과학 예술 아카데미 크로아티아와 물류 및 과학 지원을 위해 과학 베를린 - 브란 덴 부르크 아카데미, 및 ​​재정적 지원에 대한 최대 플랑크 협회의 대통령 혁신 기금 우리는 조언 M. 마이어 감사합니다. JMG는 NSF 국제 박사 교제 (우아즈 - 0754461)에 의해 지원되었다. Neandertal 1 (Feldhofer 1) FM865407, Neandertal 2 (Feldhofer 2) FM865408, Sidron 1253 FM865409, Vindija 33.25 FM865410, Mezmaiskaya 1 FM865411 : 시퀀스는 가입 번호와 에비 뉴클레오 티드 데이터베이스에 쌓인 거죠

이 방법에 보고된 연구에 사용된 <a href="http://www.sciencemag.org/cgi/content/full/325/5938/318">브릭스 외., 과학 325 (5938)가. 318-321 (2009)</a> . 시약이 필요합니다 : <ul><li> AmpliTaq 골드의 DNA 중합 효소 </li><li> 10X GeneAmp PCR 버퍼 II </li><li> MgCl 2 25mM </li><li> dNTP 혼합물, 25 MM 각 </li><li> 물에 BSA, 10 MG ML - 1 </li><li> 분자 생물학 수준의 물 </li><li> EB 버퍼 (MinElute PCR의 정제 키트와 함께 제공된), 10 MM 트리스 HCL, pH를 8.5 </li><li> MinElute PCR의 정제 키트 </li><li> M - 270 streptavidin Dynabeads </li><li> 2X 바인딩 및 씻으 (BW) 버퍼 (2 M NaCl, 10 MM 트리스 - CL, 1 MM EDTA (에틸렌 다이아 민 테트라 초산), 산도 8.0, 0.2 % 트윈 20) </li><li> 1X 바인딩 및 씻으 (BW) 버퍼 (2X BindWash 버퍼 물에 배 희석) </li><li> 핫 와시 (HW) 버퍼 (2.5mM MgCl, 1X DNA 형성 촉매 골드 버퍼, 0.1 % 트윈 20) </li></ul> 비표준 시약 및 장비에 대한 제조 업체 정보는 나중에 테이블을 참조하십시오. 1) 도서관 증폭 <ol><li> 여기서 DNA 템플릿은 낮은 사본 번호의 DNA로 설명 소스 (Rohland 및 Hofreiter, 2007a, 2007b)와 (Maricic 및 Pääbo 2009)에서 준비 454 라이브러리이다. 전체 라이브러리를 증폭하려면 다음 PCR 믹스를 준비 : <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 시약 </td><td> 반응 당 μl </td></tr><tr><td> 물 </td><td> 45 </td></tr><tr><td> 10X 진 앰프 PCR 버퍼 II </td><td> 10 </td></tr><tr><td> MgCl 2 (25mM) </td><td> 10 </td></tr><tr><td> BSA (10 MG / ML) </td><td> 2 </td></tr><tr><td> dNTPs (25 MM 각) </td><td> 1 </td></tr><tr><td> 454 emPCR fwd primer/10uM </td><td> 3 </td></tr><tr><td> 454 emPCR의 RVS의 primer/10uM </td><td> 3 </td></tr><tr><td> AmpliTaq 골드의 DNA 중합 효소 (5 U / μl) </td><td> 1 </td></tr><tr><td> 454 DNA 라이브러리 템플릿 </td><td> 25 </td></tr><tr><td> 합계 </td><td> 100 </td></tr></tbody></table></li><li> 14주기의 증폭을위한 써모 자전거 타는 사람에 다음과 PCR 프로그램을 사용하여 <ol><li> 95 ° C 12min </li><li> 95 ° C 30 </li><li> 60 ° C (또는 원하는 어닐링 온도) 1 분 </li><li> 72 ° C 1 분 </li><li> 2 (X 13)로 이동 </li><li> 72 ° C 5 분 </li><li> 10 ° C ∞ </li></ol></li><li> 제조 업체의 지시에 따라 Qiagen MinElute의 스핀 컬럼을 통해 반응을 정화. 50 μl 버퍼 EB에 Elute. </li><li> 정화 증폭 제품뿐만 아니라 qPCR (메이어 외., 2007)와 unamplified 라이브러리의 나누어지는을 수량. 증폭 제품이 UL 당 이상의 1 X 10 12 사본이있는 경우가 아니라 2 개 이상 X 10 12 복사본 뇌관 확장 반응에 추가되는 것을 보장하기 위해 다음과 같은 프리머 확장 단계에서 템플릿의 양을 줄일 수 있습니다. </li></ol> 2) 프리머 확장 <ol><li> 반응의 필요한 번호에 대한 마스터 믹스를 준비합니다. <table border="0" cellspacing="0" cellpadding="0"><tbody><tr><td> 시약 </td><td> 반응 당 μl </td></tr><tr><td> 물 </td><td> 45 </td></tr><tr><td> 10X 진 앰프 PCR 버퍼 II </td><td> 10 </td></tr><tr><td> MgCl 2 (25mM) </td><td> 10 </td></tr><tr><td> BSA (10 MG / ML) </td><td> 2 </td></tr><tr><td> dNTPs (25 MM 각) </td><td> 1 </td></tr><tr><td> 454 emPCR fwd primer/10uM </td><td> 3 </td></tr><tr><td> 454 emPCR의 RVS의 primer/10uM </td><td> 3 </td></tr><tr><td> AmpliTaq 골드의 DNA 중합 효소 (5 U / μl) </td><td> 1 </td></tr><tr><td> 454 DNA 라이브러리 템플릿 </td><td> 25 </td></tr><tr><td> 합계 </td><td> 100 </td></tr></tbody></table></li><li> 단일 뇌관 확장 반응에 대한 Thermocycler에서 다음 프로그램을 실행 : <ol><li> 95 ° C 12min </li><li> 60 ° C (또는 다른 경우 PEC의 프라이머는 온도 어닐링) 1 분 </li><li> 72 ° C 5 분 </li><li> 72 ° C 영원히! 임계 단계 72에서 연장 후 반응을 유지 ° C. 열 블록 제거하기 전에 튜브에 QIAGEN MinElute 정화를위한 다음 직접 피펫 150ul PBI 또는 PB 버퍼. 이것은 어닐링이 아닌 구체적인 프라이머를 방지하고 혼합물은 냉각으로 캡처하는 것이 중요합니다. </li></ol></li><li> 제조 업체의 지시에 따라, Qiagen MinElute의 스핀 컬럼을 통해 반응을 정화. 50 μl EB에 Elute. </li></ol> 3) 확장 제품 캡처 <ol><li> vortexing하여 M - 270 구슬 Resuspend 주식 솔루션입니다. 샘플 당 25 μl 비드 정지를 가져가라. 샘플 당 25 μl 2xBW 버퍼에 500ul 배 BW 버퍼와 resuspend로 두 번 구슬을 씻으십시오. </li><li> 단계 3.1 25 μl 구슬 중지 단계 2.3 25 μl eluate를 추가합니다. 1 회전 후 믹스상온에서 5 분. 추천 : qPCR의 부량을위한 단계를 2.3에서 eluate 5 UL을 나머지 계속. </li><li> 신선한 1.5ml 튜브에 전체 혼합물을 전송합니다 (이 아닌 대상 라이브러리 조각 carryover을 줄이기 위해 도움). 펠렛 마그네틱 입자 수집기 (MPC)를 사용 뜨는 및 뜨는 폐기하십시오. </li><li> 500 μl 1xBW 버퍼 (많은 세척은 가능한 한 많은 배경으로 제거하는 데 도움이) 5 번 씻으십시오. 마지막 세척 단계 후, 잠시 스핀 다운 및 뜨는의 마지막 흔적을 제거합니다. </li><li> 500μl 1X 핫 와시 (HW) 버퍼를 추가합니다. 65 2 분 흔들 ° C (또는 PEC 뇌관 TM 위 5C) 열 블록에. 아래로 냉각 최소화하기 위해,이 후 신속하게 뜨는을 제거합니다. (이 단계는 아직 PEC primers 아니라 실제로 확장 단계 동안에 확장과 관련된 배경 조각을 제거하는 것입니다.) 뜨는의 마지막 흔적을 제거합니다. </li><li> 30 μl EB 버퍼에 비드 펠렛을 Resuspend. 신선한 1.5ml 튜브에 전체 혼합물을 전송합니다 (이 아닌 대상 라이브러리 조각 carryover을 줄이기 위해 도움). 용출을위한 열 자전거 타는 사람 3 분 95 ° C에서 알을 품다. MPC에서 개최하고 뒤에 구슬을 떠나 아끼 뜨는을 제거합니다. </li></ol> 4) 제품 증폭 캡처 <ol><li> 샘플 필요한 번호에 대한 PCR 마스터 믹스를 준비합니다. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 시약 </td><td> 반응 당 μl </td></tr><tr><td> 물 </td><td> 45 </td></tr><tr><td> 10X 진 앰프 PCR 버퍼 II </td><td> 10 </td></tr><tr><td> MgCl 2 (25mM) </td><td> 10 </td></tr><tr><td> BSA (10 MG / ML) </td><td> 2 </td></tr><tr><td> dNTPs (25 MM 각) </td><td> 1 </td></tr><tr><td> 454 emPCR fwd primer/10uM </td><td> 3 </td></tr><tr><td> 454 emPCR의 RVS의 primer/10uM </td><td> 3 </td></tr><tr><td> AmpliTaq 골드의 DNA 중합 효소 (5 U / μl) </td><td> 1 </td></tr><tr><td> Eluted 캡처 제품 </td><td> 25 </td></tr><tr><td> 합계 </td><td> 100 </td></tr></tbody></table></li><li> 14주기의 증폭을위한 써모 자전거 타는 사람에 다음과 PCR 프로그램을 사용 : <ol><li> 95 ° C 12min </li><li> 95 ° C 30 </li><li> 60 ° C (또는 원하는 어닐링 온도) 1 분 </li><li> 72 ° C 1 분 </li><li> 2 (X 13)로 이동 </li><li> 72 ° C 5 분 </li><li> 10 ° C ∞ </li></ol></li><li> 제조 업체의 지시에 따라 Qiagen MinElute 실리카 스핀 컬럼을 통해 반응을 정화. 50 μl EB 버퍼에 Elute. 이 제품은 현재 2.1 단계에서 시작, 또는 시퀀싱을위한 454 유제 PCR 프로토콜에 직접 입력, 캡쳐의 두 번째 라운드 모두에 대해 사용할 수 있습니다. </li></ol> 대표 결과 : 권장 정상으로 혼합입니다 - &#39;긍정적인 제어 대상 시퀀스&#39;(1 picogram 쉽게 충분 예 ~ 100bp PCR 제품)의 소량 처음 캡처 반응을 수행할 수있는 PEC 프로토콜로 밖으로 시작할 때 그것은 강력하게 권장합니다 의 금액은 (단계 2.1 참조) 454 라이브러리 템플릿을 증폭하고, 캡처가 긍정적인 제어 제품을 캡처하기위한 하나의 PEC의 입문서로 수행됩니다. 모두 제어 특정 긍정적이고 454 어댑터 특정 프라이머 쌍이 제어 반응이 수행되는 경우, qPCR은 정화 뇌관 확장 반응 (1.3), 뇌관 확장 제품 (2.3의 배경 비해 &#39;관리 대상&#39;의 양을 계량하는 데 사용할 수 있습니다 )와 eluted 비드 캡처 제품 (3.6). 이러한 방법으로 귀하의 실험 조건에서 배경 제거 ( &quot;특이성&quot;) 및 대상 복구 ( &quot;감도&quot;)의 효율성의 두 효과를 직접 측정할 수 있습니다. 성공 PEC 프로토콜은 일반적으로 다음과 같은 속성을 가지고 - 감도 (프로토콜에 의해 유지 관리 대상의 금액으로 측정) : eluted 비드 캡처 제품 제어 대상의 총 금액 (3.6) = ~ 뇌관 확장 반응 (2.3) 정화 총 금액의 10~20%. 배경 (454 emPCR의 프라이머 쌍에 의해 측정) : eluted 비드 캡처 제품 (3.6)에서 배경 총 금액 &lt;정화 뇌관 확장 반응 (2.3)에서 총 금액의 0.01 %. 최종 제품에 남아있는 대상의 1 % 미만이있다면, 뇌관 확장 단계가 실패되었을 수 있으며, 조사해야합니다. 최종 제품에 남아있는 배경의 0.01 %보다 훨씬 더있다면, 세탁 단계는 잘못 수행되었을 수 있으며, 조사해야합니다.

<ol>
	<li>Briggs, A. W., Good, J. M., Green, R. E., Krause, J., Maricic, T., Stenzel, U., Lalueza-Fox, C., Rudan, P., Brajkovic, D., Kucan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+retrieval+and+analysis+of+five+Neandertal+mtDNA+genomes.">Targeted retrieval and analysis of five Neandertal mtDNA genomes.</a> Science. 325 (5938), 318-321 (2009).</li><li>Rohland, N., Hofreiter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+and+optimization+of+ancient+DNA+extraction.">Comparison and optimization of ancient DNA extraction.</a> Biotechniques. 42 (3), 343-352 (2007).</li><li>Rohland, N., Hofreiter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ancient+DNA+extraction+from+bones+and+teeth.">Ancient DNA extraction from bones and teeth.</a> Nat Protoc. 2 (7), 1756-1762 (2007).</li><li>Maricic, T., P&#228;&#228;bo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+454+sequencing+library+preparation+from+small+amounts+of+DNA+permits+sequence+determination+of+both+DNA+strands.">Optimization of 454 sequencing library preparation from small amounts of DNA permits sequence determination of both DNA strands.</a> Biotechniques. 46 (1), 51-57 (2009).</li><li>Meyer, M., AW, B. r. i. g. g. s., Maricic, T., Hober, B., Hoffner, B., Krause, J., Weihmann, A., Paabo, S., Hofreiter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+micrograms+to+picograms:+quantitative+PCR+reduces+the+material+demands+of+high-throughput+sequencing.">From micrograms to picograms: quantitative PCR reduces the material demands of high-throughput sequencing.</a> Nucleic Acids Res. 36 (1), e5-e5 (2007).</li></ol>

The authors have nothing to disclose.

PEC 방법은 간단하고 빠르고, 민감하고 구체적인 것입니다. 따라서 이러한 RNA 라이브러리의 작은 RNA 조각의 캡쳐, 풀링 샘플 또는 metagenomic 샘플에서 16 (또는 다른 loci) 다양성을 캡처에 구조적 변화의 심문으로 고대 DNA 이외 우리는 저자의 관찰하다 여러 응용 프로그램. 언급 한 지점은 캡처의 감도가 다중 캡처 반응 증가 PEC primers의 번호로 낮은 될 것입니다. PEC 따라서 이상 (예 : megabase 이상) 매우 큰 캡쳐 영역의 포착에 적합 아니지만 아주 잘 신속한 방식으로 많은 사람의 작은 대상 지역 또는 SNP 위치의 포착에 적합합니다.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
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<tr>
<td>AmpliTaq Gold polymerase with GeneAmp PCR buffer II and MgCl2</td>
<td>&nbsp;</td>
<td>Applied Biosystems</td>
<td>N8080241</td>
<td>&nbsp;</td>
<tr>
<td>QIAGEN MinElute PCR purification kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>28004</td>
<td>&nbsp;</td>
<tr>
<td>M-270 streptavidin beads</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>653.05</td>
<td>&nbsp;</td>
<tr>
<td>Tween-20</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P2287</td>
<td>&nbsp;</td>
<tr>
<td>DynaMag-2 magnetic particle separator</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>120.02D</td>
<td>&nbsp;</td>		</tbody>
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primer extension capture: targeted sequence retrieval from heavily degraded dna sources

고대 유골의 전형이므로 우리는 크게 저하 및 미생물의 DNA로 오염되어 DNA 소스로부터 타겟 DNA 시퀀스 검색하는 방법을 제시. 방법은 크게 샘플 파괴 및 PCR이나 샷건 배열 접근 방식을 직접 상대 시퀀싱 요구 사항을 줄일 수 있습니다. 우리가 그들의 지리적인 범위에 걸쳐을 다섯 Neandertals의 전체 mitochondrial DNA (mtDNA) genomes를 재구성하기 위해이 메서드를 사용합니다. 후반 Neandertals의 mtDNA 유전 다양성은 현대 현대 인간의 그것보다 약 세 배 낮은되었다. 함께 mtDNA 단백질 진화의 분석과 함께, 이러한 데이터는 Neandertals의 장기적 효과적인 인구의 크기는 현대 인간과 무언가 큰 원숭이의보다 작은 것을 권장합니다.

Watch this Scientific Journal Video about Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources at JoVE.com

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

우리는 우리가 5 Neandertal 개인의 전체 mitochondrial genomes을 재구성하는 데 사용되는 타겟으로 고대 DNA 시퀀스 검색의 방법을 제시한다. 현재 일 인간과 함께 이러한 시퀀스 비교 Neandertals가 장기 낮은 효과적인 인구의 크기 있다고 제안합니다.

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 ...

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JoVE Journal

Biology

20.0K 조회수.  Max-Planck Institute for Evolutionary Anthropology, Leipzig. 우리는 우리가 5 Neandertal 개인의 전체 mitochondrial genomes을 재구성하는 데 사용되는 타겟으로 고대 DNA 시퀀스 검색의 방법을 제시한다. 현재 일 인간과 함께 이러한 시퀀스 비교 Neandertals가 장기 낮은 효과적인 인구의 크기 있다고 제안합니다.

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

Retrieval of Mouse Oocytes

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus Genetic Stock Center (http://stkctr.biol.sc.edu), we are characterizing the salient differences needed to develop this system.  A primary goal has been to optimize oocyte/early embryo retrieval.

This protocol illustrates the technique for extracting oocytes or early-stage fertilized embryos from the oviduct of mice. The ability to identify the infindibulum and insert a blunt end needle into it is essential to correctly performing the procedure.

마우스 Oocytes의 검색

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus ...

연구

생물학

Proboscis Extension Response (PER) Assay in Drosophila

Proboscis extension response (PER) is a taste behavior assay that has been used in flies as well as in honeybees.
On the surface of the fly's mouth (labellum), there are hair-like structures called sensilla which houses taste neurons. When an attractive substance makes contact to the labellum, the fly extends its proboscis to consume the material. Proboscis Extension Response (PER) assay measures this taste behavior response, and it is a useful method to learn about food preferences in a single fly. Solutions of various sugars, such as sucrose, glucose and fructose, are very attractive to the fly. The effect of aversive substances can also be tested as reduction of PER when mixed in a sweet solution.Despite the simplicity of the basic procedure, there are many things that can prevent it from working. One of the factors that requires attention is the fly's responsive state. The required starvation time to bring the fly to the proper responsive state varies drastically from 36 to 72 hours. We established a series of controls to evaluate the fly's state and which allows screening out of non-responsive or hyper-responsive individual animals. Another important factor is the impact level and the position of the contact to the labellum, which would be difficult to describe by words. This video presentation demonstrates all these together with several other improvements that would increase the reproducibility of this method.

Proboscis Extension Response PER Assay in Drosophila

Proboscis extension response or PER is a taste behavior assay that has been used in flies as well as in honeybees. When the proboscis makes contact with an attractive substance, the fly extends its proboscis to consume the substance. Solutions of various sugars are very attractive to the fly.

Drosophila의 코 확장 응답 (당) 어세이

Proboscis extension response (PER) is a taste behavior assay that has been used in flies as well as in honeybees.
On the surface of the fly's mouth ...

Isolation of Genomic DNA from Mouse Tails

마우스 뒷면에서 게놈 DNA의 분리

Primer-Free Aptamer Selection Using A Random DNA Library

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified through an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX) to recognize a wide variety of targets, from small molecules to proteins and other macromolecules 2-4. Aptamers have properties that are well suited for in vivo diagnostic and/or therapeutic applications: Besides good specificity and affinity, they are easily synthesized, survive more rigorous processing conditions, they are poorly immunogenic, and their relatively small size can result in facile penetration of tissues.
Aptamers that are identified through the standard SELEX process usually comprise ~80 nucleotides (nt), since they are typically selected from nucleic acid libraries with ~40 nt long randomized regions plus fixed primer sites of ~20 nt on each side. The fixed primer sequences thus can comprise nearly ~50% of the library sequences, and therefore may positively or negatively compromise identification of aptamers in the selection process 3, although bioinformatics approaches suggest that the fixed sequences do not contribute significantly to aptamer structure after selection 5. To address these potential problems, primer sequences have been blocked by complementary oligonucleotides or switched to different sequences midway during the rounds of SELEX 6, or they have been trimmed to 6-9 nt 7, 8. Wen and Gray 9 designed a primer-free genomic SELEX method, in which the primer sequences were completely removed from the library before selection and were then regenerated to allow amplification of the selected genomic fragments. However, to employ the technique, a unique genomic library has to be constructed, which possesses limited diversity, and regeneration after rounds of selection relies on a linear reamplification step. Alternatively, efforts to circumvent problems caused by fixed primer sequences using high efficiency partitioning are met with problems regarding PCR amplification 10.
We have developed a primer-free (PF) selection method that significantly simplifies SELEX procedures and effectively eliminates primer-interference problems 11, 12. The protocols work in a straightforward manner. The central random region of the library is purified without extraneous flanking sequences and is bound to a suitable target (for example to a purified protein or complex mixtures such as cell lines). Then the bound sequences are obtained, reunited with flanking sequences, and re-amplified to generate selected sub-libraries. As an example, here we selected aptamers to S100B, a protein marker for melanoma. Binding assays showed Kd s in the 10-7 - 10-8 M range after a few rounds of selection, and we demonstrate that the aptamers function effectively in a sandwich binding format.

SELEX protocols comprise multiple rounds of selection, each of which require regeneration of bound ligands, which in turn require fixed primer sequences flanking the random library regions. These fixed primer sequences can interfere with the selection process (false positives and negatives). Here we present a primer-free protocol.

랜덤 DNA 라이브러리를 사용하여 뇌관 - 무료 Aptamer 선택

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified ...

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 ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

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 expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

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 ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

비례 생체 분자 비콘을 사용하여 살아있는 세포의 단일 엔지니어링 RNA 성적 증명서의 실시간 영상

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is achieved by reverse transcription of the RNA of interest using specific fluorescently labeled primers and subsequent analysis of the resulting cDNA fragments by denaturing polyacrylamide gel electrophoresis. Simultaneously, a traditional Sanger sequencing reaction is run on the gel to map the ends of the cDNA fragments to their exact corresponding bases. In contrast to 5&#39;-RACE (Rapid Amplification of cDNA Ends), where the product must be cloned and multiple candidates sequenced, the bulk of cDNA fragments generated by primer extension can be simultaneously detected in one gel run. In addition, the whole procedure (from reverse transcription to final analysis of the results) can be completed in one working day. By using fluorescently labeled primers, the use of hazardous radioactive isotope labeled reagents can be avoided and processing times are reduced as products can be detected during the electrophoresis procedure.
In the following protocol, we describe an in vivo fluorescent primer extension method to reliably and rapidly detect the 5&#39; ends of RNAs to deduce transcriptional starting points and RNA processing sites (e.g., by toxin-antitoxin system components) in S. aureus, E. coli and other bacteria.

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

We here describe a fluorescence based primer extension method to determine transcriptional starting points from bacterial transcripts and RNA processing in vivo using an automated gel sequencer.

형광 기반 프라이머 확장 기술은 전사 시작 포인트 및 RNases의 쪼개짐 사이트를 확인하는<em&gt; 생체</em

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

DNA 메틸화 분석 차세대 시퀀싱 대상

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires precise identification of cells subpopulations from a heterogeneous tissue. Identification of cells of interest for LCM is usually based on morphological criteria or on fluorescent protein reporters. The combination of LCM and rapid immunolabeling offers an alternative and efficient means to visualize specific cell types and to isolate them from surrounding tissue. High-quality RNA can then be extracted from a pure cell population and further processed for downstream applications, including RNA-sequencing, microarray or qRT-PCR. This approach has been previously performed and briefly described in few publications. The goal of this article is to illustrate how to perform rapid immunolabeling of a cell population while keeping RNA integrity, and how to isolate these specific cells using LCM. Herein, we illustrated this multi-step procedure by immunolabeling and capturing dopaminergic cells in brain tissue from one-day-old mice. We highlight key critical steps that deserve special consideration. This protocol can be adapted to a variety of tissues and cells of interest. Researchers from different fields will likely benefit from the demonstration of this approach.

Gene expression analysis of a subset of cells in a specific tissue represents a major challenge. This article describes how to isolate high-quality total RNA from a specific cell population by combining a rapid immunolabeling method with laser capture microdissection.

셀 특정 부분 집단에서 RNA 분리 빠른 Immunolabeling과 결합 레이저 캡처 이외에 Microdissection을 사용하여

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires ...