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How can I get the DNA fragment that i need!

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How can I get the DNA fragment that i need!

Postby cobi » Thu Apr 28, 2005 5:52 am

Dear everyone,
I am a new member. I am getting start working in the lab for genomics informatics so a lot of things that i have not yet understood, please explain and recommend me the sources of related documents for my following broblem, thanks a lot in advance.

When we use the kit of Bioneer or Toyobo to extract DNA from blood to do PCR and RFLP for the MC4R gene. After finishing extraction DNA step, The tube contain the DNA molecules of all chromosomes in the cells, how can we get the MC4R gene or the DNA fragment that contain the MC4R gene, we regard, to further study with it?

Please help me,
Thank you very much for your kind regards!
cobi
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Postby DevGrp » Thu Apr 28, 2005 9:01 am

Thats were the PCR / RFLP comes in.
If you want to do PCR then you design PCR primers based on the MCR4 DNA sequence. This sequence can be found at http://www.ncbi.nlm.nih.gov/gquery/gquery.fcgi. Thi sdatabase has 43 hits for nucleotide sequences described as MCR4, but I can't access any as entrez is down at the moment

Is That Melanocortin 4 receptor, the "hunger gene" btw?

So once you have your MCR4 specific primers you can use PCR to amplify the MCR4 gene from your extracted DNA and use it for what ever you want ?sequencing?

RFLP (restriction fragment length polymorphism) is a slightly older technology. First you restriction digest your extracted DNA, seperate it on a gel and then transfer that to a membrane (southern blotting). Now you hybridise the membrane with a probe designed from the MCR4 gene sequence. You can get a probe by PCR, previuosly cloned MCR4 gene in a plasmid or have a long sequence specific oligo made. The probe also has to be labelled (radioactive 32P / 33P / 35S, or biotin/ DIG/?) so that you can detect where the probe has hybridised). You then detect the probe using Xray film, or in a posh modern lab with a special digital camera. Any signal on your membrane should be from the MCR4 gene. If different individuals have MCR4 gene fragments of different sizes this is due to sequence variations in the MCR4 gene (mutations?) causing resitriction sites to be changed.

Hope this helps.


What technique you use really depends on what you are trying to do.
DevGrp
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Postby cobi » Fri Apr 29, 2005 1:17 am

Dear DevGrp,
Thank you very much for your kindly help.
I have understood alot for my lab learning base on what your help.

Is That Melanocortin 4 receptor, the "hunger gene" btw?

That's right. MC4R has known as a gene regulating food intake causing obesity or diabetes on human. It also has known as the main factor that affect the quantitative traits such as back fat thickness, marbling muscle score, average daily gain and reproductive ability on livestocks.

I will use RFLP technique to compare and realize whether have variation on MC4R or not among the pig species and the individuals in the line. The result will counsel the farmer making the breeding programme to get a good performance and least cost.

RFLP (restriction fragment length polymorphism) is a slightly older technology


As what you told, I think that you deeply understand a bout lab. biotechnology, so if it is convenient, please give me some information about the new techniques to detect the variants ( genetic markers, molecular markers) among individuals or among species to
improve the genetic in the livestocks. Are they Microsatellite and SNP ( Single Nucleotide Polymorphism) ?

Thanks again for your help.
Have a good weekend
cobi
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Postby DevGrp » Fri Apr 29, 2005 11:16 am

Sorry I have no experience with livestock genetics, my recent experience is with humans and more recently plants. I also have no experience in things like marker assisted breeding but I have recently started looking into SNP detection using TILLING (Targeted Local Lesions IN Genomes)
See http://www.ncbi.nlm.nih.gov/entrez/quer ... d=15282033

It is a method we are using to first induce (using chemical mutagenesis ie it’s a non-GM method) and then detect mutations in specific genes which might lead to improved traits in crop plants. You then PCR the gene you are interested in, mix the PCR product with wild type DNA and allow the DNAs to anneal together. Any mutations form mismatches which are then sensitive to nucleases (S1, mung bean or our favourite Cel1-which we extract from celery). We then use an old LICOR automated DNA sequencer to detect any cleaved PCR products (you can also use DHPLC but it takes longer). This then identifies which individuals have a mutation and we then go back and sequence the gene from that individual and phenotypically screen it.
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Postby cobi » Sat Apr 30, 2005 5:02 am

Thanks to DevGrp very much for your regards,

I have found some articles related in TILLING ( targeting induced local lesions IN Genomes) method. This is a new method to detect the mutations but all the articles that i found mentioned only for plant science researches, no one for animal science. After reading, I am thinking that i can be applied it to animal genetic. I will pay attention to this method! That is a potential technique for animal genetic markers!

Have fun!
cobi
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Postby DevGrp » Tue May 03, 2005 8:05 pm

TILLING was / is being applied to Zebrafish but at least one group has stopped (I know this as we bought some of their old equipment) The problem in higher organisms is all the non-coding DNA. In the planst we work on if you TILL a 1000bp region about half of it will be coding region as there are usually only a few small introns (so a typical 1.5kb transcript requires you to TILL 3kb of DNA. In animals the ratio of introns to exons is MUCH higher a gene could require you to TILL 50kb which isn't practical. Mutations in introns are also much more common ( less / no selction pressure) so in higher organisms you get far more silent mutations. This is probably why TILLING isn't appropriate for analysising traits in higher organisms. Sorry should have thought about it before I mentioned the technique.
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Postby cobi » Mon May 09, 2005 4:26 am

OK, I see the reason why TILLING have not apply for animal researches. Thanks to DevGrp for your explaining~

As my knowledge, now the scientists try to use Microsatellite ( STR, SSR) and SNP markers to discover and improve the livestock performance.

Have agood health!
cobi
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