PCR copy number calculation

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Amtekoth
Amtekoth's picture
PCR copy number calculation

I'm having a brain freeze.

I need to calculate DNA copy number of a sequence in a virally infected genomic template.

I have the following info: size of amplicon and an extrapolated ng based on a plasmid control standard curve after QPCR.

I'm using this equation to determine wt of amplicon in daltons: bp DNA x 330 Da x 2 nucleotide ds

I then divide that by avogadro's number.

I take the calculated nanograms of amplicon (based on the CT and standard curve), divide by 1000000000 (for grams). This was then divided by the derived number above.

What am I missing here?

Thanks,

SanDiablo
SanDiablo's picture
X ng * (1 nmole bp / 660 ng)

X ng * (1 nmole bp / 660 ng) * 1/N bp * 1 mole / 10e9 nmoles * 6.023e23 molecules / mole = # of molecules

Where N is the number of base pairs in your measured DNA sample (assuming this is for double stranded DNA).

I don't typically think of DNA in terms of Daltons...

Amtekoth
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That gives me even stranger

That gives me even stranger numbers...

Before, I was about 100x over expected values. Now I'm 1000000x over expected values.

Yikes...

Maybe I misdefined my original concentrations...

I'll rerun the PCR after rechecking the DNA concentrations.

SanDiablo
SanDiablo's picture
When you say 'extrapolated'

When you say 'extrapolated' do you mean your unknown did not lie on the standard curve? If so, you cannot accurately determine the copy number...

Double check that you are calculating "copies" of the amplicon within the plasmid...i.e., you need to establish a ratio between the size of the plasmid and the size of the amplicon, since you are measuring ng of the whole plasmid.

I always found it more useful and intuitive to prepare my standards based on copy number in the first place, and my standard curve typically ranged from 100 - 10e7 copies per rection, in 10 fold increments. This usually gave me a nice linear curve at the extreme ends of detection. However, at such low copy numbers, the serial standard dilutions need to be prepared fresh each time...

Amtekoth
Amtekoth's picture
Let me put in some real

Let me put in some real numbers.

I set up a DNA standard of GAPDH from 1pg down to 100ag, based on spec readings. After SYBR green qPCR, I ended up with CT values of 13.9, 17.6, 20.9, 25.3, 28.8

I ended up with an R-squared of .9983 and an equation of y=-1.6199Ln(x) + 13.82

My amplicon is 70bp long.

I added 100ng/sample of genomic DNA (2 copies of the gene per cell expected) and ended up with CT values in the 17.5-19 range. Plugging them into the equation: =EXP((B6-13.82)/-1.6199) , I came up with calculated DNA concentrations of .04-.1 pg/sample.

How do I figure out: total copies of GAPDH per sample (then dividing by 2 I get the number of cells)?

Only THEN can my real gene of interest be determined for the same samples using the same methods.

Are my equations wrong?

SanDiablo
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Amtekoth wrote:Plugging them

Amtekoth wrote:

Plugging them into the equation: =EXP((B6-13.82)/-1.6199) , I came up with calculated DNA concentrations of .04-.1 pg/sample.

OK, I'm not sure how to get my brain around what you are doing....

BUT FIRST OF ALL - I DID MAKE A MISTAKE IN THE FORMULA I GAVE YOU - I corrected my original post, but see below for an example. Sorry - it's been while since I've done this...

You cannot use ng of measured GAPDH to determine the number of copies of GAPDH in 100 ng of genomic DNA unless you already know exactly what percentage of your genomic sample IS GAPDH.

Also, I'm not sure what you are doing with the equation above, but I think there is a Log value missing...

This is why I suggested making your standard curve based on copy number. Then you are able to determine copy number of your sample directly from the standard curve...you should be able to do this using the software for your thermocylcer rather than the equation...or possibly in Excel...

So, to calculate your standard curve in Copy Number, you need to know how big your whole plasmid is (including GAPDH). See if this helps:

Let's assume your plasmid standard is 3200 base pairs + a 70bp GAPDH insert = 3270 bp. (Put in the correct numbers)

1 pg pGAPDH * (1 pmole bp/660 pg) * 1/3270 bp * 1 mole/10e12 pmoles) * 6.023 e 23 molecules/mole = 2.79e5 copies of GAPDH plasmid in your 1st standard.

Do the same calculations for each standard, then plot copy number of GAPDH vs your cT values. Generate a linear standard curve, then use that to determine copy numbers of GAPDH in your sample of genomic DNA. Viola!

Note, that I did the calculations based on copy numbers of plasmid, not amplicon...but it doesn't matter, as long as there is only one amplicon per plasmid.

I hope that helps.