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Old 12-15-2011, 12:17 PM   #1
HESmith
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Default Accuprime amplification and TruSeq incompatibility?

I'm trying to use the Broad Institute's recommendations for PCR amplification to minimize library bias (Accuprime polymerase + modified cycling parameters; described here). The protocol works fine with the old sample prep kits, but not with the TruSeq components - I can amplify the library with Illumina's TruSeq reagents, but not Accuprime. Has anyone else observed this problem?

Harold
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Old 12-15-2011, 02:42 PM   #2
Chipper
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Quote:
Originally Posted by HESmith View Post
I'm trying to use the Broad Institute's recommendations for PCR amplification to minimize library bias (Accuprime polymerase + modified cycling parameters; described here). The protocol works fine with the old sample prep kits, but not with the TruSeq components - I can amplify the library with Illumina's TruSeq reagents, but not Accuprime. Has anyone else observed this problem?

Harold
I just got the same results (very low yield). It would be interesting to know why, according to the manual it contains "a thermostable accessory protein enhances specific primer-template hybridization during every cycle of PCR". Perhaps it needs lower annealing temp, did you use the same for both kits? I used 65*C with works well with Pfu Ultra on the same samples.
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Old 12-15-2011, 03:25 PM   #3
HESmith
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I followed the published protocol exactly (e.g., annealing/extension @ 65C), except I replaced 2 ul of primers PE 1.0 and 2.0 w/ 5 ul of the primer cocktail mix from the Truseq kit (and adjusted the volume accordingly). It works well as published (with the older PE adapters and primers) but not with Truseq adapters and primers. I suspect that the primers are modified, or that the cocktail contains a component that inhibits the Accuprime enzyme. Glad to hear that I'm not the only person with this problem.
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Old 01-22-2012, 09:24 PM   #4
Dodu
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Have you tried to lower annealing temperature to 60C as described in the TruSeq protocol? It may help primer binds to template.
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Old 01-23-2012, 07:49 AM   #5
pmiguel
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Originally Posted by HESmith View Post
I followed the published protocol exactly (e.g., annealing/extension @ 65C), except I replaced 2 ul of primers PE 1.0 and 2.0 w/ 5 ul of the primer cocktail mix from the Truseq kit (and adjusted the volume accordingly). It works well as published (with the older PE adapters and primers) but not with Truseq adapters and primers. I suspect that the primers are modified, or that the cocktail contains a component that inhibits the Accuprime enzyme. Glad to hear that I'm not the only person with this problem.
The TruSeq primers migrate slower during electrophoresis on an bioanalyzer chip than one would expect from their presumptive molecular weight. (I usually see them around 80 nt.) So, I think it is likely they are modified. Possibly LNA or something LNA-like?

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