PCR and sequencing of genomic DNA - (Mar/24/2014 )
I need to do a PCR with a fungal genomic DNA to confirm the presence of a certain gene and then sequence 1.5 kb fragments upstream and downstream of the gene (to construct deletion fragments). For DNA extraction I will use Sigma GenElute Plant Genomic DNA Miniprep Kit or phenol - chloroform extraction.
I never worked with genomic DNA before but from what I heard it can be tricky. So all advices are appreciated!
How much DNA do I need to add to a PCR reaction? What are the PCR parameters - annealing temperature, denaturation time and temperature? How good are sequencing results usually? How to improve DNA purity and yield after extraction?
Using wide-bore pipette tips (or just cut the end of your normal tips) can help to avoid DNA shearing during the extraction
The PCR parameters will depend on the sequence and primers to amplify
Fungal gDNA may have specific issues (like, hard to clean of proteins or something), but othewise gDNA is just.. well DNA. I never heard it was tricky. At least I never heard of anything what would be less tricky than genomic DNA.
Anti-shearing tips (and 'tips') are good if you need a long intact stand, like for a 20kb long-range PCR. For classic < 2kb you don't need to care about that AFAIK.
PCR can usually work just fine even on "not so pure" samples (you but whole unpurified bacteria for example to PCR and it works), with an exception of PCR inhibitors. You may search web for PCR inhibitors possibly present in your samples (human blood have heme for example, soli samples have different inhibitors present, etc.), and then focus on procedures that will specifically deplete it from that inhibitors.
But only when your initial PCR don't work, because most likely it will.
Inhibitor problems can be identified by serial dilutions of the template (to even a very low concentration and having higher number of cycles), when less concentrated will amplify better than more concentrated. Also PCR aditives as BSA, can be used in such case.
On human genomic DNA, the recommended starting amount is 100 ng. Given the size of human genome, that is roughly 30 000 copies in ul. You can calculate similar starting copy number for your fungal genome. Or you can just use 100 ng as well (but this would not work on plasmid, that has so high copy number at such concentration, that it can effectivelly block amplification).
PCR parameters depend on the target sequence and primers and enzyme, as said El Crazy Xabi and is usualy very same for any DNA.
With exceptions of organisms (or amplicons) having too AT or CG rich sequence, where specific modifications of program are required.
You don't seem to have much experience with PCR itself, so you should carefully design the first experiments. It would be ideal if you could get at least some positive controls (primers that work on your gene and gDNA that was sucessfully amplified by someone else) but that's not always possible.
Good sequencing results are usually good. But that depends more on the quality of the amplicon purification, as you only rarely sequence gDNA directly. Generaly you can amplify a nice product from dirty almost nothing and then purify the PCR product and sequence with same quality as product from perfect DNA sample.
Thank you for your answers!
I only worked with plasmid DNA before but I was told (by my colleagues) that gDNA is not that easy, because of its size, possible degradation, etc. Well, I have to try and see it myself.
It would be great to sequence a PCR product but I need to sequence genomic DNA directly; regions upstream and downstream of my target gene haven't been sequenced so I can't design primer pairs for amplification.
I would say each plasmid and genomic DNA has its advantages and disadvantages.
Sequencing gDNA is tricky and generaly not recommended. Alternative approaches like inverse PCR can be used for sequencing unknown product. If you know sequences of closely related genes of other species, or protein sequence, you could design degenerate primers for amplification. You may be succesful in direct gDNA sequencing, but there are other ways.
Also since next generation sequencing is now in common use, sequencing missing regions (especially if they are longer) by this method would be handy AFAIK. In short time it's possible that Nanopore suquencing would be in use, that would make de novo sequencing of any organism, no matter how related to known genomes more convenient.