I use allele-specific real-time PCR for mapping. The only genomic DNA extraction protocol that has worked for real-time PCR has been a crude prep where mouse tails are boiled in NaOH. I have tried other protocols, which should produce cleaner DNA, - phenol-chloroform, the Qiagen DNeasy kit, the Gentra Puregene purification kit (all of which use Proteinase K). When I used this DNA in real-time PCR the lines were jagged and unlikely to produce a value above threshold (see example with Qiagen extracted DNA). However, when I ran the real-time PCR products on a gel I could see bands. Moreover, when I used this DNA for PCR in traditional thermocyclers for 28 cycles I could also see bands in the gel. Therefore, I don’t think the problem could be the purity of the DNA, but possibly something was interfering with the fluorescence.
Hi Deborah
I have a different point to make here ...give it a thought.....SYBR is a non-specific DNA binding dye and you are doing an qPCR using DNA (I'll call it as double stranded DNA, ds DNA) as a template....generally for gene expression we use either RNA (which has low SYBR binding) or single stranded cDNA (ss cDNA) generated after first strand of cDNA is synthesiized by reverse transcriptase....Both these template has low affinity for SYBR and you don't see much background when qPCR is done....additionally the SYBR in the mix is avaialbe for the ds DNA generated in each cycle of the PCR and is visible as increase in flourescence.......However in your case as genomic DNA (ds DNA) is used as template....much of it will already bind to available SYBR in the mix and there will be less amount of free SYBR left in the PCR mix to bind to the newly synthesized ds DNA in subsequent PCR cycles.....as a result backgorund will loook really high ...and the qPCR will look apparently inhibited....However actually PCR amplification is not affected (as you can see good bands when loaded on the gel)...but they are not seen as proportional to fluoresecnce increase......In my opinion this is the likely problem with your qPCR using DNA as the template.....and this can be verified if you use a specific DNA probe based on Taqman assay or molecular beacons...which will specifically bind to your fragment of interest and not everywhere in the ds DNA template...and as a consequence will show a goo amplification plot......
bye
In fig. your getting graph from first cycle it self.....is it normal? Because when i am using PCR i am getting graph after around 20 cycle
Hi Deborah,
What are these alleles!? VNTRs, SNPs!? Are you using taqman based probes to detect allele specific amplifications? The plot is coming from ABI instrument, right? Phenol chloroform isolation should provide clean enough DNA for your genotyping, unless there's something wrong with phenol, for DNA it should be saturated with alcalic TRIS. If not you might be getting an RNA or other components which will inhibit the reactions.
I agree with Rohan: it could be machine problem. What kind of instrument do you have? What is your SYBR Green master mix? Call instrument manufacturer tech support. What is the size of your amplicon? If it is small all that you see on the gel could be just primer-dimers amplification. Qiagen DNA purification typically gives very good preps without inhibitions. Phenol though may inhibit PCR reaction. If you are sure that your primer design is good and they work (make sure that the amplicon size is no more than 150 bps), make serial dilutions of your template. This may help with inhibitors. If you have inhibitors in your template control primers would not work either. Did you run your crude template that worked and purified template that did not work on one plate simultaneously?
Normally, nothing can interfere SYBR Green I signal, it just bind to ds DNA and be exited. I saw your pics and relized that you setted thresshold by your self. I don't know the other systerm, but for Roche and ABI, it should be done automatically. it help? Anyway, if you think protease K can interfere SYBR Green I, you simply could check nagative control with genomic extraction by Qiagen kit.
Hi Deborah
I have a different point to make here ...give it a thought.....SYBR is a non-specific DNA binding dye and you are doing an qPCR using DNA (I'll call it as double stranded DNA, ds DNA) as a template....generally for gene expression we use either RNA (which has low SYBR binding) or single stranded cDNA (ss cDNA) generated after first strand of cDNA is synthesiized by reverse transcriptase....Both these template has low affinity for SYBR and you don't see much background when qPCR is done....additionally the SYBR in the mix is avaialbe for the ds DNA generated in each cycle of the PCR and is visible as increase in flourescence.......However in your case as genomic DNA (ds DNA) is used as template....much of it will already bind to available SYBR in the mix and there will be less amount of free SYBR left in the PCR mix to bind to the newly synthesized ds DNA in subsequent PCR cycles.....as a result backgorund will loook really high ...and the qPCR will look apparently inhibited....However actually PCR amplification is not affected (as you can see good bands when loaded on the gel)...but they are not seen as proportional to fluoresecnce increase......In my opinion this is the likely problem with your qPCR using DNA as the template.....and this can be verified if you use a specific DNA probe based on Taqman assay or molecular beacons...which will specifically bind to your fragment of interest and not everywhere in the ds DNA template...and as a consequence will show a goo amplification plot......
bye
Thank you Deborah for posting this question, I was facing this issue all the while. And thanks to Ajay for a very reasonable answer.
This is probably a high SYBR green concetration issue. Try to make a new solution and clean the tip surface just in case u have additonal stock solution.
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