Hi!

First of all, the error could lie with the primer design. But this is hard to find out based on the information you can give us.

To me it sounds like the signal for the genomic DNA is drowned in your cDNA signal. Have you checked the concentration of your genomic DNA prior to cDNA synthesis? And how did you check it? Using NanoDrop is very unreliable.

General PCR advice: Always run a temperature gradient for annealing temperature.

@Michal J Kowalczyk Yes. There are A LOT. I tried different exons position and still can't get specific amplification so I just picked my primer pair according to the estimated product size. Right now I am sequencing my product to check what's really amplified.

@YH DNA concentration was checked with fluorescent dye. So, you're saying that my primer pair annealed stronger to the cDNA? If so, how can I prevent this?

Thanks.

I would not expect cDNA to outcompete genomic DNA from a gDNA isolation. This sounds a bit strange. Hopefully your sequencing will clear things up a bit. If you are looking specifically for cDNA amplification, then using a probe-based system could help improve your specificity (maybe UPL from Roche?). We've had good success with the mouse gapdh assay from them (it's one of their pre-validated housekeeping gene assays). Let us know how you get on, this sounds interesting!

You are most likely amplifying a pseudogene, which can have sequences very similar to the mRNA.  I would suggest designing a primer pair where one primer is in the intron and the other in an exon and see if that helps. good luck!

@Agnes Tello Thank you. Will post the sequencing result soon.

@Connie Larsson Since I want to use this primer pair for both gDNA and cDNA amplification, I tried to put them in exons. But, maybe I should have different set of primer suited to my purpose. Thanks!

Sequence result came back. It has many hits on NCBI Blast, but at the top of the result is mouse Gapdh mRNA.

Though I can still use this primer set for my purposes, I am still confused as to why this happened.

My best guess, genomic DNA extraction included some mRNA and PCR, instead amplifying the gDNA, amplified this mRNA thus the short(er) PCR product. Is it possible? Doesn't the mRNA need to be convert into cDNA first before it can be used in PCR?

Lesson learned, design one primer in intron and another in exon.

@Michal J Kowalczyk Thank you for the detailed comment. I'll check again. For your third question, published primer sets product are under 200 bp. I need between 400 to 700 bp. For that reason also I skip the early exon and the long intron.

Been a while, caught up on another experiment.

I checked the chromatogram, on the two Gs differences, they were double peak of G and A with G being the higher. The N was interposing double peaks of equal heights of G and C. These three differences, may I conclude as SNP of the template? How this will affect the primer set?

I don't have nanodrop so I cannot check the DNA template purity. I still don't understand how the amplified product is from the cDNA (?) instead of gDNA? Am I correct to assume that we can rule out pseudogenes based on the sequence result BLAST?

Hi Masita, I just looked at the sequencing data you put up. It aligns only to cDNA, and in fact spans two introns (your sequence aligns to three exons and skips two introns), so you definitely only amplified cDNA and not gDNA. Can you describe how you prepared your DNA samples? This is very puzzling.

It doesn't make sense that you would have cDNA contamination in your gDNA prep. I ran into a very similar problem when i was trying to amplify p53 exons 4-6 using gDNA. Both of my primers were within the exons of p53. I kept getting a dark band that was less than half the size of my expected product size. i found a paper by H. Tanooka et al. called Distribution of the p53 pseudogene among mouse species and subspecies and realized that i was, indeed, amplifying the p53 pseudogene and not the actual gene. The sequence of the p53 pseudogene is nearly identical to the sequence of the mRNA/cDNA of p53, which is common for pseudogenes. So I tried designed a different primer set where one primer was in exon 4 and the other in intron 6 and it fixed the problem. Hope this brings some clarification to your problem. good luck. 

Hi Masita and Connie- I think Connie is right about possibly amplifying a pseudogene here. I wasn't aware of this before, but had a quick look on pubmed and found this paper: 

PLoS One. 2012;7(8):e41659. doi: 10.1371/journal.pone.0041659. Epub 2012 Aug 22. Pseudogenes as weaknesses of ACTB (Actb) and GAPDH (Gapdh) used as reference genes in reverse transcription and polymerase chain reactions.

Sun Y1, Li Y, Luo D, Liao DJ.

They claim to have found 197 pseudgenes for Gapdh in the mouse genome, with many highly similar to the genuine cDNA. This will make it very difficult or impossible to do the type of analysis Matisa is trying to do: use one set of primers to distinguish cDNA and gDNA. May need to use another strategy in this case.

Thank you very much Agnes and Connie. This is a very enlightening discussion.

I always wonder about any cDNA in my DNA products but I guess after all this, we can conclude that it was in fact pseudogenes, not Gapdh.

Anyway, I extract the DNA using simple salt extraction method. In brief, mouse tail clippings were dissolved in NTES buffer with proteinase K for roughly 3 hours in 56 C degrees, dna precipitated with 6M NaCl and isopropanol, washed with EtOH, and resuspended in TE buffer with RNAase then used for genotyping.

Thank you very much once again.

my GAPDH primers  codes for around 510 bp, i made cDNA and used it for amplification of genes(ICAMI) with target size 135 bp, it works fine and have clear bands but I cannot get GAPDH band. Same problem in real-time PCR too,  i tired on gDNA it works but when I try with cDNA even with 400-500 ng dna, still i don't get GAPDH band but other genes band comes ok .... please help

I will recommend you to use in silico PCR to check your primers first and see if they are giving a unique product.