PCR genomic DNA vs cDNA

[gene26]

I did a nested PCR reaction with touchdown parameters for cDNA (mouse) 852bp coding region it worked well...BUT

i used the same primers and did nested PCR with increased extension time to amplify genomic DNA (mouse) which is of 4584bp but it haven't worked no bands at expected region but few bands below 3000bp in 1st PCR but nothing in second PCR reaction (as its nested PCR i said 1st PCR and second).

what could be the reason.... PCR doesn't work for larger fragments more than 3-4kb..???

[mchlbrmn]

It is definitely more difficult to PCR that size rather than 852 bp. Since the larger PCR is more difficult, conditions that are imperfect but sufficient for 852bp may need to be optimized to get 4kb, although sometimes the same conditions do work.
My first suggestion would have been nested PCR, but I guess you covered that. I suppose that it's possible that the conditions for the untested outer nested primer set are not correct. If you went at all above the actual Tm for annealing of either primer, then nothing good will happen. If the same primer can work to make a smaller product with a closer second primer, then it is annealing.
Did you check your enzyme's instructions to see how large PCR products it is recommended for? This is pushing the max for regular taq, I think. A different enzyme, probably a proofreading enzyme, might be more appropriate. I also think that 68C is often used as an extension temp for longer PCRs.
The large product including noncoding region could have difficult template with high GC% and/or secondary structures. You could try to add cosolvent to the PCR mix, such as betaine, DMSO, glycerol, and etc, or various anneal temperatures.
Would it be a workable alternative for you to PCR the sequence in several smaller pieces, and then ligate or PCR (overlapping PCR products) together?

[relaxin]

Regular Taq will not be able to amplify 4.5 kb genomic fragment. You may try polymerase mix designed for long range amplification.

Make sure the primer sequences do not lie at the junction of exon sequences. This may be important when designing nested primers, as exon sequences are sometimes rather short.

[gene26]

i just went through

my mouse gDNA conc is 3.06µg/ml and for RNA (cDNA) its 165µg/ml and also its less than 1kb for cDNA its fine yet my cDNA showed very light band for (936bp) in 1st round PCR while high intense band in the second round PCR (852bp). can my conc could be a problem as PCR requires very minute good quality sample so i dont think its a problem.

for gDNA it shows few light bands in 1st round PCR but below 3kb and nth in second round PCR...

The PCR parameters are diffrent for both of them the extension time is at 72C, 1min 30sec for cDNA while its 5min 30sec for gDNA annealing temp are 55C for 10 cycles and 50C for 20 cycles

overlapping PCR products..can u say more about it..??

[mchlbrmn]

I think I understand, after rereading the post a few times.

20 cycles + 10 cycles of possibly failed or lower efficiency cycles (at a higher anneal temp) may simply be insufficient for a genomic target at the low efficiency expected for a target this large. Assuming the same cycle was done twice in the nesting, that may be 20 + 20 - 10 (dilution) = 30 cycles. For low efficiency conventional PCR this may not be enough. If the higher anneal temp worked well then the cycle number would be plenty, however if you knew that temp worked than there would be no purpose in doing the touchdown to a lower temperature.

You still haven't mentioned the enzyme used. I'll assume that it's a high quality proofreading enzyme, or mix, and proceed. However, if this isn't the case this may be where you should changing things.

Assuming you gave the DNA/RNA concentrations in the final PCR mix (otherwise the concentrations wouldn't be useful to us) you have used 75ng gDNA/25 ul reaction, and 4.1 ug RNA/ 25ul rxn.
A one copy target in genomic DNA will be far more dilute than a gene target in cDNA at a typical level of expression, and will also be more difficult to PCR because the size is longer. However, relative to the size you use less extension time in the genomic than with the cDNA. The extend time may be OK, and may have been more than necessary for the cDNA; I don't know the enzyme and so I can't comment, but you may want to double check your enzyme manufacturer's protocol for extension times.

The anneal temp sounds low, but this is relative to the primers and enzyme mix, which I don't know. I personally would use a single anneal temp close to, but below the primer Tm, unless the enzyme's protocol directs otherwise. The nesting will give you the specificity so touchdown is not necessary. Usually with PCR from cDNA it is not very difficult to achieve a single band, and the fact that you got two bands in the first round may indicate that there is nonspecific priming so a higher temp may work better.

So, I think what I might do would be to do two rounds of nested PCR of 30 cycles each at a few anneal temperatures (primer Tm, Tm-4, Tm-8C, but not less than already worked), and maybe duplicate this with a cosolvent added in case there is difficult template present. For a cosolvent, I currently use Phusion polmerase which comes with a "GC" buffer, and/or some DMSO could be tried (perhaps 3%, this would shift the Tm down about 2 degrees C).

[gene26]

i am using BIOLINE, MyTaq Red Mix (BIO-25043 2x 200 x 50µl Reactions)

and 1µg of DNA as template in 25µl

i checked my conc again it shows 6.63µg/ml (after leaving it at room temp for 5 min)


so i need to change my anneling and extension tempratures overall.??

[mchlbrmn]

This is conventional taq polymerase. Amplifying a 4.5 kb product can be difficult. I would use a different polymerase that specifies a larger size limit. I looked at the protocol for this product, and they don't give many specifics. They do mention up to 5 kb in only 10 seconds, but that is from low complexity template. Genomic DNA also requires so much amplification that there will be a significant error rate that can affect your results, so if you require an accurate sequence as your goal you might need to sequence multiple clones to find a perfect one.
Qiagen gives 5kb as the limit from genomic template, so you are pushing close to this, ("maximum we tested", possibly meaning, "maximum we suceeded in amplifying"?)
NEB says:
The absence of 3´→5´ exonuclease activity may have ramifications other than fidelity in PCR. The lack of proofreading activity in Taq DNA Polymerase has been proposed to limit the amplicon size possible with this enzyme (7). As a generality, Taq performs best when amplifying DNA fragments < 2 kb, but works on fragments up to 3–4 kb. When kept to this amplicon size, Taq is a robust, easily optimized enzyme. However, above ~3 kb it quickly drops in effectiveness. During PCR, Taq DNA Polymerase will misincorporate nucleotides at a particular rate leading to mismatch formation. This is termed “error rate”. Taq, and polymerases in general, will stall at these mismatched bases and are more likely to dissociate before extending as compared to correctly base paired 3´ ends. Therefore, at a certain amplicon size and polymerase error rate enough mismatched 3´ ends may accumulate to effectively inhibit the PCR process. These mismatched 3´ ends are particularly problematic for Taq because it lacks the 3´→5´ exonuclease activity to remove them. By adding in a small amount of proofreading enzyme such as Deep VentR™ DNA Polymerase, amplification of fragments ≥ 20 kb can be achieved (Figure 3). Since the vast majority of the enzyme in the blend is Taq DNA Polymerase it is probably doing the bulk of the primer extension. The proofreading polymerase is most likely removing the inhibitory 3´ mismatches generated by Taq.

[dcrghujuikhjd]

Since the larger PCR is more difficult, conditions that are imperfect but sufficient for 852bp may need to be optimized to get 4kb, although sometimes the same conditions do work.