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DNA Sequencing / Cloning ?


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#1 RoseHR

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Posted 20 August 2010 - 04:12 PM

Hello!
I would just like to ask about DNA sequencing methods; for my dissertation, i spent months extracting DNA and then cloning my sequence using bacterial cultures to produce a sample that we were able to sequence from. Perhaps it was the rush of handing the project in on time that made me blank but could someone possiably tell me why DNA from a bacterial cell was sequenceable whereas it wasn't from the original mammalian cell? I guess my point is, what was the purpose of the cloning step and does sequencing via bacteria produce more accurate results from that of a mammals?

I'm guessing that perhaps the genome is smaller thus makes it more accurate to sequence from?

I would greatly appreciate some wisdom and also are there any other methods for sequencing DNA other than cloning the sequence first?

Many thanks from a fledgling scientist!

#2 HomeBrew

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Posted 20 August 2010 - 06:05 PM

Each mammalian cell contains one or two copies of your gene, whereas each bacterial cell contains 500 - 1000 copies of your gene cloned into a high copy number plasmid. Moreover, growing billions of bacterial cells, each containing many, many copies of your gene is ridiculously easy, as is recovering the vector (with all those copies of your gene) in highly pure form.

It's not that you can't sequence DNA recovered from mammalian cells -- you can (it's the same DNA, after all), it's just so much easier to recover sufficient quantities of your gene in pure form from bacterial cells than it would be to recover an equal number in as pure form from mammalian cells.

#3 RoseHR

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Posted 21 August 2010 - 04:32 AM

Oh I see! I didn't realise the plasmid contains multiple copies of the gene I only thought it contained one, well that makes it much easier to understand, thank you very much! :)

#4 HomeBrew

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Posted 21 August 2010 - 12:31 PM

Just to clarify -- the plasmid doesn't contain multiple copies of the gene; each plasmid only contains one copy. The bacterial cell, however, contains many copies of the plasmid. How many plasmids are in each cell is determined by a property of the plasmid known as "copy number", and -- for high copy number plasmids -- may run as high as 1000 copies of the plasmid per individual bacterial cell.

#5 RoseHR

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Posted 21 August 2010 - 05:04 PM

Ahh I see! thankyou again :)!

#6 Kaioshin

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Posted 31 August 2010 - 06:08 PM

Um...you guys forgot the most important part. When you clone into a vector you are cloning an unknown section of DNA into a known section. Knowing the vector sequence allows you to have primers, which are required for the sequencing reaction. You




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