Target Amplicon Length for real-time PCR - (Nov/03/2006 )

Someone once told me that ideal length of amplicon for the real-time PCR should be smaller than 500bp, but I forgot to ask why. Is this true and can someone tell me why? I'm thinking that with SYBR Green application, the longer the amplicon, the stronger the fluorescent response it will be, and thus the higher sensitivit it will get. However, this length limitation will also set the limit to increase sensitivity. Thanks.

-yms-

QUOTE (yms @ Nov 3 2006, 01:44 PM)

Someone once told me that ideal length of amplicon for the real-time PCR should be smaller than 500bp, but I forgot to ask why. Is this true and can someone tell me why? I'm thinking that with SYBR Green application, . However, this length limitation will also set the limit to increase sensitivity. Thanks.

Dear yms,
Longer amplicon consume more dNTP. Thus if your amplicon too long, your reaction may not be able to amplify exponentially because lack of dNTP.

however, regarding to your arguement that:

"the longer the amplicon, the stronger the fluorescent response it will be, and thus the higher sensitivit it will get"

I am not sure. i need to varify myself.
Or else somebody may have batter idea about this.

-Hadrian-

ideally, the amplicon for real-time pcr [especially if you're using sybr green] should be between 75 and 250 bp

-flashboy-

Dear all,

Yms seem to have his logic regarding to the amplicon length and sensitivity.
I found the following paper, every one please have a look and commend on it.

Improvement in the specificity and sensitivity of detection for the Taura syndrome virus and yellow head virus of penaeid shrimp by increasing the amplicon size in SYBR Green real-time RT-PCR.

Mouillesseaux KP, Klimpel KR, Dhar AK.

Jounal o virological methods

Best regards

-Hadrian-

My idea is that long amplicons consume PCR amplification reagent more rapidly and then shorten the exponential stage, which in fact will reduce the dynamic range of quantitation

-rye-