In addition, sometimes the expression of disease susceptibility genes itself does not change, but plays a role by regulating other genes. Therefore, the search for pathogenic genes should start from many aspects.
If there is anything wrong with my point of view, please advise me. Thank you, very good brother. My first step may just be to change the expression spectrum. If I have the chance to do it, as you said, I should consider it from all aspects. My current idea is to use the gene chip technology of expression profile to make the overall information about the difference of gene expression profile between patients and normal people. As maxon told you, finding a new disease-related gene may or may not be possible. I think it is at least one aspect (I don't know if it is right). What I can consider now is how to sort out my thoughts and come and do this job well. There are still a few questions to ask:
1. In the method of establishing gene library, for example,118 genes were selected from an article for research, which were divided into known genes, known sequences and unknown genes. Through BLAST, I don't understand how they were screened from the gene library (reverse transcription from whole cell mRNA). (or did you find it from other places? ), I understand that it seems to be direct sequencing. How to find (isolate) these genes from the gene bank and sequence them one by one?
2. how to use BLAST? For example, how to find the gene sequence of 1 1 18 trabecular cell expression profile that has been identified in the same article? Can you explain it to me? Thank you very much
Have you noticed a problem? Gene chip can only detect known genes or sequences, but it can't do anything about those unknown. Andrew is right, but the number of genes in the chip increases with the deepening of gene research. For general research, the main known channels can basically include. Thank you for your advice. Can anyone answer my above questions? I still don't understand. I haven't understood the information after reading it all day.
I would like to ask: If I customize a chip (including 1 1 18 gene) from a normal population, I find that there is a gene whose expression is down-regulated or not expressed in a patient's cDNA sample. What is the reason? Is there really no expression or something?
Thank you. Thank you. Are the samples consistent? Such as blood cells, are their cell subsets comparable?
Is there a comparison? The samples were random, and trabecular cells were homogeneous endothelial cells. As for control, do you mean negative control, positive control or internal control of transcription?
I know very little, and I may make low-level mistakes. Please give me your advice. In addition to the error of experiment and DNA chip, a gene was found to be down-regulated or not expressed in hybridization with patient's cDNA sample, which needs to be verified by RT-PCR. The down-regulation or non-expression of its expression may be regulated by its upstream gene, or it may be that the structure of the gene itself has changed, such as nonsense mutation, and the decrease of expression can be detected. After these genes are confirmed by RT-PCR, they should be sequenced to see if these genes have structural abnormalities. With the daily help of the webmaster and comrades-in-arms, I finally learned a little about the subject I am applying for, and completed the writing of the application for the Natural Science Foundation. Before tomorrow, the application that we have condensed everyone's sweat and wisdom will be sent out. I want to express my sincere thanks for your help these days. Although this is my first time to write such an application, I still feel that I have learned a lot and made many good friends, although it is almost impossible.
I have attached the text of this application. I hope interested friends can have a look and put forward some valuable opinions, because I think such a topic is still worth doing, although we may not have the opportunity and ability to do it.
Thanks again!
884 1 1-.doc (76.5k) Congratulations on your successful application! ! Thank you for your daily guidance, and thank you comrades.
Recently, the research fund began to declare, and the boss applied for the project urgently. Because I have just contacted the Foundation, I would like to ask the webmaster and comrades.
At present, 1 has collected a rare monogenic disease (epilepsy), and there is no clinical and basic report in China. Clinical work, including blood sampling, has been completed.
Since 1998, the pathogenic gene has been located and cloned, but there are genetic heterogeneity and different mutation sites of the same pathogenic gene. Many articles were published in authoritative magazines such as Nature and Genetics. The latest research shows that there are other unknown pathogenic genes.
The cooperative laboratories have successful experience in locating and cloning pathogenic genes.
The purpose of our application is to locate and clone pathogenic genes, and it is expected to find new pathogenic genes.
I want to ask you:
1 at present, can I only apply with clinical data?
2 What more work needs to be done?
If so, what are the possible reasons for the application failure?
Thank you and look forward to your advice! Thank you. If it is a monogenic disease, it depends on the family you collected. Another question is whether your clinical diagnosis is correct. I am not clinical, and this clinical diagnosis is of great significance. If some of them are wrongly diagnosed or typed, it is likely to lead to single-gene diseases in which disease genes cannot be found. The technical strategy in this field is very mature, and there are many documents for reference. There are also many research institutions in China. I want to study the relationship between gene SNP and disease. There are reports abroad that there are two links. So should I do RFLP analysis or SNP analysis? Heroes, I have recently drawn a disease-related gene of an X-linked genetic family. Now I have scanned two loci of the genome with markers (STR), but I have encountered difficulties in linkage analysis. I use linkage (version 5. 1). I would like to ask you what is the difference between the genetic parameters of neutral linkage and autosomal linkage in linkage analysis? Looking forward to your advice, thank you very much! I want to study the relationship between gene SNP and disease. There are reports abroad that there are two links. So should I do RFLP analysis or SNP analysis?
RFLP is the earliest genetic marker (first generation). With the development of genetics and the increasing number of sequencing fragments, the second and third generation genetic markers have appeared. Analysis of RFLP by enzyme digestion shows that this method is simple and cheap, but its specificity is poor and it is easy to be eliminated. SNP localization is clear and relatively expensive, and can be analyzed by sequencing, snapshot, fluorescent probe, SNP chip and other methods.
Specific RFLP analysis, or SNP analysis depends on your research objectives and economic strength. For verygood, can you introduce small sequencing (snapshot)?
I recently wanted to test the relationship between a gene and a disease. There are many exons (20), and there have been mutation hotspots in other diseases (9, 1 1, 13, 17 exons), but the diseases I want to study have not been reported. Should I sequence all exons? Kou Dan wrote:
For verygood, can you introduce small sequencing (snapshot)?
I recently wanted to test the relationship between a gene and a disease. There are many exons (20), and there have been mutation hotspots in other diseases (9, 1 1, 13, 17 exons), but the diseases I want to study have not been reported. Should I sequence all exons?
Snapshot is a small sequencing reaction, and its principle is simply to amplify a DNA template containing SNP, then purify the PCR product, add ddNTP and an intermediate probe with different fluorescence (the so-called intermediate probe is an oligonucleotide sequence of about 20 bp of SNP, and the probe and ddNTP are combined according to the template sequence, because it is ddNTP, and the combined ddNTP reacts with SNP), and then purify it for electrophoresis, so that the corresponding SNP genotype can be judged according to the different fluorescence.
This method is suitable for the confirmation of known SNP alleles and has low requirements for probes. However, there are many operation steps, so it is difficult to apply it on a large scale (the relative workload is less when using capillary sequencing method, such as ABI3 100 sequencer series).
To detect the relationship between a gene and diseases, there are many exons (20), and other diseases have mutation hotspots (9, 1 1, 13, 17 exons). I suggest you study these loci first. Of course, if the gene sequence is very short, it can also be sequenced directly, because the SNP or mutation found so far is only about 2% of the expected value.
Good luck. Thank you. Very good:)
I have been busy defending my thesis recently. I am a complete novice in this field, but my boss said I want to innovate, and my classmates gave me this idea.
At present, DNA has been extracted and genotyped But I want to know by sequencing. The snapshot mentioned above is small-scale sequencing. I have identified the mutation site, and the fragment is about 300bp. Can I sort them all?
In addition, is it possible to sequence all samples or just select a few heterozygotes and homozygotes to prove it? Where did this information come from? I'm still a novice: (nothing to look around and write:
Thank you very much:)
I have been busy defending my thesis recently. I am a complete novice in this field, but my boss said I want to innovate, and my classmates gave me this idea.
At present, DNA has been extracted and genotyped But I want to know by sequencing. The snapshot mentioned above is small-scale sequencing. I have identified the mutation site, and the fragment is about 300bp. Can I sort them all?
In addition, is it possible to sequence all samples or just select a few heterozygotes and homozygotes to prove it? Where did this information come from? I'm still a novice: (
If it is only 300bp and there are not many samples, it is better to sequence directly, because not only the known SNP genotypes can be identified, but also some unreported ones may be found by the way, that is, all samples should be sequenced.
If you only want to genotype the known SNPs, you can use snapshot method, and of course you can also use RFLP, but the specificity is poor. The obtained bands are not necessarily related to different alleles of the target SNP, and can be cut to other regions of the chromosome.
There is no certain information in this respect, and I gradually understand it after doing it. What kind of technology should be adopted according to local conditions. Verygood wrote
To detect the relationship between a gene and diseases, there are many exons (20), and other diseases have mutation hotspots (9, 1 1, 13, 17 exons). I suggest you study these loci first. Of course, if the gene sequence is very short, it can also be sequenced directly, because the SNP or mutation found so far is only about 2% of the expected value.
Thank you, very well, sir. The coding region of the gene I studied is 2930bp, the mRNA 5084 BP, and the total length of the gene is 80kb. It was intended to be sequenced directly, but there were 18 cases (paraffin) in the patient group and 20 cases in the control group (how about peripheral blood DNA? ), the cost may be 60,000! ! ! So now I want to change it to PCR-SSCP plus abnormal band sequencing. Do you agree? Verygood wrote:
If it is only 300bp and there are not many samples, it is better to sequence directly, because not only the known SNP genotypes can be identified, but also some unreported ones may be found by the way, that is, all samples should be sequenced.
If you only want to genotype the known SNPs, you can use snapshot method, and of course you can also use RFLP, but the specificity is poor. The obtained bands are not necessarily related to different alleles of the target SNP, and can be cut to other regions of the chromosome.
There is no certain information in this respect, and I gradually understand it after doing it. What kind of technology should be adopted according to local conditions.
What software detects mutations after sequencing? Are there any special biostatistics books about other statistical analysis? Or just ordinary statistics? To coldant:
In the preliminary investigation, your method should be feasible.
Nothing to do:
After sequencing, mutations are mainly screened by sequence alignment, which can be carried out by Blast. However, it is necessary to carefully determine whether it is indeed a mutation, and to expand the sample for typing research. You have too few cases and controls for disease-related research. Generally, there are 200 to 200 domestic periodicals, and about 400-500 to 500 foreign periodicals. First-class magazines are generally at least 1000 to 1000. Because you don't have enough money, you can't do sequencing, so choose the known sites directly. Because this gene is related to many diseases, it means that this gene is very conservative and probably related to the disease you are studying. Even if it is irrelevant, you can always use age, gender and risk factors of disease (that is, playing digital games) to make a comprehensive analysis and post articles.
At present, the main way to find SNP of disease-related genes is to directly sequence (DNA extracted from peripheral blood, not tissues), and to find SNP by comparing the gene sequences of patients and normal people (people who are not sick). The explosion mentioned by verygood doesn't actually apply.
A pair of primers 1 can be designed for the region where the target SNP is located, so that the SNP is located in it, and the PCR length is about 500bp. At the same time, a pair of PRIMER2 is designed in the area covered by PRIMER 1. The 3' terminal base of one primer of primer 2 must be complementary to the normal base of the target SNP site, so that if the patient mutates at this site, a pair of primers of primer 2 will not be amplified. In addition, the distance between primer 2 and primer 1 is at least 100 BP, and the product of primer 2 is more than 200 BP. In this way, when these two pairs of primers are put into a PCR reaction at the same time, four fragments can be obtained (the length of these four fragments must be different when designing primers, so as to distinguish them in electrophoresis), while the individual with the target SNP has only three fragments, and whether the individual has a mutation can be determined by electrophoresis.
I forget the specific name of this method. I hope I can help you. Maxon wrote:
At present, the main way to find the SNP of disease-related genes is to directly sequence (DNA extracted from peripheral blood, not tissues), and to find the SNP by comparing the gene sequences of patients and normal people (people who are not sick). The explosion mentioned by verygood doesn't actually apply.
A pair of primers 1 can be designed for the region where the target SNP is located, so that the SNP is located in it, and the PCR length is about 500bp. At the same time, a pair of PRIMER2 is designed in the area covered by PRIMER 1. The 3' terminal base of one primer of primer 2 must be complementary to the normal base of the target SNP site, so that if the patient mutates at this site, a pair of primers of primer 2 will not be amplified. In addition, the distance between primer 2 and primer 1 is at least 100 BP, and the product of primer 2 is more than 200 BP. In this way, when these two pairs of primers are put into a PCR reaction at the same time, four fragments can be obtained (the length of these four fragments must be different when designing primers, so as to distinguish them in electrophoresis), while the individual with the target SNP has only three fragments, and whether the individual has a mutation can be determined by electrophoresis.
I forget the specific name of this method. I hope I can help you.
Hehe, I mean to use blast to facilitate sequence alignment. Of course, applied biosystems has better software, but it is difficult to get it without buying the corresponding instruments.
As for the number of specimens, the more the better. For the pathogenic site with a relative risk of 2, the detection efficiency of 1000 case control can reach 100%, and the detection efficiency will decrease with the decrease of the number of cases. However, for the previous research, it is not clear whether the site is related to the research disease, so large-scale investment may lead to no harvest.
For reference. Today Ji Kang suggested that I directly sequence the samples to form a "pool?" Save money for the exam. Their initial suggestion was to form a 1 pool for normal patients and patients respectively, and then use TAG MAN (a new technology) of the company to detect SNP on a large scale, but I don't have that many patient samples. So we just need to rank it.
Can you take a look at this? If I divide 25 patients into 6 "pools", can I sort them and analyze them again?
Thanks in advance. Maxon wrote:
At present, the main way to find the SNP of disease-related genes is to directly sequence (DNA extracted from peripheral blood, not tissues), and to find the SNP by comparing the gene sequences of patients and normal people (people who are not sick). The explosion mentioned by verygood doesn't actually apply.
A pair of primers 1 can be designed for the region where the target SNP is located, so that the SNP is located in it, and the PCR length is about 500bp. At the same time, a pair of PRIMER2 is designed in the area covered by PRIMER 1. The 3' terminal base of one primer of primer 2 must be complementary to the normal base of the target SNP site, so that if the patient mutates at this site, a pair of primers of primer 2 will not be amplified. In addition, the distance between primer 2 and primer 1 is at least 100 BP, and the product of primer 2 is more than 200 BP. In this way, when these two pairs of primers are put into a PCR reaction at the same time, four fragments can be obtained (the length of these four fragments must be different when designing primers, so as to distinguish them in electrophoresis), while the individual with the target SNP has only three fragments, and whether the individual has a mutation can be determined by electrophoresis.
I forget the specific name of this method. I hope I can help you.
Hehe, thank you. What I saw in the relevant literature was that two primers (mutant and non-mutant) were designed, and the other antisense primers were the same. The primers designed by the normal control group are very similar to those you said about PROMER2. I want to know why I did it. Verygood wrote:
Nothing to do:
After sequencing, mutations are mainly screened by sequence alignment, which can be carried out by Blast. However, it is necessary to carefully determine whether it is indeed a mutation, and to expand the sample for typing research.
Make sure it is impossible to draw a conclusion, just put forward a prospect. After sequencing, it can be analyzed by SEQUENCEMAN software, but I want to add an RFLP later, according to relevant literature reports. This analysis seems to be supported by more data. Kou Dan wrote:
Today Ji Kang suggested that I directly sequence the samples to form a "pool?" Save money for the exam. Their initial suggestion was to form a 1 pool for normal patients and patients respectively, and then use TAG MAN (a new technology) of the company to detect SNP on a large scale, but I don't have that many patient samples. So we just need to rank it.
Can you take a look at this? If I divide 25 patients into 6 "pools", can I sort them and analyze them again?
Thanks in advance.
Hehe, did you also do it in Jikang? It seems that they use probes to detect SNP. I heard that the accuracy of probes is not as good as that of direct sequencing. I wonder what advice they gave you? :) Maxon wrote:
For disease-related research, you have too few cases and controls. Generally, there are 200 to 200 domestic periodicals, and about 400-500 to 500 foreign periodicals. First-class magazines are generally at least 1000 to 1000. Because you don't have enough money, you can't do sequencing, so choose the known sites directly. Because this gene is related to many diseases, it means that this gene is very conservative and probably related to the disease you are studying. Even if it is irrelevant, you can always use age, gender and risk factors of disease (that is, playing digital games) to make a comprehensive analysis and post articles.
5555555, but I can't accept so many cases, and the funds are limited.
What does it mean to be a known site directly? Besides, have you read any books about biostatistics? I heard that you can refer to it and do relevant analysis. Which library or bookstore in Shanghai has it? I forgot the specific method. Statistics mainly refers to two types of T-test and X2 polymorphism analysis methods:
Firstly, based on family analysis, linkage disequilibrium method is mainly used.
Secondly, based on case control, as maxon said, it is mainly t-test and X2. But pay attention to whether the control can represent the sampling population. False positive results caused by sampling errors abound in early literature, which gradually attracted everyone's attention. Nihility turned around and wrote:
Hehe, did you also do it in Jikang? It seems that they use probes to detect SNP. I heard that the accuracy of probes is not as good as that of direct sequencing. I wonder what advice they gave you? :)
It seems that the idle work is very similar to me, so we can communicate more!
Ji Kang Company proposed that 25 people in each group, 25 people in the control group (only 25 people were collected), and a group of 4 people formed a "pool", so the exons were amplified by PCR and sequenced directly. (save money)
Shenneng Company suggested that each patient directly amplify and sequence, and compare with genbank (excluding the control group, the cost is 18000 yuan/10 cases).
Beijing Dingguo Company: PCR-SSCP, (normal, 25 patients in each group)
Ask verygood, maxon, and let the comrades discuss which one is feasible.
Ask bamboo for help. Kou Dan wrote:
It seems that the idle work is very similar to me, so we can communicate more!
Ji Kang Company proposed that 25 people in each group, 25 people in the control group (only 25 people were collected), and a group of 4 people formed a "pool", so the exons were amplified by PCR and sequenced directly. (save money)
Shenneng Company suggested that each patient directly amplify and sequence, and compare with genbank (excluding the control group, the cost is 18000 yuan/10 cases).
Beijing Dingguo Company: PCR-SSCP, (normal, 25 patients in each group)
Ask verygood, maxon, and let the comrades discuss which one is feasible.
Ask bamboo for help.
I have 30 cases, compared with 12. Somebody else's suggestion is direct sequencing. I want to make an RFLP after sequencing, because I have to write a paper, so the content can't be less.