이 연구는 국립 보건원 (CH와 NS051630/MH076090/MH078972에 PJ에 GM071440)에 의해 부분적으로 지원되었다.

1. 게놈 DNA 조각 원하는 크기 범위로 sonication을 사용하여 조각 게놈의 DNA는 게놈 전체 시퀀싱 플랫폼에 가장 적합합니다. (우리는 보통 ~ 300 BP에 sonicate.) 1% 아가로 오스 겔 (그림 1)에 조각 게놈의 DNA의 크기 분포를 확인합니다. 2. DNA 준비 게놈 DNA의 5 HMC의 풍부한에 따라 시작 DNA 금액을 확인합니다. 5 HMC 수준이 다른 조직 유형에 크게 다르므로, 시작 DNA의 금액은 샘플 5 HMC 수준에 따라 달라집니다. 예제 표 1을 참조하십시오. 3. β-GT 촉매 반응 (포도당 전송 반응) <ol><li> 1 시간에 37 ° C 물 목욕의 표 2와 배양에 명시된 바와 같이 혼합물을 pipetting하여 섞는다. </li><li> 부화 후, DNA의 10 μg을 당 사용 QIAquick 염기 제거 키트와 반응을 정리열. 열 30 μl 물 Elute과 조화를 이루고 있습니다. </li></ol> 4. Biotinylation 반응 (클릭 화학) <ol><li> 150 μm의 최종 농도 (즉, 30 μl DNA 솔루션에 따라 솔루션을 작업 5 μl)에 용출 DNA 솔루션 (3 단계에서) DBCO-SS-PEG3 - 비오틴 공액 작업 솔루션 (1 MM)를 추가합니다. </li><li> 2 시간에 37 ° C 물 목욕에 pipetting 및 배양하여 섞는다. </li><li> QIAquick 염기 제거 키트와 반응을 정리. 이상적인 총 용출 볼륨은 100 μl입니다. </li><li> 마이크로 리터 규모 분광 광도계 (예를 들면., NanoDrop)를 사용하여 복구 DNA 금액을 수량화. </li></ol> 5. 5 HMC 함유 DNA의 캡처 <ol><li> 제조업체의 지시에 따라 1X B &amp; W 버퍼 1 ML과 Dynabeads MyOne Streptavidin C1의 50 μl 3 번 씻으십시오. 자기 스탠드로 구슬을 분리합니다. </li><li> 복구 biotinylated D에 배 B &amp; W 버퍼의 동등한 볼륨을 추가세척 구슬로 NA (100 UL). </li><li> 회선 근의 부드러운 회전 실온에서 15 분에 품다. </li><li> 자기 스탠드로 구슬을 분리하고 1X B &amp; W 버퍼 1 ML과 구슬에게 3 번 씻는다. </li><li> 회선 근의 부드러운 회전 상온에서 2 시간에 신선하게 조리 된 50 ㎜의 DTT의 100 μl의 구슬을 잠복기하여 DNA를 Elute. </li><li> 자기 스탠드로 구슬을 분리합니다. DTT를 제거 할 수있는 제조 지침에 따라 마이크로 바이오 스핀 6 열로 eluent와 부하를 대기음. 대상 DNA는 이제 솔루션입니다. </li><li> EB 버퍼의 10 μl의 Qiagen PCR MinElute 정화 키트 및 elute의 DNA에 의해 이전 단계에서 용출 DNA를 정화. 농도가 20 NG / UL보다 높은 경우 큐빗 Fluorometer, 또는 NanoDrop를 사용하여 DNA를 정량화. DNA는 다운 스트림 게놈 전체 시퀀싱 도서관 준비 할 준비가되었습니다. </li></ol> 6. 대표 결과 품질 O 경우F 게놈 DNA가 높은, β-GT와 biotinylation 반응 후 일반적인 복구 수율은 ~ 60-70% 있습니다. 그러나, 캡처 효율은 샘플의 5 HMC 수준에 따라 서로 다른 조직 유형 상당히 다릅니다. 일반적으로, 뇌 게놈의 DNA에 대한 캡처 효율은 ~ 4-9%이며, 일부 극단적 인 경우에 효율은 12 %까지 도달 할 수 있습니다. ES 세포의 경우 평균 캡처 효율은 신경 줄기 세포에 대한 ~ 0​​.5 %로 대조적으로, ~ 2-4%입니다. 본 최저 효율은 지금까지 암 세포의 게놈 DNA에 대한했습니다. 모든 농축 DNA는 표준 차세대 도서관 준비 프로토콜 준비가되었습니다. 또한 캡처 한 DNA는 또한 관련 프리 머를 사용할 경우 입력 DNA에 비해 몇 조각의 강화를 감지하는 실시간 PCR을위한 템플릿으로 사용할 수 있습니다. <img alt="그림 1" src="/files/ftp_upload/4441/4441fig1.jpg" /> 1 그림. 에 Sonicated 인간 게놈의 DNA 조각1퍼센트 아가로 오스 겔. 1X TE 버퍼 120 μl 인간의 IPS 세포로부터 격리 게놈 DNA의 10 μg은 sonication 장치 (Covaris)를 사용하여 sonicated되었습니다. sonication 후, sonicated DNA의 2 μl은 sonicated DNA 조각의 크기를 비교하는 DNA 마커의 100 BP를 사용하여 1퍼센트 아가로 오스 겔에로드했습니다. <table border="1"><tbody><tr><td> 구성 요소 </td><td> 음량 </td><td> 최종 농도 </td></tr><tr><td> 물 </td><td> _ μl </td><td></td></tr><tr><td> 10 X β-GT 반응 버퍼 </td><td> 2 μl </td><td> 1 X </td></tr><tr><td> 최대 10 μg의 게놈의 DNA에 </td><td> _ μl </td><td> 최대 500 μl / NG </td></tr><tr><td> UDP-6-N 3-Glc (3 ㎜) </td><td> 0.67 μl </td><td> 100 μM </td></tr><tr><td> β-GT (40 μM) </td><td> 1 μl </td><td> 2 μM </td></tr><tr><td> 총 볼륨 </td><td> 20 μl </TD&gt; <td></td></tr></tbody></table> 조직 게놈의 DNA (높은 5 HMC 내용&gt; 0.1 %)의 경우 I) <table border="1"><tbody><tr><td> 구성 요소 </td><td> 음량 </td><td> 최종 농도 </td></tr><tr><td> 물 </td><td> _ μl </td><td></td></tr><tr><td> 10 X β-GT 반응 버퍼 </td><td> 10 μl </td><td> 1 X </td></tr><tr><td> 20 μg의 게놈의 DNA에 </td><td> _ μl </td><td> 최대 500 μl / NG </td></tr><tr><td> UDP-6-N3-Glc (3 ㎜) </td><td> 1.33 μl </td><td> 100 μM </td></tr><tr><td> β-GT (40 μM) </td><td> 2 μl </td><td> 2 μM </td></tr><tr><td> 총 볼륨 </td><td> 40 μl </td><td></td></tr></tbody></table> II) 줄기 세포 게놈의 DNA (평균 5 HMC 내용 ~ 0.05 %)의 경우 <table border="1"><tbody><tr><td> 구성 요소 </td><td> 음량 </td><td> 최종 농도 </td></tr><tr><td> 물 </td><td> _ μl </td><td></td></tr><tr><td> 10 X β-GT 반응 버퍼 </td><td> 10 μl </td><td> 1 X </td></tr><tr><td> 최대 50 μg의 게놈의 DNA에 </td><td> _ μl </td><td> 최대 500 μl / NG </td></tr><tr><td> UDP-6-N3-Glc (3 ㎜) </td><td> 3.33 μl </td><td> 100 μM </td></tr><tr><td> β-GT (40 μM) </td><td> 5 μl </td><td> 2 μM </td></tr><tr><td> 총 볼륨 </td><td> 100 μl </td><td></td></tr></tbody></table> III) 암 세포 게놈의 DNA (낮은 5 HMC 내용 ~ 0.01 % 용) 표 1. 입력 DNA와 선택 화학 라벨 방식에 의한 다양한 5 HMC 수준으로 샘플을 사용하여 라벨 반응 금액의 예. <table border="1"><tbody><tr><td> 견본 </td><td> 5 HMC 수준 </strong&gt; </td><td> 시작 DNA (μg) </td><td> 라벨 후 복구 (구슬로 입력) (μg) </td><td> 복구 수율 </td><td> 풀다운 DNA (NG) </td><td> 풀다운 얻을 수 </td></tr><tr><td> 성인 마우스 소뇌 </td><td> 0.4 % </td><td> 10 </td><td> 7.5 </td><td> 75% </td><td> 236 </td><td> 3.1 % </td></tr><tr><td> 출생 후의 일 7 마우스 소뇌 </td><td> 0.1 % </td><td> 11 </td><td> 9 </td><td> 82% </td><td> 140 </td><td> 1.6 % </td></tr><tr><td> 마우스 ES 세포 E14 </td><td> 0.05 % </td><td> 60 </td><td> 42 </td><td> 70% </td><td> 350 </td><td> 0.8 % </td></tr></tbody></table> 표 2. 마우스 뇌 조직 및 ES 세포의 대표 결과입니다.

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관심 없음 충돌이 선언 없습니다.

5 hydroxymethylcytosine (5 HMC)는 특정 포유류의 세포 유형에서 상당한 양의 최근 확인 epigenetic 수정 존재입니다. 여기에 제시된 방법은 5 HMC의 게놈 전체의 분포를 결정하기위한 것입니다. 우리는 5 HMC의 수산기 그룹에 azide 그룹을 포함하는 엔지니어링 포도당 잔기를 전송하는 β-glucosyltransferase T4 박테리오파지를 사용합니다. azide 그룹은 화학적으로 포유류의 genomes에 5 HMC 함유 DNA 조각의 감지, 연관성 강화, 그리고 배열 ​​비오틴을 수정할 수 있습니다. 이 프로토콜은 hMeDIP은 간단하지만, 그 고밀도 5 HMC 지역에 대한 강한 편견을 가지고 있으며 일반적으로 독립 풀에서 일관 결과를 제공, 5 HMC 항체 기반 hydroxymethylated DNA의 immunoprecipitation (hMeDIP-Seq) 13-15 이상의 장점이 있습니다 다운. 우리의 방법은 편견을 소개 할 것입니다. 가 우리 레이블의 관점에서 두 가지 문제는, 그러나,하고 저를 캡처thod. 첫 번째 문제는 라벨과 캡처의 특이성이 될 수 있습니다. 최근 보고서는 5 hydroxymethyluracil (5 hmU)는 또한 β-GT의 기판으로 사용할 수 있다고 표시하기 때문에, 허위 - 긍정적 인 신호는 5 HMC-relatedness 16 특이 현상 아르 강화 조각으로 이어지는 도입 될 수 있습니다. 높은 활성 5 hydroxymethyluracil-DNA의 glycosylases은 지속적으로 포유류의 게놈 17,18에 본질적으로 감지 5 hmU의 결과로이 DNA 손상을 제거하는 때문에이 문제는하지만 근거가 될 수 있습니다. 두 번째 문제는 라벨의 효율성과 캡처 수 있습니다. 5 HMC 수준은 뇌 조직의 0.5 %에서 MES 세포의 0.05 %와 암 세포의 0.01 %에 이르기까지, 다양한 조직과 세포에 크게 다르므로, 우리의 방법은 하류의 관점에서 모든 조직에 적용되는 것이 중요합니다 라벨 및 캡처 게놈 DNA의 응용 프로그램입니다. 우리의 경험은 여기에 설명 된 방법은 실제로 민감하고 구체적인 것을 보여줍니다충분한 조직들 또는 깊은 게놈 19-21에서 5 HMC의 분포를 매핑하는 순서와 같은 다운 스트림 응용 프로그램을위한 5 HMC 수준 중 하나 정량화 기반의 비교의 목적이 모든 조직을 분석 할 수 있습니다. 우리의 방법의 핵심은 게놈 DNA를 시작하는 적절한 양의 사용이다. 게놈 DNA 농도가 너무 낮은 경우, 라벨 효율과 특이성 그에 따라 줄어 듭니다. 농도가 25 20 반응 μl, 라벨링 및 캡처 효율성뿐만 아니라 특이성에 μl / NG,보다 낮을 경우 5 HMC의 풍부한이 뇌 조직에서 DNA에 충분한 높은하더라도, 우리의 경험을 바탕으로 크게 감소 될 것입니다. 낮은 풍부한 DNA 샘플의 경우 농도 요구 사항에 추가하여, DNA의 총 금액은 높고 특정 캡처 효율을 얻을 것이 중요합니다. 따라서, 비록 하나는 하류 세인트​​을위한 5 HMC 강화 DNA 조각의 25 NG가 필요합니다차세대 시퀀싱 플랫폼에서 사용 andard 라이브러리 생성 프로토콜은 뇌 조직에서 게놈 DNA의 시작 금액은 2 μg (그리고 이상적인 금액 5-10 μg입니다) 이상이어야한다. 시행 착오를 통해, 우리는 표 2에 설명 된 것처럼, 높은 라벨링 효율과 특이성를 얻을 변수 5 HMC의 abundances과 다른 조직에서 게놈 DNA의 시작 양을 최적화했습니다. 요약하면, 우리의 방법은 정확하게 게놈 5 HMC 레이블을 할 자격이 있으며, 특히 그 하류 차세대 시퀀싱의 assays에 대한 민감도와 특이성의 관점에서 성공적으로 프로토콜을 만드는 5 HMC 함유 조각을 캡처합니다.

<table border="1"><tbody><tr><td> 이름 </td><td> 회사 </td><td> # 카탈로그를 </td><td> 논평 </td></tr><tr><td></td><td></td><td></td><td> 시약 </td></tr><tr><td> 5M 나트륨 염화물 (NaCl) </td><td> Promega </td><td> V4221 </td><td></td></tr><tr><td> 0.5M pH8.0 Ethylenediaminetetraacetic 산 (EDTA (에틸렌 다이아 민 테트라 초산)) </td><td> Promega </td><td> V4231 </td><td></td></tr><tr><td> 1M Trizma베이스 (트리스) pH7.5 </td><td> Invitrogen </td><td> 15567-027) </td><td></td></tr><tr><td> HEPES 1M, pH7.4 </td><td> Invitrogen </td><td> 15,630 </td><td></td></tr><tr><td> 염화 마그네슘 (MgCl 2) 1M </td><td> Ambion </td><td> AM9530G </td><td></td></tr><tr><td> 디메틸 sulfoxide (DMSO) </td><td> 시그마 </td><td> D8418 </td><td></td></tr><tr><td> 십대 초반 20 </td><td> 피셔 BioReagents </td><td> BP337-100 </td><td></td></tr><tr><td> DBCO-SS-PEG3 - 비오틴 공액 </td><td> 화학 도구를 클릭합니다 </td><td> A112P3 </td><td></td></tr><tr><td> 1,4 - Dithiothreitol, ultrapure (DTT) Superpure </td><td> Invitrogen </td><td> 15508-013 </td><td></td></tr><tr><td> QIAquick 염기 제거 키트 </td><td> Qiagen </td><td> 28,304 </td><td></td></tr><tr><td> 마이크로 바이오 스핀 6 열 </td><td> 바이오 RAD </td><td> 732-6222 </td><td></td></tr><tr><td> Dynabeads MyOne </td><td> Invitrogen </td><td> 650-01 </td><td></td></tr><tr><td> Streptavidin C1 </td><td></td><td></td><td></td></tr><tr><td> Qiagen PCR MinElute 정화 키트 </td><td> Qiagen </td><td> 28,004 </td><td></td></tr><tr><td> UltraPure 아가로 오스 </td><td> Invitrogen </td><td> 16500500 </td><td></td></tr><tr><td> UDP-6-N 3 포도당 </td><td> 활성 Motif로 </td><td> 55,013 </td><td></td></tr><tr><td></td><td></td><td></td><td> 효소 </td></tr><tr><td> β-glucosyltransferase (β-GT) </td><td> 뉴 잉글랜드 Biolab </td><td> M0357 </td><td></td></tr><tr><td></td><td></td><td></td><td> 장비 </td></tr><tr><td> Sonication 장치 </td><td> Covaris </td><td></td><td></td></tr><tr><td> 데스크탑 원심 분리기 </td><td></td><td></td><td></td></tr><tr><td> 열탕 목욕 </td><td> 피셔 과학 </td><td></td><td></td></tr><tr><td> 젤 실행 장치 </td><td> 바이오 RAD </td><td></td><td></td></tr><tr><td> NanoDrop1000 </td><td> 열 과학 </td><td></td><td></td></tr><tr><td> Labquake 튜브 쉐이커 </td><td> Barnstead </td><td></td><td></td></tr><tr><td> Labquake 튜브 쉐이커 </td><td> Thermolyne </td><td></td><td></td></tr><tr><td> 자기 분리 스탠드 </td><td> Promega </td><td> Z5342 </td><td></td></tr><tr><td> 큐빗 2.0 Fluorometer </td><td> Invitrogen </td><td></td&gt; <td></td></tr><tr><td></td><td></td><td></td><td> 시약 설정 10 X β-GT 반응 버퍼 (500 MM의 HEPES 산도 7.9, 250 MM MgCl 2) 2 X 구속력과 (10 MM 트리스 산도 7.5, 1 ㎜ EDTA (에틸렌 다이아 민 테트라 초산), 2 M NaCl, 0.02 % 십대 초반 20) (B &amp; W) 버퍼를 세척 . </td></tr></tbody></table>

selective capture of 5-hydroxymethylcytosine from genomic dna

5 methylcytosine (5-MC)는 인간 게놈 DNA의 총 cytosines의 ~ 2-8%를 구성하고 유전자 발현, 게놈 무결성 유지, 부모의 각인, X-염색체 불 활성화, 규제 등의 생물학적 기능의 광범위한 영향을 개발, 노화, 암 1. 최근 산화 5 MC, 5 hydroxymethylcytosine (5 HMC)의 존재는 배아 줄기 (ES) 세포와 neuronal 세포 2-4, 특히 포유류의 세포에서 발견되었습니다. 5 HMC는 TET 가족 철 (II) / α-ketoglutarate에 따라 달라 dioxygenases 2, 3 5 - MC 촉매의 산화에 의해 생성됩니다. 5 HMC는 배아 줄기 (MES) 세포, 정상 조혈 및 malignancies, 그리고 접합자 개발이 5-10의 유지에 참여 제안합니다. 더 나은 5 HMC의 기능을 이해하기 위해서는 안정적이고 간단한 시퀀싱 시스템이 필수적입니다. 전통 bisulfite 배열은 5 - MC 11 일부터 5 HMC을 구별 할 수 없습니다 </sup&gt;. 5 HMC의 생물학을 해명하기 위해, 우리는 특별히 5 HMC에 12 포도당 잔기를 추가 박테리오파지 효소 활용, 라벨 및 5-HMC를 캡처 할 수있는 매우 효율적이고 선택적 화학 접근 방식을 개발했습니다. 여기 5 HMC의 선택적 화학 라벨에 대한 간단한 두 단계 절차를 설명합니다. 첫 번째 라벨 단계에서, 게놈 DNA의 5 HMC는에서 5 HMC에 6 azide 포도당를 전송하는 방식으로, β-GT, T4 박테리오파지에서 glucosyltransferase로 6 azide-포도당 촉매으로 표시됩니다 수정 cofactor, UDP-6-N3-Glc (6 N3UDPG). 두 번째 단계, biotinylation에서 이황화 비오틴의 링커는 클릭 화학으로 azide 그룹에 연결되어 있습니다. 두 단계는 게놈 지역에서 5 HMC의 풍부한 관계없이 라벨 매우 낮은 배경을 제공 완료로 이어지는, 매우 구체적이고 효율적이다. 5 HMC의 후 biotinylation, 5 HMC 함유 DNA 조각을 한 후 선택적으로 캡처되어밀도 독립적 인 방식으로 streptavidin 비즈를 사용합니다. 그 결과 5 HMC 강화 DNA 조각은 차세대 시퀀싱 등의 다운 스트림 분석을 위해 사용될 수있다. 우리의 선택 레이블 및 캡처 프로토콜은 다양한 / 변수 5 HMC의 abundances과 게놈 DNA의 소스에 해당 높은 감도를, 수여. 이 프로토콜의 주요 목적은 자사의 다운 스트림 응용 프로그램 (예., 차세대 시퀀싱은 게놈의 5 HMC 배포를지도)이지만, 단일 분자입니다 실시간 SMRT (DNA) 시퀀싱과 호환됩니다 5 HMC의 단일 기본 해상도 시퀀싱을 제공 할 수.

Watch this Scientific Journal Video about Selective Capture of 5-hydroxymethylcytosine from Genomic DNA at JoVE.com

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

설명이 쉽고 밀도에 독립적 인 강화를위한 비오틴 링커를 전송할 클릭 화학에 이어, 5 HMC에 azide - 포도당를 전송하는 β-glucosyltransferase (β-GT)를 사용하여 두 단계 라벨 프로세스입니다. 이 효율적이고 특정 라벨 방식은 차세대 시퀀싱을 통해 매우 낮은 배경과 높은 처리량 epigenomic 매핑과 5 HMC의 농축이 가능합니다.

게놈 DNA에서 도보로 5 hydroxymethylcytosine의 선택적 캡처

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

selective-capture-of-5-hydroxymethylcytosine-from-genomic-dna

Universidad San Sebastian

Research

JoVE Journal

Biology

11.9K 조회수.  Emory University School of Medicine. 설명이 쉽고 밀도에 독립적 인 강화를위한 비오틴 링커를 전송할 클릭 화학에 이어, 5 HMC에 azide - 포도당를 전송하는 β-glucosyltransferase (β-GT)를 사용하여 두 단계 라벨 프로세스입니다. 이 효율적이고 특정 라벨 방식은 차세대 시퀀싱을 통해 매우 낮은 배경과 높은 처리량 epigenomic 매핑과 5 HMC의 농축이 가능합니다.

비디오: Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

Isolation of Genomic DNA from Mouse Tails

마우스 뒷면에서 게놈 DNA의 분리

연구

생물학

Large Insert Environmental Genomic Library Production

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE <a href="http://www.epibio.com/item.asp?ID=385">http://www.epibio.com/item.asp?ID=385</a>). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.

Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system.

대형 삽입 환경 게놈 라이브러리 제작

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent ...

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

소일스 및 세디먼츠에서 높은 분자량의 게놈의 DNA의 추출

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ancient bones. The method greatly reduces sample destruction and sequencing demands relative to direct PCR or shotgun sequencing approaches. We used this method to reconstruct the complete mitochondrial DNA (mtDNA) genomes of five Neandertals from across their geographic range. The mtDNA genetic diversity of the late Neandertals was approximately three times lower than that of contemporary modern humans. Together with analyses of mtDNA protein evolution, these data suggest that the long-term effective population size of Neandertals was smaller than that of modern humans and extant great apes.

We present a method of targeted ancient DNA sequence retrieval, which we used to reconstruct the complete mitochondrial genomes of five Neandertal individuals. Comparison of these sequences with present day humans suggests that Neandertals had a long term low effective population size.

프라이머 확장 캡처 : 경비 저하 DNA 소스에서 타겟 시퀀스 불러오기

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ...

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be produced to contain any desired DNA or RNA sequence and can be prepared to include a wide variety of base and sugar modifications. Moreover, oligos can be designed to mimic specific nucleic acid alterations and thus, can serve as important tools to investigate effects of DNA damage and mechanisms of repair. We found that Thermo Scientific Dharmacon RNA-containing oligos with a length between 50 and 80 nucleotides can be particularly suitable to study, in vivo, functions and consequences of chromosomal RNA/DNA hybrids and of ribonucleotides embedded into DNA. RNA/DNA hybrids can readily form during DNA replication, repair and transcription, however, very little is known about the stability of RNA/DNA hybrids in cells and to which extent these hybrids can affect the genetic integrity of cells. RNA-containing oligos, therefore, represent a perfect vector to introduce ribonucleotides into chromosomal DNA and generate RNA/DNA hybrids of chosen length and base composition. Here we present the protocol for the incorporation of ribonucleotides into the genome of the eukaryotic model system yeast /Saccharomyces cerevisiae/. Yet, our lab has utilized Thermo Scientific Dharmacon RNA-containing oligos to generate RNA/DNA hybrids at the chromosomal level in different cell systems, from bacteria to human cells.

This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.

RNA 함유 Oligonucleotides를 사용하여 변환하여 게놈 DNA에서 RNA / DNA 하이브리드의 생성

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high purity DNA. Based on multi-well filtration for bacterial lysate clearance and DNA purification, this method creates a streamlined process for plasmid preparation. Filter plates containing silica-based media can easily be processed by vacuum filtration or centrifuge to yield appreciable quantities of plasmid DNA. Quantitative analyses determine the purified plasmid DNA is consistently of high quality with average OD260/280 ratios of 1.97. Overall, plasmid yields offer more pure DNA for downstream applications, such as sequencing and cloning. This streamlined method of using AcroPrep Advance Filter Plates allows for manual, semi-automated or fully-automated processing.

This protocol is a cost effective alternative for efficient parallel clarification and plasmid DNA purification from E. coli cultures. The AcroPrep Advance process starts with an optimized lysate clarification filter plate followed by purification on a high binding capacity DNA binding filter plate.

Prokaryotic 문화 플라스미드 DNA의 유선형 정화

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high ...

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova &amp; Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).
Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

We present a public computational web site for the analysis of genomic sequences. It detects DNA sequence patterns with various non-random nucleotide compositions. This resource also generates randomized sequences with diverse levels of complexity.

게놈 MRI - 게놈 DNA 시퀀스 내의 패턴을 학습에 대한 공개 자료

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

Barcoded 효모 게놈 라이브러리의 경쟁력 화면

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Extraction of High Molecular Weight DNA from Microbial Mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA2,3 during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes4,5,6.
The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 &mu;g of HMW DNA (35-50 kb) per gram of mat sample, with an A260/280 ratio of 1.6. Furthermore, amplification of 16S rRNA genes7 suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.

We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

미생물 매트에서 높은 분자량의 DNA의 추출

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction  of high-quality, high molecular weight ...

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any two individuals is greater than previously thought3. In contrast, array-based genotyping has failed to identify a significant contribution of common sequence variants to the phenotypic variability of common disease4,5. Taken together, these observations have led to the evolution of the Common Disease / Rare Variant hypothesis suggesting that the majority of the "missing heritability" in common and complex phenotypes is instead due to an individual's personal profile of rare or private DNA variants6-8. However, characterizing how rare variation impacts complex phenotypes requires the analysis of many affected individuals at many genomic loci, and is ideally compared to a similar survey in an unaffected cohort. Despite the sequencing power offered by today's platforms, a population-based survey of many genomic loci and the subsequent computational analysis required remains prohibitive for many investigators.
To address this need, we have developed a pooled sequencing approach1,9 and a novel software package1 for highly accurate rare variant detection from the resulting data. The ability to pool genomes from entire populations of affected individuals and survey the degree of genetic variation at multiple targeted regions in a single sequencing library provides excellent cost and time savings to traditional single-sample sequencing methodology. With a mean sequencing coverage per allele of 25-fold, our custom algorithm, SPLINTER, uses an internal variant calling control strategy to call insertions, deletions and substitutions up to four base pairs in length with high sensitivity and specificity from pools of up to 1 mutant allele in 500 individuals. Here we describe the method for preparing the pooled sequencing library followed by step-by-step instructions on how to use the SPLINTER package for pooled sequencing analysis (<a href="http://www.ibridgenetwork.org/wustl/splinter" target="_blank">http://www.ibridgenetwork.org/wustl/splinter</a>). We show a comparison between pooled sequencing of 947 individuals, all of whom also underwent genome-wide array, at over 20kb of sequencing per person. Concordance between genotyping of tagged and novel variants called in the pooled sample were excellent. This method can be easily scaled up to any number of genomic loci and any number of individuals. By incorporating the internal positive and negative amplicon controls at ratios that mimic the population under study, the algorithm can be calibrated for optimal performance. This strategy can also be modified for use with hybridization capture or individual-specific barcodes and can be applied to the sequencing of naturally heterogeneous samples, such as tumor DNA.

Pooled DNA sequencing is a fast and cost-effective strategy to detect rare variants associated with complex phenotypes in large cohorts. Here we describe the computational analysis of pooled, next-generation sequencing of 32 cancer-related genes using the SPLINTER software package. This method is scalable, and applicable to any phenotype of interest.

파편을 사용하여 풀링된 장면에서 드문 게놈 변종의 탐지

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any ...