Science topic

Mutation - Science topic

Mutation is an any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations.
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In banana, we are having multiple alleles. For distrusting the function of a gene through CRISPR/Cas9 it is important to mutate all the alleles through designing specific guideRNA repeats. How to precisely design guideRNAs that target all the alleles of a gene? If any, special tools is available , please specify.
Thanks
Kumaravel.M
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Your best option would be to align the sequences of all your alleles to look for conserved regions/domains and then design your gRNAs for those. For multiple sequence alignment I like to use MUSCLE but that is just my preference.
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Olen R.Brown & David A.Hullender published a paper in Progress in Biophysics and Molecular Biology journal in August 2022 with the name ( Neo-Darwinism must Mutate to survive ) : https://www.sciencedirect.com/science/article/abs/pii/S0079610722000347
the writers doubt macroevolution or the ability of known mechanisms of evolution to explain macroevolution as they say :
The central focus of this perspective is to provide evidence to document that selection based on survival of the fittest is insufficient for other than microevolution. Realistic probability calculations based on probabilities associated with microevolution are presented. However, macroevolution (required for all speciation events and the complexifications appearing in the Cambrian explosion) are shown to be probabilistically highly implausible (on the order of 10−50) when based on selection by survival of the fittest. We conclude that macroevolution via survival of the fittest is not salvageable by arguments for random genetic drift and other proposed mechanisms.
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Hello Boudjema; Your last reply to Mr. Dsugutov is perfect! The Grants' research is the clearest demonstration of the phenomenon available to a nonspecialist audience. Well done! Best regards, Jim Des Lauriers
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Hello,
So my query is for knocking down a gene. Will one guide serves the purpose, or does it require more than one guide RNAs in the CRISPR-Cas12a system in bacteria?
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Usually cut near the start gives you a better chance of a proper KO since it has a higher chance to cause an early frameshift. If you have only one guide then I would sequence multiple clones as the repair is not identical in all of them and some will be better than others. The only downside if you only have one, is that some sequences simply do not work efficiently and hence multiple guides are usually designed. As I said above the best option for you right now is to try and see.
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Chicken or Egg?
Considering the outcome of 183 patients admitted to Tongii Hospital in Wuhan in January, mean age, 54 when all patients received supportive care and antivirals. 41% had comorbis chronic diseases. 45.9% remained as inpatients with an overall mortality of 11.5%.
The patients were tested for prothrombin time, activated partial thromboplastin time, antithrombin, fibrogen, D-dimer and fibrin degradation products every 3 days for the first 2 weeks as inpatients. 71.4% succumbed to the virus as non-survivors and 4% showed no evidence of disseminated intravascular coagulation.
Speculation as to whether this is caused by the virus directly has been presented in the news bulletins giving COVID 19 an even more sinister characteristic or may the observed blood clotting be more due to the comorbidity conditions with a pre-existing thrombophilia tendency, than purely COVID 19. A chicken or the egg discussion of this situation needs to be addressed.
Thrombosis can simply occur due to immobility, particularly postoperatively and, although patients are regularly turned intensive care treatment, this level of activity may exacerbate blood clotting complications. Clearly pre-existing cardiovascular conditions are an important factor such as arterial thrombi in MIs and Strokes.
Genetic factors that interfere with the human coagulation cascade are relatively common. Thrombophilia can be caused by a severe deficiency of inhibitors (type I) or a severe elevation of coagulation factors the can be congenital or acquired also arterial, venal or combined. Venal thrombosis can be portal, renal, hepatic, Paget-Schrotter disease (upper extremity) and Thoracic outlet syndrome (unrelated to trauma).
Of the congenital conditions 5% of the population have the Factor V Leiden thrombophilia condition where 95% carting this genetic mutation develop a blood clot during their life.
• Prothrombin mutation (G20210A, 5’UTR)
• High homocysteine levels due to MTHFR mutation
(High homocysteine levels also due to vitamin deficiency B6, B12 and folic acid)
• Factor VIII promoter polymorphism (high FVIII levels)
• Other factors causing blood clotting are autoimmune disorders such as Anticardiolipid antibodies
• Lupus anticoagulants
• Renal disease (renal loss of thrombin)
• Budd-Chiari syndrome
Some rarer coagulation abnormalities include
• Plasmogen and fibrinolysis
• Paroxysmal nocturnal haemoglobinuria (haemolytic)
• Protein C deficiency
• Protein S deficiency
• Antithrombin III deficiency
Lillicrap D. Disseminated intravascular coagulation in patients with 2019-nCoV pneumonia. J Thromb Haemost. 2020;18(4):786‐787. doi:10.1111/jth.14781
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Prof. Annwyne Houldsworth: Please let me ask the following three questions:
  • Is there a lack of interpretability and transparency related to this virus?
  • Did we reach a state with this pandemic that is hard to control and monitor?
  • Is COVID-19 one of nature's secretions or it has been fabricated in the laboratories of one or more countries?
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Hello everyone,
I have recently designed a droplet digital PCR assay for a detection of a SNP (G->C) which uses a single primer pair (Forward and reverse sequences) and two competing, fluorescently-labelled probes differing in a single nucleotide in order to distinguish between DNA templates containing either a mutated (G) or non-mutated (C) allele.
I have a few well defined isolates, which i have performed allele specific PCR on as well as sequenced and know a priori their genotypes (let's name these 'wild-type' for isolates presenting only the non-mutated allele and 'mutant' for isolates containing only the mutated allele).
The problem occurs when i test the DNA from these well-defined isolates with my ddPCR assay:
1. Firstly, the isolates that i previously found to contain ONLY the MUTATED allele (G), that is mutant individuals, produce only the droplets that are positive for the mutated allele (channel 2 droplets). This is exactly what i expected and thus seems to suggest the assay works in distinguishing the alleles based on a single SNP. (Picture 1)
2. However, when i test isolates positive ONLY for the NON-MUTATED (C) allele, i.e., wild-type individuals, the ddPCR assay returns droplets for both alleles, suggesting that my isolates are perfectly heterozygous individuals (Picture 2). This is strange because it should not be the case, since both allele specific PCR and sequencing suggest they are indeed homozygous for the 'C' nucleotide and not heterozygous.
I would like to get some input on what could have gone wrong with the ddPCR assay that i have developed: it appears that it distinguishes the mutant allele found in the mutant individuals, but not the wild-type allele in only wild-type individuals.
P.S. I have double checked the sequences; alignment to the templates; annealing temperatures seem OK; contamination has never been an issue previously;
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Thank you for the answers thus far. As regards to the contamination of the materials, no template control samples return only negative droplets.
In addition, standard, single probe assay also returns the results described above (wild-type DNA having samples show up as heterozygous, whereas mutant are only positive for mutant DNA).
I suppose i could try moving the probe sequence a little up/downstream, but i am not sure it will generate a different outcome, since in the end, the difference between the probes is in the same nucleotide (C->G) at the same position.
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What is the best way to determine a genetic mutation? Is gene expression methods better or polymorphism?
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Altered gene expression can be a result of gene mutation or changes in gene regulation (epigenetic, transcription, post-transcription, translation, or post-translation).
So, the best way to identify a genetic mutation would be to categorize the mutation you are interested in. Are you going to identify a genetic mutation that is known or unknown?
Many different approaches have been used for the identification of known mutations as well as unknown mutations. You may usually start with the polymerase chain reaction (PCR), and additional assay steps may be performed based on the type of mutation.
For known mutations:
You may use Reverse transcriptase PCR (RT-PCR) in which the mutations are studied at RNA level.
Multiplex PCR in which multiple selected target regions in a sample are amplified simultaneously using different pairs of primers.
Nested PCR which includes two successive PCRs to amplify templates in low copy numbers in specimens.
Amplification refractory mutation system (ARMS) PCR is a general technique for the detection of any point mutation or small deletion.
DNA microarray which includes DNA “chips” or microarrays used for testing of multiple mutations.
DNA sequencing may also be used as it can provide analysis of genes at the nucleotide level, though many laboratories may not be able to afford it owing to its high cost.
For mutations that are unknown:
Single Strand Conformational Polymorphism (SSCP) may be used which is one of the simplest screening techniques for detecting unknown mutations such as unknown single-base substitutions, small deletions, small insertions, or micro inversions.
Denaturing Gradient Gel Electrophoresis (DGGE) may be used for screening of unknown point mutations. It is based on differences in the melting behavior of small DNA fragments (200-700 bp). Even a single base substitution can cause such a difference.
Heteroduplex analysis in which a mixture of wild-type and mutant DNA molecules is denatured and renatured to produce heteroduplices. Homoduplices and heteroduplices show different electrophoretic mobilities through nondenaturing polyacrylamide gels. Nearly 80% of point mutations have been estimated to be detected by heteroduplex analysis.
Best.
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I will perform the mutations using osprey software.
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Dear Dr. Rob Keller ,
Your response was very helpful, thank you for your time.
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A patient with desminopathy (mutation Thr341Pro DES in a heterozygous state) with the progression of the disease has a decrease in taste and smell, immunosuppression, and an increase in IgA in the blood.
Oddly enough, but all this is characteristic of infections, including viral ones. For example, it is known that if the hepatitis C virus is not treated, then death will occur in 20 years.
In the identified case of late onset desminopathy, muscle weakness manifests itself at the age of 30, and death occurs 20 years after the onset of the disease.
Could the desmin mutation in myofibrillar myopathy be caused by an infection?
Perhaps the infection contributes to the progression of desminopathy?
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A patient with desminopathy survived Covid-19 six months ago without pneumonia, but with a temporary loss of smell and taste. After Covid-19, we note an accelerated progression of desminopathy, penetration accelerates, new muscles are quickly involved in the pathological process, muscle mass decreases, and heart function worsens. Perhaps the infection or its consequences are somehow connected with the mechanism of progression of desminopathy?
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Can anyone suggest the answer for the question above mentioned?
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Hi Nitya
in genets/genomics everything is possible, just take as example the t9/22 leading to the BCR/ABL oncogenic protein. as mutation in oncogenes (as PDGF, ERB, RET, KRAS, NRAS, MYC, P53...) or in cancer suppressor genes (as BCRA genes....) leading to a dysregulation of metabolisms in cells, every change in life cycle will have an impact, good but mostly bad.
all the best
fred
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Hello,
I am currently doing a site-directed mutagenesis experiment. I made my mutation, and then designed primers for this mutation. For example one of the mutations is N32S and the primer lengths were 28bp for the reverse and 25bp for the forward primer. I followed our protocol to the T and for that mutation, after running a gradient PCR from 50 C to 72 C (temp), ran my agarose gel and I got a band in every lane (from 50C-72C), however, when I did the same for a different mutation, there were no bands whatsoever, all I saw were the primers that ran through.
I double-checked the primers to ensure that they were ordered properly, that the melting temperature of the overlapping region is 5 C higher than the non-overlapping but the two non-overlapping temperatures are within 1.5 C of each other.
I am truly stumped when it comes to how I could adjust them, or why some of them are working and others aren't.
We are using K296R MYC (PCDNA 3.1- ) tag for the DNA template, I made it fresh when I prepared my master mix.
I am just stumped. And my apologies if anything is unclear, I am just starting out in my program, so I still have a lot to learn.
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Your primers look a bit long (usually 20 bp) and if there are any missmatch, it is not going to work, especially at high annealing temperature. As Paul Rutland mentioned, It is better to sequence the fragment that cover your mutation to make sure the reference sequence you used to design your primers is correct. For that work, you may design primers for bigger amplicon.
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How is it possible to identify random mutations induced by environmental factors in a small cohort by the sanger sequencing method without knowing any specific target genes?
What might be the best and simplest method for this purpose without use NGS?
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I agree entirely with Katie A Burnette that the genome is just too large and for random changes NGS is absolutely necessary these days.
There are many methods described for mutation detection like SSCP, DGGE ,TGGE, chemical cleavage of mismatch, enzymatic cleavage of mismatch but all of these are limited to fragments of less than 1000 bases and often less than 500 bases so are all suitable for targeted mutation detection is likely gene candidates but inadequate for whole genome screening of any genome greater than 30,000 bases. It has been done with Sanger sequencing but took 13 years and billions of dollars.
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Spontaneous Mutations are the mutations that are unpredictable and occur due to errors in replication of DNA.
genetic engineering/Induced mutations are the mutations that are caused by known physical, chemical or biological agents.
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Both spontaneous mutation and genetic engineering leads to heritable changes in the individuals. Whereas most spontaneous mutations are random and often deleterious, genetic engineering is targeted towards a specific direction, and the expected result is desirable.
The molecular basis of mutation relates to mis-pairing between nucleotides of the DNA molecule irrespective of whether the mutation is natural or artificial. However, classical genetic engineering aims at creating heritable changes by inserting a transgene into the genome of the recipient individual. However, the present day gene editing that includes addition, deletion or substitution of one or a few nucleotides is also included in genetic engineering. For details, you can access:
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If a mutation occur in noncoding DNA does it affect phenotype.
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Changes in the sequence of regulatory elements and any changes in introns or before the coding sequence of genes that introduce a new splice sites may change the expression of genes and these may affect phenotypes
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Hi all!
I have found specific mutations in dengue genome sequences apparently related to the infection outcome. Here my questions:
How can I find if this mutation is synonymous or non-synonymous?
How can I verify if this mutation affects immunogenic/biologic properties of the protein?
I have some ideas but wondering if there's a standardised way to do this.
Kind regards,
B
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First part is easy. Go to GenBank and open a genome for your virus and then check if your mutation is within a known protein by nucleotide position.
If your mutation is within a protein paste the genomic region including the mutation into Expasy to get the translation for each frame. You can then do an amino acid sequence alignment using an online tool like MUSCLE against the reference amino acid sequence from GenBank.
The checking for changes in immunogenicity or function is the hard part.
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Hi!,
I am trying to figure out the connectivity between subpopulations of a parrotfish species along the coast of western Africa by analyzing the differences of neutral mutations in SNPs in the COI region between the subpopulations and trying to correlate these to the coastal currents.
I have done sanger sequencing for around a hundred samples, I have bi-directionally sequenced these and then aligned the pairs to get one consensus sequence per sample. However, I have now gotten stuck since most analyzing programs and packages use the newer VCF format rather than the FASTA format that I got the results in.
Is there a way to convert the FASTA sequences to VCF?
There are several sequences of the same gene in Genbank that I could use as a reference if needed.
Thankfull for any help or input!
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Thank you for your answer!
I'm sort of a beginner with bioinformatics as you probably understand, but I was thinking to use a reference sequence from genbank to do the alignments. Should I align/map the consensus of the reads from the two primers of each sample? and do you recommend any good tool to do that and to generate the SAM file?
Thank you so much for the help!
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These mutations cause a large number of genetic diseases due to which many are loosing their lives.
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Insertions/deletions are not necessarily harmful in the same way that mutations are not necessarily harmful. If the indel changes the number of bases to a number not divisible by 3 then the whole coding sequence downstream of the indel will be completely different from the original sequence until a stop codon is produced so over a long range of bases the protein sequence is greatly changed and the stop codon may also produce a longer or shorter version of the protein so many indels will produce a protein of greatly changed size, structure and activity. In the case of a single base change only one codon is likely to be changed and if this is in an unimportant part of the protein the change will have little effect
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In Evolutionar Biology, Epigenetics has become part of the explanations for changes in the phenotype across generations. But can these changes directed to specific phenotypic traits along many generations be converted into DNA mutations?
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Epigenetic changes via expression of appropriate long non coding RNAs serve advantageous answers to harmful inner and outer environmental stimuli as a quick means of adaptation. When the danger of DNA stability is high along many generations, the defensive epigenetic changes may be converted into DNA mutations. In extreme danger of DNA derangement quick (defensive) mutations may occur even in tumor cells exposed to endocrine disruptors (Suba Z. 2017, Suba Z. 2018, Suba Z. 2020).
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Dear collegus
I need to construct barch chart TOP 5 countries exactly in that order Chine","Egypt","Turkey","Indonesia","Spain") (Chine is first and largest) and put a numerical value on the chart
and add a flag icon for each country
May I ask you please to help me with a code
Thank you very mcuh
# Library
library(ggplot2)
library(dplyr)
# Dataset 1: one value per group
data <- data.frame(
name=c("Chine","Egypt","Turkey","Indonesia","Spain"),
val=sample(seq(1,5), 5 )
)
# load the library
library(forcats)
# Reorder following the value of another column:
data %>%
mutate(name = fct_reorder(name, val)) %>%
ggplot( aes(x=name, y=val)) +
geom_bar(stat="identity", fill="#E69F00", alpha=.6, width=.4) +
coord_flip() +
xlab("") +
theme_bw()
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Does anyone have some good suggestions to add the amino acid substitution information for the GISAID download SARS-COV-2 metedata.csv? Are these suggestions work?
1; Use the fasta data for pairwise alignment? But how to get the location and the mutation information?
2: By the leverage of the pangolin-lineage and mutation information from the cov-lineage wed? But there is a problem that this cannot auto-update if there are new mutations happening on one sequence.
3, Derive the mutation information using an UShER tree and the fasta data? But how?
Could someone give some comments and suggestions?
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This can be a bit tricky. Unfortunately, GISAID does allow provide that sort of metadata for in-bulk download.
You can use the following steps to get the AA substitution data:
1) Download FASTA files from GISAID.
2) Run Nextclade. For ease of use, you can perform this on usegalaxy.com (or usegalaxy.eu, either one should work). Make sure you select the SARS-CoV-2 option and "tabular format report" as the output.
Advantages of this approach: In my experience, this should run quickly and does not require you to align the downloaded sequences beforehand.
Disadvantages: It is my understanding that Pango and Nexclade report deletions slightly differently. You will need to keep this consistent across your analyses.
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there is a research namded : ( Genetic Redundancy Eliminates the Dream of Beneficial Mutations ) : pdfs.semanticscholar.org/99ab/9f57dc2a76af45d04652a69b295344cc208a.pdf
the writer of this research claims it disproves the Theory of Evolution ! what do you think about this ?
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Hello Amr; Nicolo has it right! I'll address only the premise. Whether a gene is "beneficial" or not depends entirely on the conditions of the environment in which it occurs. If the bearer of the "responsible" gene produces more than the average number of offspring in the population, then the gene is beneficial. That's all. It has essentially no bearing on the validity of the theory. Best wishes, Jim Des Lauriers
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Hello All,
Our group has identified a recurrent mutation in a retinitis pigmentosa family. Though our identified mutation was already reported but our phenotype was different. We did whole exome sequencing and Sanger sequencing to confirm our mutation. We also performed some Bioinformatics analysis to support our findings.
This is so unfortunate that after spending so much money, journals always look for novelty in the study. They never showed interest in our paper as the identified mutation is not a novel one.
I would like to hear your comments on this, how we can publish this finding.
Thanks
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Hi you can publish it as a letter to editor.. most journals now look for novel variants resulting in a new phenotype.
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Dear all,
I have a problem and I do not know why it is happening. When I do my KRAS mutation analysis most of the samples come out as in the image without the predominant red line. However, some samples, as seen in the other image, show that high thymine contamination.
What I don’t understand is, why that high contamination of thymine only shows up on the KRAS codons. The rest of the sequence is clean and does not shows thymine spikes like that.
Thank you for your help.
Kind regards
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The broad peak looks like unincorporated dye from the sequencing kit which usually happens when purification of the sequencing reaction is not good. It is worse when there is too little primer or too little pcr product in the sequencing reaction so there is a lot of unused dye to purify away. It usually happens at 2 positions near the start of the sequencing.If you attach one of the .abi sequence files with this problem it would help.
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I am trying to clone a gene sequence - I have tried the takarabio software but I have not had success in the primer design. Any software that you could suggest will be highly appreciated.
Froylan
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Serial cloner is the best soft wear .iused in my PhD .you can download fere
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We have seen recently that past 4 months , Vaccine derived paralytic polio (VDPP)has been observed in the (UK), London sewage .Oral polio vaccine , P2 strain which is mainly implicated in VDPP , has been stopped in UK since 2004. The reason may be some virus may have been imported from endemic country like Nigeria or traveller recently vaccinated from other country and moved to UK. Interestingly recent reports suggest cluster of genetically linked viruses (VDPP) obtained from sewage which may indicates viral mutation. VDPP is vaccine derived(Oral), which is transmissible and can mutates
In India National switch day was declared on 25th April 2016 in which P2 strain was removed from OPV to avoid VDPP cases. With neighbouring countries like Afghanistan and Pakistan still endemic for Polio. Should we consider complete switch from OPV to IPV .Another concern is should we consider booster of IPV for susceptible adults?
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The immunological rules to change vaccine strain ,route in any community ,country depends number of clinical cases ,immunological status and gene identity between vaccine used and newely isolates from clinical cases.For India i think they are fameliar in such situations
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I am aware of GISAID but I can only find the full original Wuhan strain and amino acid mutations in the Spike protein for BA.4 and BA.5. Does GISAID share mutations in other regions of SARS CoV 2's genome?
Edit: clarified whether the mutations in the S protein are Nucleotide or Amino Acid
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Tahreer M. Al-Thuwaini Frederic Lepretre Thank you for your replies!
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One positive for mutation and the other one is negative. I am calculating mean for example mean Hemoglobin. And also percentages like 12% had fever.
What tests should I use to compare these two groups one positive for a mutation and the other one negative.
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for difference berween means use two sample t test
for difference between proportions use normal approx with
z = (p1 - p2 )/sqrt( pq(1/n1 +1/n2)) where p =( r1 +r2)/(n1+n2) ; q = 1-p
n1, n2 size of group, r1,.r2 number positive for characteristic eg fever p1= r1/n1 p2 = r2/n2
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Since both alleles of the same gene are getting edited in Bi-allelic mutants of any particular organism, Can we consider that organism as a homozygous mutant for the gene?
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Thanks to both of you John Hardy Lockhart and Sven Reister :)
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Hi
I am working on a project where I have specific segments of interest. Almost 18 region of TB positive samples were selected from where the mutation can be occurred. For that mutation the specific drug could be resistant. So i wanted to sequenced that specific region applying mini seq illumina sequencing. So i extracted the DNA from left over through Qiagen extraction kit. But when I go for quantification through qubit fluorometer i was surprised that the quantity of the sample is very low . I am confused why this happens. Even though when i pcr that product the quantity is also low . But my pcr was good because the positive control result is perfect. So anyone know or have better solution on that ,please kindly help me
Thanks
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Thank you so much .But we are using pcr kit. All the necessary thinks are mixed up together . So its not possible to check the primer concentration. I think changing the cycle can be a good option although we are maintaining the standard protocol .
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Hello my Dear,
I have a question about CALR chromatogram analysis. I sequenced and aligned the amplified sequences with the reference sequences but I'm having trouble identifying the anomalies there. The problem being that I can have any sorte of modification (ins/del or del or ins or transversion see all at once). I tried to BLAST the potential pathological sequences but I'm having trouble understanding what comes out of it.
When I align with the normal protein reference sequence I have 20 aa aligned 100% and when I do the same thing with the mutated protein I have 36 aa aligned 100%. The expected protein is 60 aa and the affected portion is exactly where the two proteins align 100%.
A little help would woud not be refused.
Sincerely!
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Paul Rutland I'm comming back to you cause I couldn't analyse the chromatograms as you did. Can you please send me the document you mentioned ? Thanks
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I'm looking into using inverse PCR as a QuikChange PCR tended to have high primer dimerisation. I've been looking through protocols for Q5 mutagenesis but am unsure how it results in mutations on both strands if only 1 primer contains a mutation.
Would love any help in explaining how this happens.
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Recently I needed to introduce more than 10 mutations (substitutions, deletions and insertions) on a stretch of 30bp in my plasmid. I used NEBaseChanger with their Q5 and KLD and it worked perfectly alright in 1 step. Out of 6 clones that I picked for sequencing 5 contained all the needed mutations. I just followed their standard protocol https://nebasechanger.neb.com/
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We need a review paper about mutations & anatomy of succulent spiny plants.... all the best
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The folllowing articles can give you a clue on the issue you raised, Thank you!
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As I know, smoking can cause lung cancer and body cells turn into cancer cells due to mutations. However, what is the name of the carcinogenic substance in cigarettes and how can it make body cells mutate?
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Benzo[a]pyrene (BP) is one of several ring-shaped chemicals called polycyclic aromatic hydrocarbons that are produced when organic matter, such as a tobacco leaf, is burnt. When it enters the body, BP becomes a powerful DNA disruptor, producing mutations that can lead to cancer.
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I have mutated plant DNAs and want to detect off targets in the plant lines . WGS is an expensive way to do it when you have so many lines to check. Could anyone suggest other methods to detect them?
#CRISPR
#CRISPR/CAS9
#GENEEDITING
#offtargets
#mutations
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The best way is NGS but you can use T7E1,RFLP or high resolution melting curve for off target predicted regions.
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Spread of MONKEY POX is due to mutational etiology? Monkeypox was first discovered in 1958 when two outbreaks of a pox-like disease occurred in colonies of monkeys kept for research, hence the name ‘monkeypox.’ The first human case of monkeypox was recorded in 1970 in the Democratic Republic of Congo during a period of intensified effort to eliminate smallpox. Since then monkeypox has been reported in humans in other central and western African countries,
then why its being now spreading all over ?
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Researchers are racing to find the origin of the monkeypox outbreaks that have now been linked to some 600 confirmed or suspected cases worldwide. They are also investigating whether the virus is spreading differently compared with previous outbreaks, whether it has accrued unusual genetic changes and how to contain it. One obstacle is that the monkeypox genome is enormous relative to those of many other viruses — more than six times larger than that of the SARS-CoV-2 coronavirus, for example — making it harder to analyse...
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Shall we have previous genetic variation in explant tissues used in in vitro approaches, how can we estimate an adequate ratio for somaclonal variation among regeneration events?
In tissue culture we use several plant tissues and “reprogram” the cell development to obtain somatic embryos, calluses, shoot meristems, whatever.
Considering that the raw material we start with would not have a chance to follow another developmental sketch (E. g. a spongy parenchyma cell) is expected to die as originally planned, a spongy parenchyma cell.
It is clear that some stress triggers, frequent cell divisions, growth regulators and other substances, may increase the mutation ratio differently. We may also consider that there is a tiny probability of this mutation events occur by chance.
My reasoning is, how can we correctly attribute a somaclonal variation ratio being aware of the possibility of mutations that would not jeopardize a cell developmental path already settled in an explant (a chlorenchyma cell for instance) that, was not for the tissue culture detour, would “kick the bucket” as such (a chlorenchyma cell)?
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You can use molecular markers
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Hey everyone,
I am looking for a way to show truncating mutations in BRCA1/2 via IHC. The idea is to use two slides per sample for each of BRCA1/2, respectively. In theory, samples that have a truncating mutation should show expression on the N-, but not C-terminal IHC slide.
I also found the following publication (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2799150/) by Watson et al. (2009), where they developed a IHC antibody that specifically targets the BRCA2-C terminal region, and showed that this method worked for them.
Unfortunately, the paper is a couple years old - and I cannot find this antibody anywhere - or any other BRCA2-C antibody. There are some that use aa 2400 as the immunogen, but I am looking for something more in the region of aa 3000 to really include all truncations.
Has anyone implemented a similar assay to allow for quick and easy IHC of FFPE samples to check for truncating BRCA1/2 mutations?
Thank you for your help!
Sincerely,
Marcel
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Fortis Life Sciences (Bethyl Labs antibodies) has Rabbit pAb anti-BRCA2 where they describe the immunogen as "between 3350 and C-term": http://www.fortislife.com/p/A300-005A
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I made several combinations of mutations in a 747 amino acid-protein based on prediction tool as well as some additional mutations in non-predicted lysines including one affecting all 42 lysine of the protein. I did not observe a phenotypic change specific to K-R mutation, but I think the effect of fluorescent tag used here is more dominant in the protein stability. I am wondering if anyone has also observed the effect of changes in amino acid side chain geometry and electrostatic interactions in such R variants.
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Annemarie Honegger Thank you for your suggestion! I appreciate it. Hopefully David Vukovic will be able to provide me specific answer.
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Hi Everyone!
I have performed western blot for X protein modifications (4 different modifications) and control is unmodified X protein. The total sample are only two, out of which one is control (unmutated sample) and the second one is mutated sample. I did image analysis in Biorad Imager and quantified and normalized the RFU intensity. Now I am not able to understand which statistical test I can apply. As I don't have replicates at all. Which test I can perform to show the difference between mutated sample and unmutated sample.
Thanks
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I had a similar thinking approach
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I am using CRISPR Cas9 to introduce a point mutation in a gene to get patient-like cells. I am applying a plasmid with cas9 and my gRNA inserted. I used a ssODN as a template for introducing the desired mutation. After puromycin selection and limiting dilution to get single-cell clones and after almost two months when I get the sequencing results it seems that there are no indels etc, it seems that there is no double-stranded break in the desired site in the genome.
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If you are lucky and putting in your point mutation cause a restriction site to form or be lost, you can do as Farheen Shafique is probably going to suggest. Do a PCR and try cutting with the RE to determine the percent of cells with the mutation in the whole population, to see how many clones you need in order to get one with the mutation, and if it is worth even doing the cloning.
Otherwise, after selection but before limiting dilution, grow some of the cells up to confluency in a 35mm dish, do a quick and dirty lysis with something like the mouse Extracta kit (which works fine on tissue culture cells) and an Exo-SAPIT cleanup, and then send it in for Sanger sequencing in both directions. Use one primer upstream of the cut followed by the mutation and the other from the other direction, upstream of the point mutation followed by the cut.
The sequence from the primer on the upstream side of the point mutation might show the mutation peak, if you get a good percentage of HDR, but for both primers, the sequence downstream from the cut should show two or more extra peaks in the same position for most peaks, due to NHEJ causing the sequence to shift. That tells you how well the guide cuts. The peak height of the non-WT peaks relative to WT peak height gives you a rough idea of the fraction cut (can use Synthego's ICE program to figure out actual percentages). The peak height of your single point mutation relative to the WT peak tells you how well your HDR worked.
In both cases, if you can't see these non-WT peaks, it means less than 5% of the cells are being cut or carrying the point mutation. For 5% of the cells carrying the mutation, you have to have at least 10 clones to get possibly 1 clone with the point mutation (2 alleles per cell). Also, the site on the other allele may have gone through NHEJ with imperfect repair, in your clone with the allele containing the good mutation so actually you need a lot more clones, if you want one WT and one point mutated protein in your clone.
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Hello,
I have sequenced my KO clones.
I have two different mutations, one insertion and one deletion at same position in same clone,
What I want to know is it possible that same gRNA did two different mutations in 2 alleles,
Can anyone provide an explanation
thanks
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Yes. Once Cas9 cut the DNA the repair is random so in each allele you will get a slightly different indel.
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I am interested in evaluating the role of silent/synonymous mutation using the CRISPR-Cas9. However, I am unable to find any literature related to it. Can you please provide data related to it?
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Muhammad Abrar Yousaf do you want to completely shutdown the gene or you just want to insert point mutation, in which such gene might/might not be functional?? in case of knockout, CRISPR would be great. However, for silent/synonymous mutation, wouldn't "site directed mutagenesis" work fine? using CRISPR for single base sub, check this papers: https://www.nature.com/articles/s41598-019-41121-4
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I recently sequenced (Sanger) an exon from single alleles of a CRISPR-Cas9 mutated cell line (diploid) to find whether the mutation is homozygous or heterozygous and what the mutation is. What I got is 3 different results, a WT, and two diff mutations, a 1 base del and a 7 base del. My lab has come to the conclusion that it must somehow have 3 alleles instead of 2, and suggested looking into this. I can't really find anything relevant to this find, so I'm wondering if anyone might know of such research articles or point me in the right direction, and if this is even possible?
My original thought was that perhaps the 7 base deletion is somehow an error due to the crispr process? But more experienced people than me have suggested it means there are 3 alleles, although like I said I'm struggling to find relevant literature to support this.
Any help would be much appreciated, thanks.
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Another option is that your population is not 100% pure and you have a mix of cells with different mutations.
How did you isolate single clones after the CRISPR treatment?
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I recently sequenced (Sanger) an exon from single alleles of a CRISPR-Cas9 mutated cell line (diploid) to find whether the mutation is homozygous or heterozygous and what the mutation is. What I got is 3 different results, a WT, and two diff mutations, a 1 base del and a 7 base del. My lab has come to the conclusion that it must somehow have 3 alleles instead of 2, and suggested looking into this. I can't really find anything relevant to this find, so I'm wondering if anyone might know of such research articles or point me in the right direction, and if this is even possible?
My original thought was that perhaps the 7 base deletion is somehow an error due to the crispr process? But more experienced people than me have suggested it means there are 3 alleles, although like I said I'm struggling to find relevant literature to support this.
Any help would be much appreciated, thanks.
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My thought is that you have a mixed population of cells, each with their own alleles. Streak/dilute them out to single cell colonies and screen them again.
No, your cells are not magically triploid.
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Hello!
I got the 23s rDNA of my enterococcus strains and found some skeptical mutations. Now I'm trying to number it like G1234T. I read from some documents that E.coli 23s rRNA numbering is widely used, but I don't know how to do it. So my problem is, what is E.coli 23s rRNA numbering and how can I number the mutation correctly? Do I need to align my sequence with the reference E.coli sequence?
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the following thesis "although in Arabic" BUT got good references for numbering the 23S rRNA , I hope it will be helpful
"The Development of linezolid Antibiotic in Staphylococcus haemolyticus Bacteria Resulting from Modifications in the Ribosomal RNA: it is on ResearchGate
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Hello all, what I'm wondering is can CRISPR-Cas9 induce different mutations in the same targeted cell line or would we expect to see the same mutation?
I have a knockout myoblast cell line which in which two particular exons were targeted with CRISPR-Cas9 when creating it. this cell line was sequenced (Sanger) to determine the type of mutation. The DNA obtained from the cells was PCR amplified with the primers for that Exon, and then used to transform bacteria cells in order to isolate the two alleles (TOPO-TA Cloning).
when analysing the sequenced data against the WT exon I noticed that some samples had the same type of mutation while others were WT (therefore the original cell line must be aheterozygous for that mutation). However some other samples also had a mutation but a different one. So is this possible with the CRISPR-Cas9 process? for different types of mutations to be introduced into the cell line? Or would we expect to see only the same type of mt, whether that be a deletion or a point mutation or whatever? Thank you kindly for your help!
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Yes, it is possible to get more than one type of mutations from CRISPR Cas9 at the same time. I am also working on CRISPR Cas9 to knockout few viral genes by multiple gRNA and I got different mutations including deletion substitution, INDELS at the same time in one sample after performing amplicon sequencing (NGS) and Sanger sequencing as well.
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Hiii,
I try to make mutations with QuikChange II XL Site-Directed Mutagenesis Kit. When I did first mutation, I got colonies and also mutation was succesfull. However, although I tried everything such as new stock of compotent cell, another compotent cell strain, different plasmid even puc18, different shaker rpm, new lb agar plates, empty compotent cell growth in without amp agar and convert compotent again; there was no transformation or colony. I tried all troubleshooting and we cannot to obtan any colony. Lastly, we tried puc18 transformation as positive control and didnot happening.
Can you give any advise or suggestion?
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We checked heats with thermometer every step. Maybe we can try electroporation method, thank you.
On the other hand, we thought maybe out component cells is dead, but we streaked them on the agar plate without amp and they growed. However, we can try another plasmid stock. Thanks for answer. :)
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Mutated miR-21 of glioblastoma.
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The first thing that came to mind, and this would be a good resource for variant-gene-cancer matching in general, is the COSMIC database (https://cancer.sanger.ac.uk/cosmic).
However, since you are specifically asking for a microRNA in this case, it may be more cumbersome to find the information you need. I'm not aware of a cancer specific miR sequence database. You can find the sequence on mirbase (https://www.mirbase.org/cgi-bin/mirna_entry.pl?acc=hsa-mir-21), but then may need to cross reference with glioblastoma specific publications to see where they mention any mutations.
It looks like that as of today, there are 135 publication on PubMed which include miR-21 and glioblastoma. I suspect only a smaller subset would have actually done sequencing of the miR. Hopefully that narrows your search somewhat but maybe others will have a better answer or link to different resources for you. Good luck!
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Hi
I have a challenge with a somatic mutation in the beta cells. I think I know the disease-causing gene GCK due to the histological picture, but how can detect the mutation?. I have tried to use LCM and purify RNA from the islets but this is difficult on FFPE tissue and it requires a lot of islets and the yield is small and unstable. I think that only some of the islets are hyper-functional concerning the GCK mutation.
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Thank you!
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I need to run molecular dynamics simulations to test hundreds of mutations in two proteins which bind to one another to assess the effect of these mutations on the binding interaction and protein stability. What is the best molecular dynamics software for this purpose? Are there any options with a graphical user interface? Or ones that can be run on the web? This would be highly preferred.
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Software such as GROMACS, Desmond, NAMD, AMBER, CHARMM can be used to perform protein-ligand MD simulations. Among them, Gromacs is quite easy to understand at the beginner level.
Yours truly
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Hello! For example, I want to design a primer for a SARS-CoV 2 gene using bioinformatic tools such as Primer BLAST. How would I ensure that my primer will work on future variants of the virus in which the target sequence mutates?
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Hi
For some critical mutations you may also synthesize short stretches of synthetic templates and actually test your primers and PCR assays.
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Hi there I'm just a student working on my research and need suggestions for free and webserver Bioinformatics tools that I can use for post translational stage of mutated protei
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Among the cultivated primroses, I observed a plant with 5 stigmas and styles. I could not find a report on this. Does anyone know the reason for this feature? Is there a report on this?
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It seems normal and okay to me :)
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There should be an element, property, characteristic, etc, that is common to all the mutations that COVID-19 has and will have, once this characteristic is identified, a vaccine could be created that, in the presence of this common property, will block all types of synapses with human cells preventing infection for all possible mutations.
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Like the flu virus, the COVID-19 virus may have a new mutation each year, and a vaccine may be made that only reduces the symptoms but is not suitable for prevention.
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Hello,
I am struggling to interpret couple of splice site mutations I have encountered and I would like to ask if there is a way to predict the outcome of a given splice site mutation (does it lead to exon skipping, or truncation, for example). I have a splice site mutation I need to interpret which occured in the exon 14 splice site of the MET gene. MET exon 14 skipping is critical parameter in cancer patients for therapy decisions. The mutation I am working on has not been shown in the literature however, a splice site mutation just 1 nucleotide upstream was detected and classified as pathogenic in some studies. I am aware there are some in silico tools for prediction however I am not sure which one would be the best fit for my purposes (predicting the outcome of the mutation on the protein level, does it lead to exon skipping? truncation? etc). I understand that in vitro testing is the gold standard however I have no access to lab at this moment.
Thank you very much in advance and please let me know if something is not clear.
Cheers!
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you can test VEP from Ensemble to test its prediction on RNA and protein structures (https://www.ensembl.org/info/docs/tools/vep/index.html), but there is no way to predict the impact of a variation on RNA and protein levels. those levels depends of course on the genomics, but also on cell states, pathologies, compensation and regulation phenomenon, copy number of the gene in the genome.....lab is the only way to be sure on what you're thinking it should, sorry.
stay safe
fred
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I am using cas-offinder to find mismatches. I found 1000s of sequences that differ from my guide RNA with minimum 4 nucleotides. I was thinking to consider the guide RNA with max 3 bases difference but could not find any. I am not sure will the gRNA bind if the difference is more than 3 bases? should i just check or off targets with only sequences with 4 bases mismatch?
Could anyone suggest me how should i proceed with this?
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Off-targets have been reported by guideRNAs having as high as six mismatches, the process is not fully understood. However, in most instances 1 or 2 mismatches is enough to prevent recognition. I designed 5 different guideRNAs each based on a gene of interest but with 2 mismatches, and none of the five guideRNAs can cut the gene of interest. When i introduce the target site (containing the 2 mismatches) into the genome, all five guideRNAs can cut these sites.
It matters where the mismatches are located, specifically the relative distance to the PAM site (where a mismatch in the guideRNA close to the PAM site is more ‘severe’, thus leading to no recognition). I have personally used CHOPCHOP to predict my guideRNAs, so far always happy and without causing off-targets in my filamentous fungi (even confirmed no off-targets after 5 rounds of crispr with whole-genome sequencing). Note that most of these experiments were done in a ku70 deletion background (impaired non-homologous end-joining DNA repair). See
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In Sanger sequence, after having sequencer output (ab1 file), I would like to apply a free software to check the mutation on level of transcript. I was already working with “mutation Surveyor” software, but it is not freeL. Is there any free software? Thanks a lot.
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Check DNA Baser tool
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qPCR, capillary sequencer
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Shreyasri Dutta you can go for QS5, AB 3500.
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Hi all!
Could you remind me any database where I can find protein sequences of MUNB mutations (NUMB20, 15 and 1) in D. melanogaster?
Regards, Tatiana
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you could try flybase at https://flybase.org/
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how to find out a Tumor cell lines whose p53 gene is not missing or mutated ?
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@Weiping Weiping. Hopefully, this book will help you.
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For instance, RPE65 mutations can be missense frameshift etc, but is there an efficient process to know whether the vast majority of mutations lead to a functional loss?
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It may help you to look at our recent paper on single-site mutations – variants of unknown significance – where we used structure (which could be an AlphaFold model) and sequence conservation in terms of evolutionary relationships.
See Front. Mol. Biosci., 14 December 2021 | https://doi.org/10.3389/fmolb.2021.791792
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I want to ask if there is a website to determine the percent of covid variants have a combination of mutations/deletion? For example, what is the percentage of different variants that has L452R, T478K, E484K and N501R based on the database in GISAID? I assume the algorithm will look for all the variant sequences that has all these mutation and then calculate the percentage based on number of sequences of each variants that have all these mutations. If not a webiste, it is ok if you know any open source bioinformatics tool can do that. Thank you.
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I have some interest in the interaction between protein A and B but I barely know about proteomics so I leave the question here.
To specify the exact interaction sites, I made four site-directed mutated plasmids having a GST tag using the quick-change method. The mutated sites are on the cold shock domain of protein B. Because I read this phrase "systematic alanine scanning mutagenesis has revealed that the substitution of an amino acid residue by alanine in these hot spot regions lowers the binding affinity by at least 2 kcal/mol (Bogan and Thorn 1998).", I changed every mutagenesis site into alanine.
And then I did a GST pull-down assay after co-expression of MYC-A and GST-B (treated with RNase, DNase, and MNase). This is the question. I could not understand the results I got from this experiment. The affinity between protein A and mutated B(all four mutations!!!) is so much higher(>100 folds) than that between protein A and wild-type protein B. They interact much stronger via these mutated sites. Do you have any idea what it is? And can it be a clue for finding exact interaction sites?
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Reading about biotechnology database and bioinformatics directory in this article may help you find your way through your research
This article published by Stanford university
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Hi, I wanted to ask two questions that would save me a lot of work.
Is there a specific function that gives me the set of PDB codes for single point mutations in the sequence of a specific protein? The idea is to filter the list directly without doing the manual filtering in Protein Feature View.
Is it possible to obtain with some search function, the PDB of a single point mutation of a specific protein? For example: to know if there is a PDB for the mutation of a cysteine to a proline at position 144 of a specific protein.
Thank you very much for your time and attention
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I asked PDB directly and they replied as follows:
Thank you for your email message. Here is a query for any mutation using the API
I am afraid that searching for positional features isn’t possible.
Please let us know if we can be of additional assistance.
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I am doing targeted sequencing on colitis-associated cancer sample, and found some mutations. So I need to validate 3 synonymous mutation with additional samples. Unfortunately, samples are limited and out of 6 samples, I only found the mutation in 1 sample. Is it strong enough? Or do i need to do further validation with sanger? I am also planning to do functional study of that mutation via in vitro.
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If you only have the empty dna sample tube left of your original sample then whole genome amplification will generate large amounts of dna from the small amount coating the bottom of the tube and then you can amplify and sequence. If that is too expensive then just add 10ul of water to the sample that you have left and pcr amplify but add 4 more cycles than usual and you should get enough amplimer to sequence or restriction digest or whatever method that you want to try next
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Some drugs/ carcinogen cause mutation in DNA, Which carcinogen causes highest mutation in genome?
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Hi Amrit,
The most commonly mutated gene in people with cancer is p53 or TP53. More than 50% of cancers involve a missing or damaged p53 gene. Most p53 gene mutations are acquired. Among most active carcinogenic mutagens the championship title belongs to polycyclic aromatic hydrocarbons (PAHs). These are the most abundant indirect-acting carcinogens to which humans are exposed to on a daily basis. Exposure has been associated with the development of breast, skin or lung cancer. Bioactivation of PAHs is required in order for these agents to exhibit mutagenic properties, which is primarily mediated by cytochrome P450 enzymes (among many others, our data on this subject - in http://www.xenobiovir.com/). Bioactivated metabolites target multiple genomic sites, including guanine and adenine bases via PAH diol epoxides. This results in the generation of bulky BPdG chemical DNA adducts; examples include quinone-mediated cross-linking of N7 position of guanine and N3 of adenine.
All the best,
Ilya
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Hello,
I am trying to mutate a NheI restriction site on a ~11.2 kb circular plasmid but nothing I have tried seems to work. I have tried modifying the PCR reaction by adding and removing DSMO, MgCl, changing the PCR parameters (as of now I am using 98ºC for denaturing, 55ºC for annealing, 72ºC for extension), and modifying the quantities in the reaction. I have also tried running the annealing step for 10 mins at 98º, 80º, 70º, ..., 30º but nothing seems to work. I have also tried using different primers but it is unclear if one type works better than the other, as I cannot seem to get any PCR product at all. I have also tried transforming the DNA into bacterial cells but have determined that the cells are not the issue (as colonies were successfully grown in another experiment). The running theory is that the size of the plasmid is causing issues so I have thought to use other restriction enzymes to cleave a fragment of the plasmid containing the NheI site, mutating it separately from the plasmid, and then re-inserting it. I would greatly appreciate any ideas or advice anyone may have!
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We have tried running the annealing step with just the plasmid and the primers, starting at 98º, 80º, 70º, ..., 30º, and got no results, so I suspect this to be the issue.
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What wattage and distance from plated E. coli does a UV light need to be to induce mutation without killing the whole colony. I would like to induce mutation and then test for antibiotic resistance so I will need some surviving colonies.
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For the method you can use the attached article below
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All tumors have DNA mutations, and a predictive understanding of those mutations could inform clinical treatments. However, 40% of the mutations are variants of unknown significance (VUS). So the challenge is to objectively predict whether a VUS is pathogenic and supports the tumor or whether it is benign. We are working on this problem and would welcome feedback on our efforts (see doi: 10.3389/fmolb.2021.791792 ) and also alternative ideas and insight.
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I am very interested in this because of the multiple mutations in Papillary Ca of Thyroid which are VUS and how best to discern if they are more likely to be pathogenic. So far I look at Clin Var and a few other sources so I will take a look at your paper- thank you!
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I have a protein that is easy to aggregate when the temperature is lower than 4℃ or the concentration is more than 5 mg/ml. As we know, protein often is stored on ice or 4℃ during experiments.
Except for mutating amino acid sequence, how to protect proteins from aggregation? Does any one know the mechanisms for this kind of protein aggregation?
Thank you in advance.
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It could be that in a non-ideal buffer, the proteins are likely to unfold, resulting in aggregates of insoluble proteins. If additives are added, they can either stabilize the proteins from partially unfolding, preventing protein-protein interaction or they can aid as chemical chaperones, leading to a properly folded and non-aggregated state.
Addition of additives can serve two purposes,
1) increase protein solubility either prevention of aggregation via stabilization, or
2) assistance to the stable folded state.
So, buffer supplemented with small molecule additives like osmolytes may help overcome this problem of protein aggregation. Osmolytes like sucrose and detergents exert a stabilizing effect and prevents aggregation. It favors proteins in their native state. If proteins contain cysteine residues you can consider adding reducing agents like beta mercaptoethanol to prevent oxidation and protein aggregation.
Some ionic stabilizers like salts also enhance solubility of proteins. So, try using different salts to achieve protein stability and prevent aggregation.
I hope this may help.
Best.
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Typically exposing plant parts such as seeds, stems, pollen grains etc. to radioactive isotopes (e.g. gamma radiation and x-ray) and chemical mutagens (e.g. ethyl methanesulfonate [EMS]) induce vital mutations for plant breeding programs.
BUT Natural radiation and microgravity in space induce genetic mutations for selection. Below is a summary:
  • Grow plants in the space station to maturity for one or few cycles.
  • Space grown plants are exposed to natural stresses (reduced soil moisture, nutrients, and carbon dioxide during reproduction).
  • This stress is induced by natural cosmic radiation (cosmic rays and effluvia from the sun) and earth’s gravity both enhancing genetic mutations.
  • Seeds harvested from space-grown plants can be grown on Earth to select novel mutants under glasshouse and field conditions for various traits (e.g. tolerance to drought and heat stress, resistance to insect pests and diseases, early maturity, flower colour, plant architecture, reduced plant height, improved gas exchange, better root growth etc.).
  • The new mutant varieties can be bred further or the seed deployed to farmers for commercial production.
Satellite missions and subsequent selections have produced some 200 improved crop varieties in China.
StarLab Oasis, a private organisation, is set up to raise plants in space for this purpose.
It reads like science fiction, but it is a fascinating, optimistic and complementary tool to conventional breeding.
I hope this service will be available for major crops in the near future. I cannot wait to send my seeds to the International Space Station (ISS), and I hope you do too.
The above read is extracted from
Shimelis Hussein
Professor of Plant Breeding
University of KwaZulu-Natal
South Africa
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Dear @Hussein Shimelis Yes, there are several reports and reviews that have thrown lights on space breeding:
I don't know whether the space bred varieties of crop plants have been tested in other countries. I doubt varieties bred under microgravity (zero gravity) will perform similarly under the influence earth's gravity. Moreover, the basic philosophy remains the same, that is, radiation induced genetic alteration which leads mostly to recessive and deleterious mutations. Although application of artificial mutagenesis to crop improvements started as early as 1928 by Stadler in barley, it never gained much importance in practical crop improvement. Even through site directed mutagenesis, not much has been achieved so far. If China has achieved so much in a short span (just in 3 decades), I call it highly impressive achievements! Let China share its space bred varieties to other countries to assess their performance!
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perhaps the most useful aspect of epigenetic processes is that they are readily reversible. Unlike genetic effects that also play a role in cancer and aging, epigenetic aberrations can be relatively easily corrected. One of the most widespread approaches to epigenetic alterations in cancer and ageing is dietary control. now, I would like to know more about the types and mechanisms of that nutrition that have a positive impact on epigenetic alteration during cancer???
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thank you, dear Shin Murakami for your sugession
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I have question about efficacy of new vaccines against new, mutated strain of COVID-19.
Is new vaccines against traditional COVID-1 also effective against new, mutated strain of COVID-19?
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I have experience in phylogenetic analysis, but not virology. I would like to develop a substitution model for the mutations that occur in SARS-CoV-2, and would like feedback from virologists. A substitution model provides values for the 12 mutation rates A->C, A->G, ..., T->G at a particular site. There are lots of substitution models used in phylogenetic analysis, from the simplest Jukes-Cantor model, which says all 12 rates are equal, to one with 12 individual rates, and more complicated ones still, which take into account neighbouring nucleotides.
Here are some observed values:
A C G T
A - 52 308 68
C 58 - 18 1098
G 255 46 - 437
T 56 327 52 -
These may be mutations produced by the virus, or by host editing, and there are sequencing errors which can confuse matters. Note the large number of C->Ts and G->Ts, and high C->T/G->A and G->T/C->A ratios.
Please correct me if I am wrong, but this is how I understand the mechanisms within a cell:
Virus-mediated mutations. If the polymerase mismatches a pair like G:T instead of G:C, at a certain rate, it will do this when copying positive sense to negative sense, and negative to positive, at the same rate. (If not, why not?) Every virus genome that exits a cell must be the result of an equal number of positive-to-negative and negative-to-positive copies. This implies a symmetry among the rates like this:
A->C ~= T->G
A->G ~= T->C
A->T ~= T->A
C->A ~= G->T
C->G ~= G->C
C->T ~= G->A
For example,
C mispaired with A, A correctly paired with T produces a C->T.
G correctly paired with C, C mispaired with A produces a G->A.