l**********1
发帖数: 5204 | 1 LZ pls try go to
//www.sanger.ac.uk/genetics/CGP/cosmic/
or Cell article:
by
Nik-Zainal S et al. and Stratton MR (2012)
Mutational processes molding the genomes of 21 breast cancers.
Cell. 149: 979-93.
Abstract
All cancers carry somatic mutations. The patterns of mutation in cancer
genomes reflect the DNA damage and repair processes to which cancer cells
and their precursors have been exposed. To explore these mechanisms further,
we generated catalogs of somatic mutation from 21 breast cancer... 阅读全帖 |
|
E*****m
发帖数: 25615 | 2 查了下 wiki, 跟我想的差不多， mutation 有許多種
http://en.wikipedia.org/wiki/Mutation#Causes
Four classes of mutations are (1) spontaneous mutations (molecular decay), (
2) mutations due to error prone replication by-pass of naturally occurring
DNA damage (also called error prone translesion synthesis), (3) errors
introduced during DNA repair, and (4) induced mutations caused by mutagens.
Spontaneous mutation 是四種裡的一種， 沒人說只有 Spontaneous mutation 才能造
成evolution 吧？
如果教徒硬要說這些 mutation 都是神幹的， 那也可以， 就是沒證據罷了， 還是與
理論相... 阅读全帖 |
|
l*****a
发帖数: 38403 | 3 发信人: littletshirt (小仙鹤), 信区: TrustInJesus
标 题: Re: 从某些基督徒攻击进化论的行为看其信仰的虚妄 （原创）
发信站: BBS 未名空间站 (Tue Jul 12 21:54:02 2011, 美东)
thanks for the reply.
most carcinogenic mutations do not lead to new functional genes as we all
agree. But evolution from lower to higher life form need introduction of new
genes. I think even
with sequential mutations (instead of simultaneous multiple mutations), the
joint probability (having the sequential mutation & favorable
environments) is no better than simultaneou... 阅读全帖 |
|
m*********D
发帖数: 1727 | 4 1. Crispr载体选择， px330, px335(nickase), px459(puro+), px462(nickase,
puro+)
Nickase的系列在做KO的时候效率会高些，不知道做knock-in mutation哪种效率更高？
你所说的selection marker是crispr载体上带的selection marker(eg.
Puro+), 还是donor上带的selection marker? 我准备用ssDNA做donor, 做点突变的
时候好像也不方便在donor上引入selection marker
**我用的459，主要是要它的puro-selection。这个是筛选transfected cells的。也可
以用带GFP的vector,作cell sorting. 我原文说的selection和这个无关，是指有HDR发
生后的筛选。是，这个selection marker是一个头疼事，我也不太熟悉。
2. 你是在donor 上引入酶切位点（通过silence mutation）, 然后PCR酶切鉴定吗？
*... 阅读全帖 |
|
E*****m
发帖数: 25615 | 5 如果一個人以為 random 就是 mutation， 那麼這人
讀到 random mutation， 應該理解成 mutation mutation。
大致這樣。 |
|
m*********D
发帖数: 1727 | 6 先道歉--我上面回答网友的havala的第二个问题时搞错了：
”2. 你是在donor 上引入酶切位点（通过silence mutation）, 然后PCR酶切鉴定
吗？
**是，PCR后梅切。但没有silence mutation.我需要只改变一个AA。“
我理解silence mutation错了。是，我在donor上通过silence mutation引入两个酶且
位点，然后用这两个酶作geno-typing的。大约是10M细胞，lipofectamine3000作
transfection,三天的puromycin sellection，除掉non-transfected的细胞。约有1%的
细胞能survive,也就是100，000细胞。我用mutant的phenotype作下一步筛选，就是
mutant在一种特殊medium里可以生长，但wild-type不能。这个筛选了三个礼拜后，大
约拿到100 colonies.这其中约25%为double-replacement，50%为single-replacement,
还有约25%在酶切上看不出来，也没有sequenci... 阅读全帖 |
|
j********x
发帖数: 2330 | 7 啥问题，mutate的应该有一个参数不mutate
不mutate的应该有个参数mutate？ |
|
a*****v
发帖数: 128 | 8 Who made such mutations before? Is that possible to make four base mutations
at same time?
Here is my mutated part, CagGcGA (the capital letters are mutated), on both
side I have already added about 15 bp and the Tm is calculated at 78. But I
did not get any PCR products.
Thank you in advance |
|
h****a
发帖数: 65 | 9 我是新手，正准备做crispr point mutant, 很多问题想请教
1. Crispr载体选择， px330, px335(nickase), px459(puro+), px462(nickase,
puro+)
Nickase的系列在做KO的时候效率会高些，不知道做knock-in mutation哪种效率更高？
你所说的selection marker是crispr载体上带的selection marker(eg.
Puro+), 还是donor上带的selection marker? 我准备用ssDNA做donor, 做点突变的
时候好像也不方便在donor上引入selection marker
2. 你是在donor 上引入酶切位点（通过silence mutation）, 然后PCR酶切鉴定吗？
3. 一般knock-in mutation挑克隆都挑一百个克隆以上，具体操作的时候，是不是
在大板上有单克隆后，转96孔板里面，然后duplicate， 其中一板用来做PCR鉴定？做
PCR鉴定的模板是96孔板里面的细胞提取的g... 阅读全帖 |
|
G***G
发帖数: 16778 | 10 from DNA-Seq, a mutation happens in gene BRAF.
what should we do in RNA-SEQ?
1) detect gene BRAF
2) detect mutated gene BRAF.
if we do the second, what do we need?
1) normal transcript file
2) customized transcript file for mutated gene BRAF.
if we use 1, we can identify the gene BRAF, but can we say we find mutated
gene BRAF? |
|
e*******n
发帖数: 872 | 11 【 以下文字转载自 NextGeneration 讨论区 】
发信人: endaimien (endaimien), 信区: NextGeneration
标 题: 求助：一年内流产两次，MTHFR C677T和A1298C基因有mutation
发信站: BBS 未名空间站 (Tue Jan 13 06:51:33 2015, 美东)
每次都是2个月的时候流的，症状是先出血，然后胎心停了
流产后作了各种检查，发现MTHFR C677T和MTHFRA1298C基因有mutation
请问这两个基因的mutation是造成流产的原因吗？如果是应该如何治疗？有相似的情况
吗？ |
|
j*******2
发帖数: 309 | 12 流产后做了整套凝血的检查，查出是two copies of C677T mutation in MTHFR gene.
这种genotype会引起increased homocystein level，a risk factor for venous
thrombosis.但我的homocystein是正常的，所以ob不认为C677T mutation是引起流产的
原因。
想问一下，单纯C677T mutation 会造成流产吗？ |
|
m******t
发帖数: 109 | 13 i am going to do mutations. kit and primers are ready. however, still have
questions. please help.
1. i got 2 PAIRS of primers (sense and anti-sense for each pair). when i
read the manual, i found that for the control, they used 3 primers for 3
mutations, one single primer for one mutation. so i am confused now. should
i use pairs of primers or just sense or anti-sense primer?
2 may i use common SOC medium or i have to use NZY+ both for XL10 competent
cells?
any other suggestions are greatly a |
|
h********n
发帖数: 4079 | 14 请教一个Cre-LoxP 的问题.
I have a construct with a pair of LoxP sites, which I need to make mutation/
deletion so the LoxP sites do not work upon Cre. What is the minical
required mutation to let LoxP not work? And should I make mutation on both
LoxP site or just one?
谢谢谢谢. |
|
A***i
发帖数: 50 | 15 Sorry that I cannot type Chinese on this computer.
I have an ion channel gene mutation which was found in an atrial
fibrillation patient. I made the mutated DNA. And in western blot experiment
, the protein level dramatically reduced. In transfected culture cells,
there was no current recorded for the ion using patch clamp.
I wonder what else I can do to make a story out of the mutation. Your help
is highly appreciated! |
|
n********k
发帖数: 2818 | 16 shall be an easy job---I would use fusion PCR---less sequencing and always
works...order two overlapping primers with the mutations in the middle, just
make sure there are at least 16-20bp on the 3 primer side. The overlapping
between the two primers (for fusion) should be at least 16-20bp too. Your
primer should be about 6-12bp+CagGcGA+18-20bp about 30-40bp for both primers
. try to keep the amplicons short 100-500bp. Longer is ok but need more
sequencing and also could get more challenging...... 阅读全帖 |
|
m*********D
发帖数: 1727 | 17 有一个月了，还没有来得及seq.确认。但酶切pattern表明了我拿到了。
本来以为可以发篇方法文章的。但Biotechniques已经出来了。作者里还有认识的老朋
友呢。方法上我们基本是同一个思路和设计，他们的统计数据和我的很接近。他们没有
作point mutation,就是方法上证明可行。基本上，没有selection marker, 拿到
replacement的概率很低。我的统计数据就是transfected cell里面，单copy
replacement的概率是千分之一左右，双copy replacement概率更低。
刚劝说了一个教授放弃他们的point mutation计划（一个博士后的project)。没有
selection marker，基本上指望不大。
生物人太多，竞争太强了。我要不是这个mutation有别的用途，我这几个月时间就是白
花了。
谢谢上次给我出主意的人。我现在也算CRISPR的“专家”了。：）。 |
|
t*******w
发帖数: 107 | 18 https://www.ncbi.nlm.nih.gov/pubmed/28557981/#comments
FYI all.
Xiaolin Wu2017 May 31 2:11 p.m. (6 days ago) 9 of 9 people found this
helpful
This paper raises important safety issue for gene therapy application of
CRISPR-Cas9. However, there are serious doubts about the results or
interpretation. First of all, the authors listed Top-10 predicted off-target
sites. But all genes are wrong! looking at the sequence they listed (supp.
figure 3), you will not be able to find it in the genes! After ca... 阅读全帖 |
|
w*********s
发帖数: 2136 | 19 【 以下文字转载自 Medicine 讨论区 】
发信人: whiteclouds (/ 参考消息 /), 信区: Medicine
标 题: Polio surge in Nigeria after vaccine virus mutates
发信站: BBS 未名空间站 (Sat Aug 15 00:09:33 2009, 美东)
Polio surge in Nigeria after vaccine virus mutates
By MARIA CHENG, Associated Press Medical Writer Maria Cheng,
– Fri Aug 14, 9:36 am ET
LONDON – Polio, the dreaded paralyzing disease stamped out in the
industrialized world, is spreading in Nigeria. And health officials say in
some cases, it's caused by the vaccine used to fight |
|
d******0
发帖数: 1598 | 20 稍微有点生物学基础的同学都知道what the hell's mutation。
一点没有基础的同学可以去google扫盲。
anyway,许多时候，只要DNA序列上一个碱基发生mutation，整个基因就有可能twisted
或彻底完蛋。
在语言上也有类似的情况。
比如说：
卖火柴的小女孩（正常序列，安徒生）
卖女孩的小火柴（突变序列，porno）
再比如说：
人中吕布，马中赤兔（正常序列，三国）
人中赤兔，马中吕布（突变序列，XXOOXXOOOOO）
再再比如说：
唇红齿白（正常序列，美女）
唇白齿红（突变序列，吸血鬼）
再再再比如说：
穷则独善其身，达则兼济天下（正常序列，儒家理想）
穷则独骟其身，达则鸡奸天下（突变序列，现实reality）
所以，大伙有没有类似的突变序列，拿出来共享一下？ |
|
j********x
发帖数: 2330 | 21 mutating：
def foo(dict_arg):
dict_arg.pop("xxx")
if dict_arg == yyy:
# do something
non-mutating:
def foo(dict_arg):
if DictEqualExcept(dict_arg, yyy, "xxx"):
# do something
哪种比较好，需要其他细节我可以补充 |
|
c*t
发帖数: 1063 | 22 一个dominant的老鼠，heterozygous breeding female根本不怀孕，所以无法得到
homozygous的老鼠。现在只能用heterozygote来找mutation。并且位点在比较大的
intro上，如何找这个mutation呢。。
大概图（exon 2 and 3 deletion）
exon1（70） （intron 60k） exon2（1800） exon3（400） （intron 20k） exon4
（140）。
欢迎各位前辈指教.
谢谢！！！ |
|
p*****c
发帖数: 116 | 23 Intron mutation 有什么意义吗？
最近找到一个intron的mutation, 不知道有什么意义没有？ any comment is welcome!
谢谢先！ |
|
a********k
发帖数: 2273 | 24 细胞株还怕intron mutation？！那只能说这个mutation太太太强大了 |
|
m*p
发帖数: 226 | 25 You saw point mutation and you also see lower translation of the gene. You
also mean this is a dominant negative mutation. Kind of confusing. Which is
or are responsible for the phenotype?
Which ion channel is affected? Put your gene to the whole ion channel story
and make a story. |
|
n***1
发帖数: 45 | 26 在测某个特定基因的mutation，请教怎么定义splicing site mutation，谢谢 |
|
j****x
发帖数: 1704 | 27 太懒啦...
From Wiki:
A splice site mutation is a genetic mutation that inserts or deletes a
number of nucleotides in the specific site at which splicing of an intron
takes place during the processing of precursor messenger RNA into mature
messenger RNA.The
abolishment of the splicing site results in one or more introns remaining in
mature mRNA and may lead to the production of aberrant proteins. |
|
n***1
发帖数: 45 | 28 谢谢，这个我当然知道啊，我是说在EXON的5＇和3＇端多远，哪些conserve sequence
的mutation算splicing site mutation, 看了半天文献也没看明白。。。
做遗传的请指教下
in |
|
o*****r
发帖数: 156 | 29 I suspect your primer is too short and the way you calculate Tm is not right
.
go to their website and use the online primer design tool.
I used to mutate 4-6 bases, and always worked (the primers are usually 40-50
bp).
mutations
both
I |
|
u*********1
发帖数: 2518 | 30 如果你不是做技术开发，就直接的PCR+sanger sequencing
尤其因为你的样本不多，而且只做一个基因（当然一个基因或许有multiple的exon）
如果你要做比如3000个sample，同时做40个基因，那这个方法肯定就不行了，
最先进的办法请看：
Multiplex Targeted Sequencing Identifies Recurrently Mutated Genes in Autism
Spectrum Disorders
http://www.sciencemag.org/content/338/6114/1619.abstract
当然未来whole exome测序的成本变得非常非常低的时候，这方法估计也要淘汰
btw，上面的science paper测了几千个样本的44个基因，最后也就发现6个recurrent
de novo mutated gene，也只能解释1%的sporadic ASD，所以我觉得disease genetics
还是任重道远 |
|
T****u
发帖数: 424 | 31 1)expressiion
GEO
Arrayexpress
NIH oncogenomics
oncomine
2)mutation:
COSMIC: Catalogue Of Somatic Mutations In Cancer
NIH TCGA
good luck |
|
x*****d
发帖数: 704 | 32 比较稳妥的是nomral tissue和cancer patient都用hg19做reference，然后在cancer
patients里面的mutation排除掉normal tissue里面的mutation |
|
L*D
发帖数: 3966 | 33 【 以下文字转载自 Medicine 讨论区 】
发信人: whiteclouds (/ 参考消息 /), 信区: Medicine
标 题: Polio surge in Nigeria after vaccine virus mutates
发信站: BBS 未名空间站 (Sat Aug 15 00:09:33 2009, 美东)
Polio surge in Nigeria after vaccine virus mutates
By MARIA CHENG, Associated Press Medical Writer Maria Cheng,
– Fri Aug 14, 9:36 am ET
LONDON – Polio, the dreaded paralyzing disease stamped out in the
industrialized world, is spreading in Nigeria. And health officials say in
some cases, it's caused by the vaccine used to fight |
|
w*********s
发帖数: 2136 | 34 【 以下文字转载自 WorldNews 讨论区 】
发信人: whiteclouds (/ 参考消息 /), 信区: WorldNews
标 题: Polio surge in Nigeria after vaccine virus mutates
发信站: BBS 未名空间站 (Sat Aug 15 00:11:29 2009, 美东)
Polio surge in Nigeria after vaccine virus mutates
By MARIA CHENG, Associated Press Medical Writer Maria Cheng,
– Fri Aug 14, 9:36 am ET
LONDON – Polio, the dreaded paralyzing disease stamped out in the
industrialized world, is spreading in Nigeria. And health officials say in
some cases, it's caused by the vaccine used to fig |
|
|
w*********s
发帖数: 2136 | 36 Polio surge in Nigeria after vaccine virus mutates
By MARIA CHENG, Associated Press Medical Writer Maria Cheng,
– Fri Aug 14, 9:36 am ET
LONDON – Polio, the dreaded paralyzing disease stamped out in the
industrialized world, is spreading in Nigeria. And health officials say in
some cases, it's caused by the vaccine used to fight it.
In July, the World Health Organization issued a warning that this vaccine-
spread virus might extend beyond Africa. So far, 124 Nigerian children have
been paralyzed |
|
w*********s
发帖数: 2136 | 37 Polio surge in Nigeria after vaccine virus mutates
By MARIA CHENG, Associated Press Medical Writer Maria Cheng,
– Fri Aug 14, 9:36 am ET
LONDON – Polio, the dreaded paralyzing disease stamped out in the
industrialized world, is spreading in Nigeria. And health officials say in
some cases, it's caused by the vaccine used to fight it.
In July, the World Health Organization issued a warning that this vaccine-
spread virus might extend beyond Africa. So far, 124 Nigerian children have
been paralyzed |
|
m******e
发帖数: 9 | 38 it seems i have the same problem with you, except my OB is testing my
homocystein today. but the OB said homocystein test is just for my own
health. C677T mutation itself will cause miscarriage. he prescribed me
forlic acid, baby asprin and preg vit. |
|
l**********t
发帖数: 5754 | 39 no. and not interested. thanks.
new
the
mutations |
|
x*a
发帖数: 4968 | 40 你不会是文科生吧？
这一贴内容在说 mutation 的分类。
那 spontaneous 的定义有讲么？ |
|
E*****m
发帖数: 25615 | 41
你閱讀哪裡學的？ 我有說我在講定義嗎？
wiki 上下一節就是講 spontaneous mutation 是啥意思，
你既然沒興趣， 吵啥？ |
|
m******t
发帖数: 109 | 42 i plan to construct a plasmid with 3 amino acid mutations. anyone with
experience? please help. i appreciate it very much..... |
|
k*******s
发帖数: 214 | 43 Contact me. We can finish multiple mutation in once cloning. Thanks |
|
m******t
发帖数: 109 | 44 2 questions: i was provided 2 pairs of primers for 3 mutations by the
stratagene program. 1' i should use the MULTI kit cause there are several
kits. 2' should i do 1 or 2 PCR? thx |
|
o**i
发帖数: 1165 | 45 你要想不让cre 去binding 还是不去recombine
你不让cre去work 你干嘛放loxP上去
mutation/
both |
|
M******t
发帖数: 555 | 46 要做个targeting construct,准备clone一个~15kb的bac片断到retrieval vector，如
何做我们要的那个point mutation最
有效？有经验的同修说说。非常感谢！尽量自己做了，帮老板省点。 |
|
p*****c
发帖数: 116 | 47 好像细胞株活的好好的？
跟intron mutation和谐共存呢。 |
|
p****p
发帖数: 3360 | 48 I think ss mutation potentially could also leads to alternative splicing and
therefore missing of exon?
in |
|
p****p
发帖数: 3360 | 49 广义地说，影响splicing的mutation都应该算吧。比如说polypyrimidine tract的突变
应该也算。
sequence |
|
l*****a
发帖数: 1431 | 50 do you have a cell line that carries the potential splice mutation? if yes,
do a RT-PCR. |
|
