- Cingeetham Vinod added an answer:Can someone suggest a protocol for primary breast cancer cell culture for Karyotyping?Seeking an established protocol to culture short term primary breast cancer cells for karyotyping. I request you to suggest a best mitogen to enhance the cell division and its conc. to be used.
@ Betty G Dunn: Madam i have succeeded in culturing the primary breast cancer cell but now im stuck at hypotonic solution, I have treated cells with various concs of KCl and also used detergents along with KCl as suggested by various papers but all affords gone into vain. a very few no. of plates are obtained like 5-6 metaphase plates per slide, so i need your suggestion to increase the plate number.Following
- O. Solovyov added an answer:Can the monozygotic twins have different blood types?I really want to know the answer.
In fact, if very rarely might be in monozygotic twins different caryotype and even different sex, why could not be different blood type? :) Look Enrico Lopriore answerFollowing
- Thorsten Gerstner added an answer:Correlating epilepsy with chromosomal aberration?We are conducting a review study on a relationship between chromosomal aberrations and childhood epilepsy. Has anyone had registered cases with chromosomal abnormalities and epilepsy?
and more and more of the early encephalopathic epilepsies are known for a genetic background. I do a genetic panel of encephalopathic epilepsies in all my patients who are suspect for that kind of epilepsy. That includes ARX, CDKL5, FOXG1, KCNQ2, MECP2, PCDH19, PNKP, POLG, SCN1A, SLC9A6, STXBP1, SYNGAP1, UBE3A. And i have found more and more of these genetic epilepsies the last to yearsFollowing
- Kirsi Vaali added an answer:I am looking for anyone that is either conducting active research or has completed research on the AUTS2 gene 7q11.22 interuptions.I am in the process of compiling as much information as possible on AUTS2. If you are actively conducting research and need participants please contact me. If you have completed any research published or unpublished I would like to compare data and possibly see if any new findings have been made that I have not yet come across. I am particularly interested in the syndromic qualities that are not autism related. That information is well documented I am looking for things like: Microcephly, Short Stature, facial dysmorphisms, systemic findings, immunological, otolaryngological (specifically hearing losses), visual abnormalities, brain malformation, and neurocognitive deficits, Gastro findings, and any other connections to this gene's interuption that are considered syndromic in nature.Hi Cheri,
We are trying to start a study of autism in which the role of diet will be studied. We will take cases that are permanently living 24/7 in health care units, many of these are aggressive in behavior and the intention is to affect the behavior with gluten-free and milk-free diet. We will do electro encelographical measurements, behavioral evaluations, assay f-calprotectin, fecal microbiota (still not confirmed), serum anti-MAG, anti-gangliosides, S100, Vit D and B12, T3, Fe and ferritin, amylase-trypsin inhibitor IgG Abs, milk protein-specific Abs, and eventually metabolomics with 100 different metabolites. At the moment we are applying ethical permission (the first meeting will be at the end of July), and we do not yet have funding for these assays. We can run the study and collect the samples (when we have received the ethical permission) but will need to leave most of the assays for later if we will not get funding for this.
University of HelsinkiFollowing
- Bhairavi Srinageshwar asked a question:Incorporation of ddGTP?Why does ddGTP gets incorporated faster than other three ddNTPs during sequencing PCR?Following
- Jafar Mohseni added an answer:Why is mtDNA particularly susceptible to mutation?Can anybody please explain to me why mitochondrial DNA is particularly susceptible to mutation? I know that in the most organisms mtDNA lacks intron and repetitive DNA. Can anybody explain further? Thanks in advance.The POLG low fidelity and higher exposure rate to Oxidative agent made mtDNA pron to mutations. Despite of them, mtDNA is the most informative in phylogentic studies and sequencing based mutation rate for mtDNA in control region is .0043 per generation.Following
- Corrado Testolin added an answer:Does anybody have information about the genes involved in Aicardi syndrome?We have a clinical case and now we're doing a review article in molecular topics in Aicardi syndrome.I suggest this linK:
WWW.ORPHA.NET is a very good syndrome database where you can find information about syndromes and links to genetic Lab almost all over the worldFollowing
- Mert Burak Öztürk added an answer:Is there any software or website for matching sequences and diseases?For example, you got some DNA sequences from patient and you saw that there are some mutations. Thus, you wanted to check that sequences with possible diseases. Wıth this software/website you enter that sequences and it gives you possible diseases. Do you know like that program?Thanks to everyone for your interest. I decied to use firstly, UCSC to understand that sequences belong to which gene and secondly, HGMD to see the possible diseases with using that gene symbol.Following
- Bhairavi Srinageshwar added an answer:What is the difference between Mosaicism and Chimaerism?--Thank You!Following
- Hadi Bayat added an answer:Can somebody suggest a protocol for DNA extraction from amniotic fluid ?.Dear Bhairavi
Here is a good protocol:
1. Divide the sample of amniotic fluid into labelled 2 mL eppendorf tubes (the number of tubes will depend on the volume of sample received). Spin in microcentrifuge at 13,000 rpm for 10 min. alternatively, centrifuge the cells in a larger tube, remove supernatant and transfer entire pellet to single eppendorf using PBS and then centrifuge eppendorf as above.
2. Remove supernatant.
3. Add 375 uL salting out buffer, 6.25 uL 20% SDS and 125 uL proteinase K (5 mg/mL)
4. Incubate Over night in 37C water bath or 30 - 60 min at 65C (flicking to resuspend constantly) to digest protein.
5. Label 3 X 1.5 mL microfuge tubes
6. To the digested sample add 400 uL phenol, invert and spin at 10,000 rpm in microcentrifuge for 5 min.
7. Transfer aqueous layer to another tube and add 400 uL 1:1 phenol:chloroform. Mix gently by inversion, then spin 10,000 rpm in microcentrifuge for 5 min.
8. Transfer aqueous layer to a new tube and add 400 uL chloroform. Mix gently by inversion, then spin 10,000 rpm in microcentrifuge for 5 min.
9. Transfer aqueous layer to a new microfuge tube and add 1/10 volume 3 M NaAc, pH 5.2 (40 uL) and 2.5 volumes of 100% ethanol (1 mL). Invert tube gently to precipitate DNA.
10. Spin at 10,000 rpm for 5 min and pour off ethanol.
11. Rinse the pellet once with 70% ethanol, centrifuge 10,000 rpm for 5 min. (Do not include this step if the pellet is very small)
12. Dry DNA pellet in laminar flow cabinet and resuspend in 20 - 50 uL of sterile water depending on size of pellet.
Salting out Lysis Buffer
1M Tris pH.7.5 10 mL
5M NaCl 80 mL
0.5 M Na2 EDTA 4 mL
Add dH2O to 1lt. and mix reagents with magnetic stirrer for short while. Label and date. Autoclave. Store at RT.
- Francesco Saverio Dioguardi asked a question:Arginase 1, is it only a liver's matter?Very nice paper, and thanks to cite one of my first papers!
But, I am sorry to notice that in this paper, again, arginase 1 activity is defined as limited mainly to liver. Arginase 1 expression is controlled by exogenous arginine and arginase 1 is present also in red blood cells (RBC), not only in liver! Therefore, Arginase 1 concentrations in RBC and availability of arginine as substrate for eNos and/or iNOs in may be of pivotal importance when evaluating peripheral vasomotor efficiency. Moreover, recycling of citrulline to arginine in peripheral endothelium is dependent both on availability of aspartate exported from citric acid cycle, which is blunted if beta-oxydation becomes prevalent on glucose full oxydation, and maintained expression of the enzymatic path.Following
- Felix L Nunez-Santana added an answer:Can somebody share their GC rich kit protocol for PCR amplfication of difficult templates?see aboveFor GC-rich fragments and difficult templates, try using Taq DNA Polymerase with Q-solution.
The Q-Solution facilitates amplification of GC-rich templates or templates with a high degree of secondary structure by modifying the melting behavior of DNA. Use of this reagent often enables or improves suboptimal PCR. Unlike DMSO and other PCR additives, Q-Solution is used at a defined working concentration with any primer–template system and is not toxic.
Handbook: Table 5. Reaction Composition Using Taq DNA Polymerase, Q-Solution, and 10x PCR Buffer.
We also achieve far better results for this kinds of templates by replacing dGTP with 7-Deaza-2'-dGTP
You can obtain 7-Deaza-2'-dGTP from several vendor. i.e.
- Joep De Ligt added an answer:Is possible to perform a batch search of SNPs in the 1000 genomes browser in order to get individual genotypes for the variants?I need to perform some ancestry analysis and I'd like to use the individuals from 1000 genomes as proxies for the test.Hi, the complete dataset underlying the browser can be downloaded in different formats, or accessed through their data slicer tool.
The 1000 genomes DataSlicer tool is available here; http://www.1000genomes.org/data-slicer.
And raw dat file locations are listed here; http://www.1000genomes.org/data
From your question I think vcf files with genotypes are what you are looking for, which are available here;
The files can be downloaded per chromosome and using tabix (http://samtools.sourceforge.net/tabix.shtml) you can extract the desired genomic positions.
Answers to these types of questions are often also listed on the projects FAQ page;
- Jose A Lopez-Escamez asked a question:Would you recommend BIOgranat software for pathway-based analysis of WES data?We are trying to use this software to find novel variants in genes in proteins in the same pathway. Do you think that this strategy could be useful to find molecular targets in the same pathway?Following
- Ashraf Al ghzaly added an answer:How to calculate genotype relative risk for AA and Aa using Genetic Power Calculator?When using Genetic Power calculator, besides inputting the high risk allele frequency A (I used the hapmap data), the value of genotype relative risk for AA and Aa were required. How could I get the value? Should I use the data of previous literature with positive association to calculate?you can use CaTS Power Calculator program.
-Downloaded from Michigan University Center for Statistical Genetics web site: http://www.sph.umich.edu/csg/abecasis/CaTSFollowing
- Zarrin Basharat added an answer:How to predict nucleotide changes causing amino acid changes cause disease or not?I have already checked by using SIFT and Poly-Phen softwares. Is there any other software?Following
- Jaimin Patel added an answer:How to check amino acid change from an identified SNP?I have found an SNP in a gene under my research. In mutationtaster, it appears to be disease causing. How can I check for amino acid change caused by single bp change? Any online tool?Following
- Alka Ekbote added an answer:Why does polymerase slippage occur when there are homopolymeric repeats during sequencing?-Slippage occurs when the growing strand temporarily dissociates from the template,In case of homopolymers it can reassociate at a different spot - say, one nucleotide forward or back from where it started. This will make every individual band becomes a family of closely-spaced peaks giving a 'roller coaster' look to the chromatogram. Which is often regarded as slippage.Following
- Charles E. De Bock added an answer:Is there any possibility that retrovirally induced animals can shed the virus, which can in turn infect other animals in the lab?We are planning to induce chronic myeloid leukemia in mouse. Our plan is to induce CML by infecting bone marrow cells from donor mouse with retroviral vector, and then implanting into recipient mice. My question is, do these recipient mice pose any threat to other mice (non-transplanted) by shedding retroviral particles by any means? What I understood is that induced animals are not a threat to other animal, but I just wanted to confirm it from some literature before any final conclusions for lab ethical approval.In our laboratory we routinely carry out such experiments but for genes involved in ALL.
When you generate the retrovirus you will transfect two vectors into a packaging cell line. For example you will have pMIG or MSCV encoding your oncogene and you will transfect together with a packaging vector such sa Ecopac. This ecopac vector encodes the envelope proteins to generate the complete viral particle allowing infection of mouse/rat cells. However, the virus itself is replication defective - as the components of the virus were split between the two vectors you used.
You will then harvest the virus from the packaging cell line, transduce you lineage negative cells from donor mice and then inject these into your irradiated recipient mice. These donor cells are transduced but will not be able make virus.
So rest assured that your transduced mice will not be able to infect other mice at all.
All the best with your work.Following
- Sabine Strehl added an answer:Retroviral vector (MSCV-BCR-ABL-ires-GFP) - Is it possible to buy complete vector with gene of interest?We are planning to induce chronic myeloid leukemia in mouse model with the MSCV-BCR-ABL-ires-GFP retroviral vector, but I am unable to find any company who is selling complete retroviral vector (MIG) with gene of interest (BCR-ABL). Does anybody know if any company sell MIG vector where we can order with specific target gene ?
or if after buying empty vector, we have to insert our gene of interest?Another option would be to contact authors of published papers, in which this construct was already used, and ask them to provide you with an aliquot.Following
- Mohammed Ali Al Abri added an answer:How to correct a p value for genomic inflation factor?I found a case control sample's genomic inflation factor by Eigenstrat analysis. Now I have genotyped one SNP in this sample and calculated the p value for the allelic association test. How can I calculate the corrected p value for the genomic inflation factor ?I suppose that you can just divide your test statistic by the inflation factor (if the inflation factor is larger than one) then you can obtain the corrected p value
- Alberta liesbeth Paul asked a question:Does anybody have experience with Invivoscribe IGH Somatic Hypermutation assay?I want to use it for CLL analysis.Following
- Michael Mannen added an answer:What is meant by the Geneotype-Phenotype relationship?Except the one that is simple Geneotype codes for Phenotype. I am studying a paper on Mitochondrial Mutations and there it says "The geneotype-phenotype relationship of diseases caused by a Mutation is highly complex and poorly understood, although genetic heterogeneity between cells and tissues appears to be of great importance." What does this mean?A particular geneotype does not necessarily guarantee a particular phenotype. Understanding what factors can change this expression is quite complex.. What is what i believe that above statement meant.Following
- Ru-Jeng Teng added an answer:Why to use chemotherapy to treat leukopenia?If chemotherapy lowers bone marrow activity and thus reduces the white blood cell count, why would it be used as a treatment?Do you mean leukopenia secondary to hemophagocytosis? Some viral infections can manifest like a malignant disease with pancytopenia and chemotherapy may help. I have not taken care of hemophagocytosis patients for decades and can not remember the name of chemotherapeutic agents. Yu should be able to find them in literature.Following
- Amr Halawani added an answer:How to design a targeted NGS inherited disease panel?We are looking to set up a first tier phenotype specific disease panel for inherited conditions. The aim is for low cost, first pass in a diagnostic lab, after which exome or targeted mendeliome could follow. We are heading towards a Life Technologies Ion Torent custom panel. Most genes have 99% coverage, and we are planning on 100 depth. Most colleagues have indicated a preference for Illumina platforms, but cost is a big factor in tiered testing. We have been told that Life Technologies are overcoming the issues with repetitive sequences. Does anyone have any advice as to what pitfalls/ hurdles we can expect to face on the Ion Torrent?Hi Yemima,
we used to sequence the entire genes using Ion Torrent PGM and we are happy about the results. To be honest, we don't have an access to Illumina but it looks more accurate than Ion Torrent. But last month I believe Life Technologies has launched two technologies to reduced the indels and homopolymer errors.
Please see the following link:
- Pia Hermanns added an answer:What is the benefit of detecting a mutation which contributes in a disease or causes that disease?I want to know that when are going to detect a mutation in a population and its frequency in any disease, and when it does it then what is its benefit? As we can't revert it in a functioning body. Kindly explain the benefit of evaluating/screening or detecting a mutation?Following
- Chong Ge You added an answer:How do I adjust the 'p' value of haplotype association study for multiple testing?I genotyped several SNPs of a gene and investigated the association of all possible two and three marker haplotypes with the disease. In this case, how do you adjust the ' P' value for multiple testing? These tests are related.Including P value in the software of SHEsis.Following
- Helmuth Haslacher asked a question:Is there any reliable method for genotyping of Chido/Rodgers blood group status in human complement 4 alleles?Genoytping Chido/RodgersFollowing
- Vadieh Ghodsinejad asked a question:Primers that can differentiate the GBA from the GBAP?Alternation in the GBA gene leads to Gaucher disease and this gene has 98% homology with its pseudogene, GBAP. In several papers that I have read, researchers applied combination of different methods such as PCR, RT-PCR, and Long PCR to differentiate the GBA from the GBAP gene.
I need primers that can differentiate the GBA from the GBAP. My problem is that the primers that I found for PCR-sequencing and RT-PCR, they amplify both of the GBA and GBAP genes and cannot cover the entire GBA cDNA respectively. Also, we only can use either PCR-sequencing or RT-PCR, and Long PCR is not set in our lab.Following
- Vikki Stefans asked a question:There are extensive GWAS and genomic studies on myopathic side effects of statins. Is anyone aware of similar studies on cognitive side effects?This was controversial at one time, but it happened to me and possibly to my parents, and the FDA has validated cognitive dysfunction as a known side effect. It is clearly less common than the myopathy, but I think worthy of attention; it seems possible that some genes predipsosing to dyslipidemia could be involved in these negative effects on brain function as well. It is possible also that pravastatin has lower risk for this than the other statins.Following