I would like to use GAPDH on some DNA samples I extracted to test for human DNA. The GAPDH I have at hand is mainly used for cDNA. Since GAPDH is a housekeeping gene that is always present. Do you think that GAPDH will work on DNA (not cDNA) samples?
Hi Diana,
They may not work. You have to check their position on the genomic DNA. GAPDH has several introns. Your oligos could be part of two exons and non-functional. If this is not the case your CDNA oligos should span at least one intron which means that you expect a larger PCR fragment.
Here as an example the human gene structure:
http://www.ensembl.org/Homo_sapiens/Transcript/Exons?db=core;g=ENSG00000111640;r=12:6533927-6538374;t=ENST00000229239
yes it would.. i dont understand y it wont? primers are for cDNA..if u align those primers to the DNA sequence it would definitely bind and work.. however the converse may not be true cuz cNDA is the CDS of the gene. if the primers are designed in such a way tht they lie intron exon junctions that those primers wil not be suitable for use for cDNA amplification
Hi Diana,
They may not work. You have to check their position on the genomic DNA. GAPDH has several introns. Your oligos could be part of two exons and non-functional. If this is not the case your CDNA oligos should span at least one intron which means that you expect a larger PCR fragment.
Here as an example the human gene structure:
http://www.ensembl.org/Homo_sapiens/Transcript/Exons?db=core;g=ENSG00000111640;r=12:6533927-6538374;t=ENST00000229239
you must control where your primers fall within the gene:
usually primers for amplification from cDNA (especially for quantification purposes) are designed with the aim of avoiding amplification from genomic DNA.
Therefore, as Uwe said, check that one or both of them are not designed on exon-exon junctions (otherwise they will never work on genomic) OR that the 2 primers are not too far from each other on genomic DNA (because they are separated by a big intron, let's say > 1kb) otherwise PCR will not be efficient enough to let you detect the product.
If your primers are designed to one exon (or into two with some short intron), it should work. Check whether your primers don´t bind over exon/intron borders.
Yes. It works but check the position of your primers in aim to be sure not amplifying a fragment that you don't want and still be specific.
Hi Diana,
At first try to align the primers with template sequence. If the amplicon size is similar to that of cDNA sample you can use same PCR conditions otherwise, you need to optimise the conditions to amplify the template.
cDNA primers should really be designed so at least one of them actually is spanning an exon-exon border (half in one exon-half in the following exon, so would not bind genomic DNA as this still contains the introns; if it is a particularly big gene, you would design more towards the 3 prime end; and keep in mind alternative splice forms!). Sometimes that is not possible because of the specific sequence around the exon borders, then the second choice is to design the primers so they are in different exons, with an intron of at least 1 kb in between.
In theory, you should then only get amplification from your cDNA, as short PCR products are favoured over longer ones, particularly if your extension time is very short.
So if you do get amplification from the cDNA GAPDH primers on the genomic DNA, your lab has a BIG problem with all their RT-qPCR data! Presumably these GAPDH primers were used to normalize RT-qPCR data? Think about what it means if there was amplification of your reference gene from genomic DNA - is any of the RT-qPCR data still valid?
Addendum:
In case your GAPDH primers are in two different exons with an intron between and not spanning exon borders, you could in theory check and see how big the intron is - and adjust your extension time in your genomic PCR accordingly. However, to get good quantitative data from PCR you have to amplify small fragments of 200 bp or less. Certainly not more than 1000 bp which your amplicon would be if the cDNA primer design rules were followed. Therefore back to the point I made above.
Really, designing some new primers and ordering them in the US should be no problem and cost $5-10.
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