-28。细菌和古细菌的基因的拷贝数是基于标准曲线上计算出的，对数转换，以占治疗之间不等方差，并通过ANOVA评估。
群落的组合物通过使用流动池和桥扩增技术测序16S rRNA基因，并与“定量分析上市微生物生态学'（QIIME）29分析该社区确定。正向和反向读取被连接在一起，然后序列进行过滤，索引，并选择高品质的代表从头操作分类单元（OTU）通过序列比对分配有一个参考数据库。比对的序列进行了比较，对分类分配一个单独的参考数据库。一个门级OTU表的建立是为了确定一般群落组成。
Protocol
Representative Results
Discussion
冰雪圈提供了保存完好的生物，过去的环境条件下坚持访问。虽然恢复类群可能不是完整的历史社会，来自冰川和冻土样品的分析中回收可产生约选择时间段15-16宝贵的历史资料。举例来说，有意义的生物信息已经从冰研究在格陵兰冰盖考察20厌氧活性和冻土试验研究了碳循环过程的解冻33的结果绘制的，并已在白令陆桥16提供了深入了解真菌的多样性冻土。之前进…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was funded through the U.S. Army Engineer Research and Development Center, Basic Research Program Office. Permission for publishing this information has been granted by the Chief of Engineers.
Materials
| Auger | Snow, Ice, and Permafrost Research Establishment (SIPRE), Fairbanks, AK | N/A | |
| 70% Isopropanol | Walmart | 551116880 | |
| 95% Ethyl Alcohol (denatured) | Fisher Scientific, Pittsburgh, PA | A407-4 | |
| DNA decontamination solution, DNA Away | Molecular Bio-Products, Inc., San Diego, CA | 7010 | |
| RNase decontamination solution, RNase Away | Molecular Bio-Products, Inc., San Diego, CA | 7002 | |
| Light Duty Suits | Kimberly-Clark Professional, Roswell, GA | 10606 | |
| Nitrile Gloves | Fisher Scientific, Pittsburgh, PA | FFS-700 | |
| Antiviral Masks | Curad, Walgreens | CUR3845 | |
| Sterile Sample Bags | Nasco, Fort Atkinson, WI | B01445 | |
| Steel Microtome Blade | B-Sharp Microknife, Wake Forest, NC | N/A | |
| Metal Rack | Fabricated at CRREL, Hanover, NH | N/A | |
| Tray | Handy Paint Products, Chanhassen, MN | 7500-CC | |
| Aluminum Foil | Western Plastics, Temecula, CA | N/A | |
| 500 ml Bottle with 0.22 μm Filter | Corning, Corning, NY | 430513 | |
| Serratia marcescens | ATCC, Manassas, VA | 17991 | |
| Biosafety Hood | NuAire, Plymouth, MN | NU-425-400 | |
| Petri Dish | Fisher Scientific, Pittsburgh, PA | FB0875712 | |
| ATCC Agar 181- Tryptone | Acros Organics, NJ | 61184-5000 | |
| ATCC Agar 181- Glucose | Fisher Scientific, Pittsburgh, PA | BP381-500 | |
| ATCC Agar 181- Yeast Extract | Fisher Scientific, Pittsburgh, PA | BP1422-500 | |
| ATCC Agar 181- Dipotassium Phosphate | JT Baker, Phillipsburg, NJ | 3252-01 | |
| ATCC Agar 181- Agar | Difco, Sparks, MD | 214530 | |
| NanoDrop 2000 UV Vis Spectrophotometer | Thermo Fisher Scientific, Wilmington, DE | ||
| Lightcycler 480 System | Roche Molecular Systems, Inc., Indianapolis, IN | ||
| Halobacterium salinarum | American Type Culture Collection (ATCC), Manassas, VA | ||
| Pseudomonas fluorescens | American Type Culture Collection (ATCC), Manassas, VA | ||
| Microbial DNA Isolation Kit | MoBio Laboratories, Carlsbad, CA | 12224-50 | |
| Ear Protection | Elvex | EP-201 | |
| Hard Hat | N/A | N/A | |
| Kimwipes | Kimberly-Clark Professional, Roswell, GA | 34705 | |
| Glass Wool | Pyrex | 430330 | |
| Ruler | N/A | N/A | |
| Weighing Tin | Fisher Scientific, Pittsburgh, PA | 08-732-100 | |
| Sodium chloride | Sigma Aldrich, St Louis, MO | S-9625 | |
| Potassium chloride | JT Baker, Phillipsburg, NJ | 3040-04 | |
| Potassium phosphate, monobasic | JT Baker, Phillipsburg, NJ | 3246-01 | |
| Potassium phosphate, dibasic | JT Baker, Phillipsburg, NJ | 3252-01 | |
| Sodium phosphate dibasic, anhydrous | Fisher Scientific, Pittsburgh, PA | BP332-500 | |
| 50 ml Centrifuge Tubes | Corning, Corning, NY | 4558 | |
| 2 ml Microcentrifuge Tubes | MoBio Laboratories, Carlsbad, CA | 1200-250-T | |
| 2 ml Ceramic Bead Tubes (1.4 mm) | MoBio Laboratories, Carlsbad, CA | 13113-50 | |
| Scoopula | Thermo Fisher Scientific, Wilmington, DE | 1437520 | |
| Balance | Ohaus, Parsippany, NJ | E12130 | |
| Diethylpyrocarbonate (DEPC) | Sigma Aldrich, St Louis, MO | D5758 | |
| Hexadecyltrimethylammoniabromide (CTAB) | Acros Organics, NJ | 22716-5000 | |
| Polyethylene glycol 8000 | Sigma Aldrich, St Louis, MO | P5413-1KG | |
| Phenol-chloroform-isoamyl alcohol (25:24:1) (pH 8) | Fisher Scientific, Pittsburgh, PA | BP1752-400 | |
| Centrifuge | Eppendorf, Hauppauge, NY | 5417R | |
| Chloroform-isoamyl alcohol (24:1) | Sigma Aldrich, St Louis, MO | C0549-1PT | |
| TE Buffer | Ambion (Thermo Fisher), Wilmington, DE | AM9860 | |
| Pipets | Rainin, Woburn, MA | Pipet Lite XLS, 2, 10, 200, 1nd 1000ul pipets | |
| Pipet tips | Rainin, Woburn, MA | Rainin LTS presterilized, low retention, filtered tips, 10, 20, 200, 1000ul | |
| Vortexor | Scientific Industries, Bohemia, NY | G-560 | |
| Vortex Adaptor | MoBio Laboratories, Carlsbad, CA | 13000-V1 | |
| Clear Bottle | Corning, Corning, NY | C1395500 | |
| Amber Bottle | Corning, Corning, NY | C5135250 | |
| Bottle Top Filters, 0.22um | Corning, Corning, NY | 430513 | |
| 60 mL Syringe | Becton, Dickenson and Company, Franklin Lakes, NJ | BD 309653 | |
| Millex Syringe filters, 0.22 μm | EMD Millipore, Billerica, MA | SLGV033RB | |
| 70% Ethanol | Fisher Scientific, Pittsburgh, PA | BP2818-500 | diluted & filter sterilized |
| Isotemp 100 L Oven | Fisher Scientific, Pittsburgh, PA | 151030511 | |
| Cell Spreader | Fisher Scientific, Pittsburgh, PA | 08-100-10 | |
| Disposable Inoculating Loops | Fisher Scientific, Pittsburgh, PA | 22-363-602 |
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