m***e
发帖数: 482 | 1 DNA repair pathways:
-direct repair
e.g. thymine dimers (DNa photolyase),
DNA adducts caused by alkylating agents (methyltransferases)
-excision repair
base excision repair(BER)
mismatch repair(MMR)
nucleotide excision repair(NER)
-recombinational repair
DNA double-strand break repair: 2 distinct system in eukaryotic cells
1. homologous recombination system
2. non-homologous end-joining (NHEJ) system
en, DNA-dependent proptein kinase is required for normal double-strand break
rejoin |
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M****e
发帖数: 70 | 2 For such microarray analysis, typically the mRNA is reverse-
transcribed into complementary DNA (cDNA) and labeled with
fluorescence dye or radioisotope. and the single-stranded
cDNA is hybridized to the complementary strand that is already
spread on the matrix with known position. i have only used
microarray from Clontech. theoretically, the RT enzyme with
gene-specific primers can reverse-transcribe the mRNA into
same amount of cDNA. otherwise, you cannot make the comparison
quantitatively.
fo |
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c***y
发帖数: 615 | 3 I have read that protocol. Single stranded DNA is preferred. Mine is double-
stranded. So just wondering the efficiency
89818 |
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a****o
发帖数: 1786 | 4 if you use double strand DNA, it is more likely one or both of single strand
DNA may bind to your protein too. you can test this.
My titration meant to use lower concentration of your protein. |
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c******r
发帖数: 3778 | 5 what marker did you use? and what marker did they use?
if you used DNA marker which were double stranded, your single strand RNA should run faster than the marker of the same sized DNA.
so my guess is you used DNA markers, but they used RNA markers. |
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S*******m
发帖数: 34 | 6 I found the following paragraph on this webpage. I agree with him.
http://www.molecularstation.com/forum/microbiology-forum/20624-pfu-
polymerase-mixed-taq-polymerase.html
Pfu polymerase mixed with Taq polymerase
That won't work. Pfu is a polymerase. It's not capable of
proofreading without polymerizing. So, it won't follow Taq around and
act as a high fidelity proofreader. If you mix the two enzymes, you'll
have some strands of DNA that are high fidelity (which were synthesized
by Pfu), and oth... 阅读全帖 |
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g*********d
发帖数: 233 | 7 Genetic history of an archaic hominin group from Denisova Cave in
Siberia
David Reich, Richard E. Green, Martin Kircher, Johannes Krause,
Nick Patterson, Eric Y. Durand, Bence Viola, Adrian W. Briggs, Udo
Stenzel, Philip L. F. Johnson, Tomislav Maricic, Jeffrey M. Good,
Tomas Marques-Bonet, Can Alkan, Qiaomei Fu, Swapan Mallick, Heng
Li, Matthias Meyer, Evan E. Eichler, Mark Stoneking, Michael
Richards, Sahra Talamo, Michael V. Shunkov, Anatoli P. Derevi... 阅读全帖 |
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b****y
发帖数: 105 | 8 Neural network computation with DNA strand displacement cascades.
Qian L, Winfree E, Bruck J.
Nature. 2011 Jul 20;475(7356):368-72. doi: 10.1038/nature10262.
Scaling up digital circuit computation with DNA strand displacement
cascades.
Qian L, Winfree E.
Science. 2011 Jun 3;332(6034):1196-201. |
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v*****s
发帖数: 20290 | 9 【 以下文字转载自 Joke 讨论区 】
发信人: mutouhui (mutouhui), 信区: Joke
标 题: Re: 麻省科学家发现病毒克星“天龙”
发信站: BBS 未名空间站 (Sun Nov 13 19:08:54 2011, 美东)
http://web.mit.edu/press/2011/antiviral.html
New drug could cure nearly any viral infection
Researchers at MIT’s Lincoln Lab have developed technology that may someday
cure the common cold, influenza and other ailments.
CAMBRIDGE, Mass. — Most bacterial infections can be treated with
antibiotics such as penicillin, discovered decades ago. However, such drugs
are useless... 阅读全帖 |
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x**y
发帖数: 46 | 10 很有创意，只是还不够完善。看看其他的评论：
Broad-spectrum antiviral strategy
Peter Hare
Journal name:
Nature Biotechnology
Volume:
29,
Page:
885
Year published:
(2011)
DOI:
doi:10.1038/nbt.2014
Published online13 October 2011Most antiviral therapies are pathogen-
specific and likely to select for resistance if the virus can mutate the
drug target. Rider et al. couple the ability to detect long double-stranded
RNA (dsRNA)—a telltale indicator of viral infection in mammalian cells—
with procaspase-activated apoptosis ... 阅读全帖 |
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a******k
发帖数: 1190 | 11 计算机预测蛋白结构有三个level。
研究最早的是二级结构预测，就是给你一个蛋白序列，找出其中的alpha helix, beta strands
和loop区。最早的一些算法出现在上个世纪70年代。基本的idea很简单，从已有晶体结构中统计
helix, strands和loop区氨基酸的出现概率，然后根据这个进行预测。效果一般。后来出现的一些
基于贝叶斯、神经网络等等的算法考虑到了序列前后的相互影响，结果改善很多。现在二级结构预测
的准确度很高，大概80%左右的样子。说起来，二级结构预测可以算是机器学习在生物学中最早的成
功应用之一。也是bioinformatics最早热起来的方向之一。
三级结构预测就困难很多。从方法上大概可以分成三个类别：ab initio, homology modeling和
threading. ab initio预测基于力场，计算蛋白折叠构象的最小能量状态。大家都知道David
Baker是这方面的代表人物。要注意的是，Baker的ab initio预测可不是一个原子一个原子地搭
建，而是一个片断一个片断的搭建。用的力场也是low resolution的... 阅读全帖 |
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e****p
发帖数: 354 | 12 请教SSCDNA TO DS CDNA 除了OD260 跑胶看看 有没有其他办法， |
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A********2
发帖数: 107 | 16 Should be transcribed in the same direction as replication since they are
highly expressed genes |
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k*******3
发帖数: 1909 | 17 我指的是测genome的DNA啊， DNA不是双链吗？ 我想知道出来的read的哪条链的。 |
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l**********1
发帖数: 5204 | 20 OMG
LZ used degenerated primer for cDNA single strand synthesis
R U Seriously? an amateur molecular biologist LZ?
Please go to
//www.protocol-
online.org/prot/Molecular_Biology/RNA/Reverse_Transcription__RT____cDNA_Synthesis/index.html
RT reaction is also called first strand cDNA synthesis. Three types of primers can be used for RT reaction:
oligo (dT) primers, random (hexamer) primers and gene specific primers with each having its pros and cons.
or
/media.affymetrix.com/support/technical/usb... 阅读全帖 |
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l**********1
发帖数: 5204 | 21 oligo dT 没错 如target gene 只有一段很短的碱基序列(<120bp) 已知
而没有任何其上游5' 或下游3'的序列信息
只能用oligo dT qRTPCR 从poly A tail 揪出那条mRNA to single strand cDNA
then to double strand cDNA the 附加带酶切位点的各种adapter
再用那些人工加入的带确定碱基序列的 adapter 5' to 3' 对应的 反链 3' to 5' 的引物
PCR cycling
具体步骤请看图:
snap copied and pasted from
//www.ncbi.nlm.nih.gov/pmc/articles/PMC50225/ |
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f*******e
发帖数: 628 | 22 如果不扩增，很难保证能够重复多次的测某个特定分子的某个特定片段吧，这样就很难
判断错误是来源于测序错误或者 real 吧？
看了看好像不能像 PacBio 一样不停的轮回对同一个分子测序。Nanopore 的技术好像
至少用那个 exonuclease 的是测序同时也摧毁分子。Strand sequencing 也许还行，
不过很难保证这次测了之后还能再 capture 到同一个 strand 来测啊？
到最后 error 大概只能靠 coverage 来解决。
了。 |
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f*******e
发帖数: 628 | 23 一般的 sample amplification 是把一个单分子原始 DNA 在一个 bead （或者其他介
质）上扩增成序列一样的的足够多数量的分子。 这个过程产生的 error 很小，比如 <
0.1%, 可以忽略不计。说这个过程可以纠正 error, 是因为在后续 sequencing 的时候
是读取的 amplification 之后整体产生的信号，所以即使这个集合里面某些少量的分
子产生错误的信号, 也淹没在 noise 里，不影响集合读出的正确序列.
相比较之下，single molecule 的方法只读取一个原始分子的信息，比如 nanopore,
读错了就错了，没有这么一个纠正的机制，所以就只能靠大量的 coverage 来弥补。
根据 nanopore 的网站，他们用某种 dsDNA binding enzyme 在 nanopore 的端口来
unzip double strand, 所以最后通过 pore 读取的是 single stranded
trinucleotide。一个分子被测序之后不会回来反复被测。所以要靠测序的 coverage
来弥补错误的话，就需... 阅读全帖 |
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l**********1
发帖数: 5204 | 24 Proposal just:
Oxford nanopore based NGKOT Next Generation Knout Out Technique:
用某种 dsDNA topoisomerase single molecular level cut and relink dsDNA with
magnetic tweezers
NB: NGKOT 能BACbase 300KP 内任意地knoct out 比起20-35 kp Vector的片段内PCR knoct out还是效率高啊
最佳的是可以Knock in again within BACbase 300KP 内任意的 based Oxford nanopore platform.
References:
//www.ncbi.nlm.nih.gov/pubmed/21809210
//www.ncbi.nlm.nih.gov/pubmed/19377505
//en.wikipedia.org/wiki/Topoisomerase
//www.mitbbs.com/article_t1/Biology/31... 阅读全帖 |
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p*****e
发帖数: 332 | 27 我想主要原因是因为有些ORF是反向的原因，所以那种定义就不太准了，后来慢慢
的就不太用watson和Crick了，这篇review写的相对比较清楚了，总的感觉是，w
atson是通常写法中的上链（5‘-3’），Crick是下链（3'-5'），而通常上链是
用来做转录模板的,便被定义成了了antisense了。共同进步，hand!
http://www.biology-direct.com/content/6/1/7
Definition Watson Crick
Original cytosine-poor cytosine-rich
Compositional pyrimidine-rich purine-rich
Transcriptional antisense sense
Replicational lagging leading
Arbitrary this that
Database top/plus ... 阅读全帖 |
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k*******3
发帖数: 1909 | 28 thanks! I will read that review. |
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b******y
发帖数: 627 | 29 If affecting the folding of flanking regions is your only concern, just use
a flexible linker.
On the contrary, I would worry about folding if you literally link two
compact domains without any linker. For instance, domain A ends with a helix
and domain B starts with a beta-strand. If you create an A-B polypeptide,
it is conceivable the last helix of A induces the first beta-strand into a
helix and therefore destabilizes the core of domain B. |
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a****k
发帖数: 1130 | 30 哦，R2D2是fly里面的name
你没说具体要做什么，human system有个比较tricky的事情是human Ago2结合single-
strand RNA的能力很强，siRNA如果有一点single strand在里面，只用Ago2 itself就
可以看到activity。如果你用stringent的duplex siRNA，除了human Ago2，还需要
C3PO (Tanslin & Trax)。human system不需要Dicer-TRBP for siRNA loading
建议你可以看下这篇文章：http://www.ncbi.nlm.nih.gov/pubmed/21552258 |
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i**y
发帖数: 71 | 31 presumably not, especially if BER (single-strand break) is involved. the
plant demethylases have higher activity towards mCpG/mCpG than hemi-
methylated CpG, so it won't induce BER on both strands at the same time.
in animals, it is unclear. the problem now is that there is no good in vivo
system to study bona fide demethylation anymore, after the discovery of hmC,
fC and caC. |
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m***o
发帖数: 272 | 32 Is it a silly question or nobody know? |
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m***o
发帖数: 272 | 33 Thank you! that is very helpful! You are very smart! |
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G****a
发帖数: 10208 | 34 【 以下文字转载自 Missouri 讨论区 】
发信人: Geisha (朔风如解意 容易莫摧残), 信区: Missouri
标 题: 钱mm的文章都是ERIC的， ERIC让她当第一作者
发信站: BBS 未名空间站 (Sun Jan 27 19:16:10 2013, 美东)
http://www.qianlab.caltech.edu/Qian_CV.pdf
Refereed Publications (* co-first authors)
1. L. Qian, E. Winfree, and J. Bruck. Neural network computation with DNA
strand displacement cascades. Nature, 475:368-372, 2011.
News and Views: “DNA and the brain” by Anne Condon, Nature, 475:304-305.
Media coverage: msnbc News, CBS News, Discovery News, CNET News, D... 阅读全帖 |
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G***G
发帖数: 16778 | 35 negative strand 上的snp的起始位置和终止位置
相当于正带上的什么位置？
negative strand上的exon的起始位置和终止位置
相当于正带上的什么位置？ |
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p*****c
发帖数: 20445 | 36 Michael Rape, Ph.D.
University of California, Berkeley
Michael Rape’s love of science began in the basement of his parent’s house
, where he used to conduct rudimentary biochemistry experiments. Today, he
uses far more sophisticated methods to understand a complex process critical
to nearly all organisms: ubiquitylation. The term describes the attachment
of a regulatory protein called ubiquitin—named for its ubiquity—onto other
proteins. Ubiquitin tags communicate a wealth of information to the ... 阅读全帖 |
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k*****8
发帖数: 91 | 37 I do have ape on my laptop. However, when I tried to copy-paste the whole
double strand sequence into ape, it only remove part of the labels. There's
no way to get single strand sequence either. Is there something I was
missing from ape? Thanks |
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f*****f
发帖数: 195 | 38 mission sirna的话，不用annealing。只要订的是duplex就不用退火，公司都是退火好
的。除非是单独的single strand，我没听过哪个公司订购sirna还要自己退火。而且
duplex is more resistant to nuclease than single strand. |
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g****0
发帖数: 425 | 39 Can I get the abstract as well?
How do you comment on this report?
Nucleic Acids Res. 2013 Mar 1;41(5):e63. doi: 10.1093/nar/gks1446. Epub 2012
Dec 28.
Differential integrity of TALE nuclease genes following adenoviral and
lentiviral vector gene transfer into human cells.
Holkers M, Maggio I, Liu J, Janssen JM, Miselli F, Mussolino C, Recchia A,
Cathomen T, Gonçalves MA.
Source
Department of Molecular Cell Biology, Leiden University Medical Center,
Eithovenweg 20, 2333 ZC Leiden, The Nether... 阅读全帖 |
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D**********e
发帖数: 41 | 40 My understanding is in acidic pH, because RNA is single stranded, the bases
of RNA ionize with H+, and is soluble in aqueous phase. While DNA is double
stranded, it's base is not exposed, it's not soluble in either aqueous or
organic phase. In basic pH, the phosphate backbone of DNA form salt with Na+
, and become soluble in aqueous phase. |
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T*****e
发帖数: 247 | 41 http://www.nature.com/nature/journal/vaop/ncurrent/full/nature1
标题 DNA-guided DNA interference by a prokaryotic Argonaute
摘要
RNA interference is widely distributed in eukaryotes and has a variety of
functions, including antiviral defence and gene regulation1, 2. All RNA
interference pathways use small single-stranded RNA (ssRNA) molecules that
guide proteins of the Argonaute (Ago) family to complementary ssRNA targets:
RNA-guided RNA interference1, 2. The role of prokaryotic Ago variants has
r... 阅读全帖 |
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G***G
发帖数: 16778 | 42 I mapped a read into a gene.
The read falls into the range of starting and ending of the gene.
But the strand of the read is negative and the gene's strand is positive.
Can we say the read belongs to the gene?
How to annotate the read? |
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G***G
发帖数: 16778 | 43 two genes are from a to b in the same chromosome.
But one is from positive strand.
another is from negative strand.
What is relationship between the two genes?
Are they actually one gene or two different genes? |
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p*******i
发帖数: 449 | 44 最近在用CRISPR来删除一部分基因组，就在试验细胞系上成功地看到一次正确的删除。
其他目的细胞系尝试全部都是未删除。具体就不说了，只说一个问题：
我使用下列两个CRISPR来做double strand break. 两个CUT之间不想引入任何序列。然
后通过系列稀释和PCR筛选正确的删除细胞克隆。
CRISPR1 gRNA特异部分: XXXXXXXXXXXXXXXXXGT()TAA
CRISPR2 gRNA特异部分: XXXXXXXXXXXXXXXXXAG()TAA
具体序列保密，不方便透露。 两CRISPR相距400bp. 括弧是double strand break位点。
我发现重组修复后，如果精确剪切和连接，会产生一个完全相同的CRISPR位点。 如果
我做的是瞬转， 这个位点会不会再次被“CRISPR1"识别和剪切？ 如果如此，是不是大
大降低了我得到正确克隆的可能性？是不是可以解释为什么我重复4 5次，筛几百克隆
无一正确的原因？
请大家讨论指教，非常非常感谢！ |
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x********e
发帖数: 35261 | 45 就是在replication fork上同时复制的两条链，有一条一直是leading strand, 另一条
一直是lagging strand。如果新旧链的区分是在复制时区别开的，unidirectional
replication是一种可能机制。我只是打酱油的 lol |
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y***n
发帖数: 109 | 46 Although this prize is ”for mechanistic studies of DNA repair“, it is in
fact only single－strand damage. There is another big field of double-
stranded DNA damage repair, including non-homologous end jointing and
homologous recombination. It is so unfair. |
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h*********9
发帖数: 35 | 47 这些分分析定制的东西比较多，我还不知道那些软件可以，不过用R的话，半天就可以
搞定。
基本办法是把 ChIP－Seq peak region 和 methylated sites 都编码成 R GRange
objects， 然后用 R 提供的 operations on GRanges ， find and count overlaps。
如果要计算 differentially methylated sites, you can use beta-binomial
regression. If you want to identify differentially methylated regions,
hidden Markov model is a good option.
最近写了一个 regression hidden Markov model for methylation data, 还没来及测
试。
Below is some sample code:
library(GenomicRanges)
library(GenomicFeatures)
library(da... 阅读全帖 |
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l*****7
发帖数: 8463 | 48 主导，控制和利益的对决:
韩春雨的阿狗,
不管结局如何，
都已牵涉到了各方,各路大小神仙们的利益,
决不是什么小事
References:
1
CRISPR/Cas9 - Academics fight over patent rights to an important technology
Stephanie Wroe
Two groups of academic researchers are battling in various jurisdictions
around the world to secure patent rights to a revolutionary gene-editing
technology. For the reasons discussed below, it is possible that there could
be a different winner in different jurisdictions. One group is lead by
Professor Doudna (University of California)... 阅读全帖 |
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c********n
发帖数: 225 | 49 【记录历史】谁重复验证了NgAgo？
项目 结题
date： 2017年8月2日
updated: 2017年8月4日
Note2017080401: added a few notable reviews/summaries
---------------------------------------
- NBT retraction note：
http://www.nature.com/nbt/journal/v34/n7/full/nbt.3547.html#correction2
原文引用
“Retracted online 02 August 2017
We are retracting our study because of the continued inability of the
research community to replicate the key results in Figure 4, using the
protocols provided in our paper. In this figure we report that the
Natro... 阅读全帖 |
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c********n
发帖数: 225 | 50 【记录历史】NgAgo'2016 - Milestones draft 第一阶段时间点补充：
（added on 2016年8月30日）
由于这则信息和以后第二阶段里的Addgene updated protocol有些相似之处
为了承前启后，也一并收入 第一阶段的milestones
Date：2016年06月28日
(NBT paper 上线近两个月以后）
(方舟子第二篇文章发表两天之前）
Title：please transfer to Biology. RE:Ng-Ago 重复
（发布于WaterWorld版）
Link:
http://www.mitbbs.com/article_t/WaterWorld/2680181.html
Key Points：
1. 提到3个关于optimize ss DNA “transformation tricks"
“Try these single stranded DNA transformation tricks.
1) transformation done at a low pH of ~6. Low pH significant... 阅读全帖 |
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