Genomic DNA copies

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harrypbs
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Genomic DNA copies

how can one determine the number of copies of chromosome present in a cell?

roudi
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harrypbs wrote:how can one

harrypbs wrote:

how can one determine the number of copies of chromosome present in a cell?

Genes are found in specific segments along the length of human DNA, neatly packaged within structures called chromosomes. Every human cell contains 46 chromosomes, arranged as 23 pairs, with one member of each pair inherited from each parent at the time of conception. After conception, these 46 chromosomes duplicate again and again to pass on the same genetic information to each new cell in the developing child. Human chromosomes are large enough to be seen with a high-powered microscope, and the 23 pairs can be identified according to differences in their size, shape, and the way they pick up special laboratory dyes.

Do you need to know name of dyes used to stain chromosomes

harrypbs
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sorry sir

sorry sir
but i need to know the number of copies present in bacteria, not human beings.I want to know that the chromosome present in this bacteria is in single copy or is in multiple copies?

labrat
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That would depend on the

That would depend on the materials etc you have available and exactly why you don't know the chromosome numebr - is the chromosome number unknown because you don't know how many that species has? or is it because you think there have been cellular changes? do you think one particular chromosome has increased in number.

Here are a few ideas -

Arrest the cells in metaphase and produce metaphase chromosome spreads and then count the chromosomes in say, 20 cells and average them - I'veonly done this in human cells so I have no idea how easy this would be in bacteria.

If you're looking at a possible increase in copy number of one (or only a few) known chromosomes as compared to the normal cell line, you could do quantitive pcr on the normnal vs the altered bacteria on a gene known to lie on that chromosome.

Maybe a bit more info on why you don't know the chromosome number already would help.

harrypbs
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Sir

Sir
thanks for the reply
what i know about my bacteria is that it has one chromosome and six mega plasmids.But the problem is that i heard from many people that in this bacteria chromosome itself is present in multiple copies, but i didnt found any literature. So i want to confirm that the one chromosome which is present per cell, is that present in multiple copies or single copy?

Tony Rook
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harrypbs:

harrypbs:

Do you know the size of the bacterial genome? If so, you should be able to extract and purify the bacterial genome from the plasmids. From there you can use a number of methods to quantitate the purified genomic DNA. Then you can back calculate how many copies of this may be present. Try working with some companies such as Promega or ABI, there tech service dept may be able to suggest some kits to make your life easier.

Tony Rook

Sandy
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Tony Rook wrote:harrypbs:

Tony Rook wrote:

harrypbs:

Do you know the size of the bacterial genome? If so, you should be able to extract and purify the bacterial genome from the plasmids. From there you can use a number of methods to quantitate the purified genomic DNA. Then you can back calculate how many copies of this may be present. Try working with some companies such as Promega or ABI, there tech service dept may be able to suggest some kits to make your life easier.

Tony Rook

Bacteria are prokaryotic. Rather than a nucleus, each possesses a single main chromosome in the cells cytoplasm. That sounds simple enough. But to make life more interesting for microbiologists, bacteria may also possess small, circular genetic elements, called plasmids, that can pass from one bacterium to another. Plasmids carry several nonessential genes that sometimes confer antibiotic resistance on the bacteria that receive them.

Several companies, among them Qiagen, Roche Applied Science, Sigma-Aldrich, and Stratagene, provide kits and reagents for isolating and purifying plasmid DNA. Amersham Biosciences have a product called TempliPhi that allows you to amplify extrachromosomal DNA such as plasmids. Virtually every major genome center is using it today and they are about to launch GenomiPhi, a product that allows one to amplify genomes of all types, including bacteria, by a factor of 10,000. With a small amount of sample one can amplify the whole genome without the sort of skewing that amplifies one region more than another.

harrypbs
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thanks sir for the reply

thanks sir for the reply
but i still didnt get my answer.my bacteria has one chromosome of 6.4 Mb and six mega plasmid ranging from 5 kb to 800kb. My doubt is that this one chromosome is present in more than one copy per cell probably. With plasmids i dont have any problem, they are there in one copy per cell total six plasmids per cells and they have essential genes also thus forming an essential part of my bacterial genome. But that one chromosome is probably present in more than one copy per cell. so how can i confirm this, tell me if anybody can.