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how to determine certain gene copy number? - (Jun/22/2009 )

I kow southern blotting is one of the techniques capable to determine the gene copy number but how? I am new for this technique. Do I need to set up a control to determine the interested gene copy number? Thanks in advance for your help!

-zx0819-

zx0819 on Jun 22 2009, 01:59 AM said:

I kow southern blotting is one of the techniques capable to determine the gene copy number but how? I am new for this technique. Do I need to set up a control to determine the interested gene copy number? Thanks in advance for your help!


Copy number can be determined from a Southern pretty easily. Homologs can be detected too. And if you're working with transgenics, the southern will also identify multiple insertion sites in an animal.

Copy number is calculated by comparing the signal intensity of your unknown copy number to your known copy number. Your known will typically be the transgene in plasmid form, and it is blotted using picograms equal to 1 copy, 10 copies, 50 copies, etc.

-eldon-

eldon on Jun 23 2009, 08:50 AM said:

zx0819 on Jun 22 2009, 01:59 AM said:

I kow southern blotting is one of the techniques capable to determine the gene copy number but how? I am new for this technique. Do I need to set up a control to determine the interested gene copy number? Thanks in advance for your help!


Copy number can be determined from a Southern pretty easily. Homologs can be detected too. And if you're working with transgenics, the southern will also identify multiple insertion sites in an animal.

Copy number is calculated by comparing the signal intensity of your unknown copy number to your known copy number. Your known will typically be the transgene in plasmid form, and it is blotted using picograms equal to 1 copy, 10 copies, 50 copies, etc.

But if my interest plant is a wild type one and there is not so much information on copy number of certain gene?

-zx0819-

zx0819 on Jun 22 2009, 06:12 PM said:

But if my interest plant is a wild type one and there is not so much information on copy number of certain gene?


Same procedure applies. Have you cloned the cDNA or your gene? This will be your control.

If you know the haploid size of the plants genome, the gene size and a known amount of gDNA you can assess gene copy number.

Here is an example...just substitute your plant numbers into the haploid genome size:

Assumption: the Haploid content of a mammalian genome is 3 X 10^9 bp
Assumption: you have 2 micrograms of gDNA available

You want to determine the amount of your gene to assay by Southern. Since transgenic founder mice are hemizygous:

mass of transgene/1 ug of gDNA = N bp transgene DNA/3 X 10^9 bp gDNA


Now, for a 5,480 bp gene/transgene..re-arrange the equation to:


mass of gene DNA = 2 x <(5,480 bp cloned DNA) X (1 g gDNA)/ 3 X 10^9 bp gDNA>

mass of transgene DNA = 3.66 picograms

Thus, to prepare a 1 copy standard: use 3.66 pg of gene DNA and compare signal intensity to 2 microgram of gDNA
0.1 copy 0.366 pg
1 copy 3.66 pg
10 copy 36.6 pg
50 copy 183 pg
100 copy 366 pg

-eldon-