you are amplifying very large genomic fragments. I would be less optimistic and amplify 4 overlapping fragments then use these to get the full length product. Answering your last question yes I think that some primers are person specific if the primer is designed with its 3' end over a polymorphism in the sequence of the template then only that allele of the template will amplify.

Such long amplimers may have high GC regions so using dmso or betaine in the pcr mix may help but I think that shorter amplimers would be the greatest chance of success

you are amplifying very large genomic fragments. I would be less optimistic and amplify 4 overlapping fragments then use these to get the full length product. Answering your last question yes I think that some primers are person specific if the primer is designed with its 3' end over a polymorphism in the sequence of the template then only that allele of the template will amplify.

Such long amplimers may have high GC regions so using dmso or betaine in the pcr mix may help but I think that shorter amplimers would be the greatest chance of success

Hi Sijia,

in addition to Paul Ruthland advices, I would ask if you tested your template quality? since it's a long range PCR, quality of the DNA will give you the possibility to amplify whole fragments. If DNA is damaged and fragmented (as in FFPE samples), it will be hard to get the right amplicon.

fred

I would add to prefer primers couples with Tm difference under 2°c.

fred

Paul Rutland Thank you for your suggestion, Paul. It never occurred to me amplifying more fragments. I'm trying using nanopore sequencer which can read ultra long length, and I'm worrying about more amplicons with different sizes makes it difficult to get close read depths in sequencing. But I think your suggestion is very worth trying. Could you give me some more suggestion for primer design? As the enzyme I use （PrimeSTAR）suggests a annealing temperature no higher than 68℃, I wonder what is the best range I should set for Tm value.

Thank you so much for your help.

Frederic Lepretre Hi, fred. I check my samples of gDNA (extracted from blood) using nanodrop. Simply make sure my samples meet the requirments: A260/280 1.8-2.0 & A260/230 ~2.5. But I didn't check other things. That reminds me another question: I stored the gDNA in -20℃，but every time when I thaw and do re-quantification (after 1 week), the nanodrop results are quite different. So Everytime before I do PCR with those samples, I need to requantified them. Is the quantificaiton result reliable only for a short time like that? Any similar experiences? Thank you.

Hi Sijia,

quality controls should not differ time to time with DNA. I see 2 possible problems: are your DNA not too concentrated, or is your NanoDrop working well? Do you have a commercial DNA with known quality and concentration to be sure you're on the right way?

To know approximately the best Tm, make an in-silico PCR (use the tools from UCSC or NCBI primer-blast).

fred

Hi Xu,

primers usually work well a few degrees above their Tm so about 64 will be fine....the high temperature helps primer annealing for those amplimers that have high Gc regions and tend to self anneal quickly but your care in primer design looks good. 3 points

You are possibly using too much dna so there are inhibitors present.Try using less ( 50ng-100ng) which may give better amplification.

If the nanodrop gives poor readings ( should not change after degradation as the ntp concentration has not changed) then clean the optics with water or HCl as suggested in the handbook and make absolutely sure that you zero the nanodrop with exactly the diluent that the dna is dissolved in or you may even get negative dna values. You can try adding betaine ( final 1M) and/or DMSO up to 6% in the reaction pcr mix to make the template melting easier. I susoect that only 1 dna working well indicates that too much dna is being used and this one may be the lowest concentration dna rather than a special sequence. Ads well as diluting the dna a gradient of Mg may be interesting as many pcr inhibitors remove Mg from the reaction mix. Ithink that nanopore technology works well on short fragments as well as long ones just make sure of a good overlap between fragments but I have to admit that I know nothing about nanopore technology but my feeling is that it should work nearer to the primers than dideoxy technology

good luck

Paul

Hi Sijia

I think this reference useful

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=2ahUKEwifpOmx2drfAhVHZFAKHd7xAyoQFjAAegQICRAC&url=https%3A%2F%2Fwww.takarabio.com%2Fassets%2Fdocuments%2FUser%2520Manual%2FR050A_e.v1509Da.pdf&usg=AOvVaw2N1g2auMC4wkiKYulDPLQt

Ref.

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=6&cad=rja&uact=8&ved=2ahUKEwjTzcbL2trfAhUQiKYKHV54CVAQFjAFegQIBxAB&url=https%3A%2F%2Fwww.nature.com%2Farticles%2Fsrep05737&usg=AOvVaw2tCLf8Cofoi_zplJYn3XGL

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=17&cad=rja&uact=8&ved=2ahUKEwjupMad29rfAhVHM-wKHfoYBAU4ChAWMAZ6BAgFEAI&url=https%3A%2F%2Fjmd.amjpathol.org%2Fcms%2F10.1016%2Fj.jmoldx.2013.10.005%2Fattachment%2Fe88ade0a-3710-46f4-9753-4d3be1c0737e%2Fmmc1.doc&usg=AOvVaw2yUvWHwE12ZDre12YA4EAW

Thank you all for those suggestions.

Hi, Paul Rutland .Sorry for late reply. I took your suggestion and tried amplifying shorter lengh. Since I want to use the Qiagen purification kit to clean the PCR products (up to 10kbp), I tried the length no longer than 10k this time.

The first 2 parts of the gene have successfully been amplified. And I decided to move on the rest of the gene. I need more suggestions about:

1. As you can see in the picture, I got smearing in the 2nd part amplicon. Increasing the annealing tempreture helps, but not completely. And the limit for primestar GXL polymerase is 68 degree according to the guidebook. How should I improve that? Or it's doesn't matter much?

2. Take part 1 in the picture for example. The background in 68 degree is clearer than that in 66 degree. But 66 seems have better enrichment. For sequecing library preparation, which one would be better?

3. If I could successfully amplify all the parts of the gene (~10kb x5), when I do PCR product purification, is it ok that I mix all the amplicons (5 parts) and purify them at the same tube? Later they would be given a same barcode for sequecing. It's a lazy question, but I'm just trying to get a better efficiency.

Thank you very much!

Hello Xu,

It is good that you have successful amplifications so possibly just a little tweaking on the 3rd amplimer. Smearing is often either genomic degraded dna ( less likely here I think) or one primer annealing either in a repeat sequence or just randomly and extending to random lengths because there is no reverse primer near it to stop the extension….I think that this is the likely situation here,

Since temperature helps the equivalent of increasing temperature is to add DMSO which makes primer binding more stringent. Try a gradient of 1%, 2% up to 7% DMSO at 66C . high amounts will decrease the amount of amplimer and over 10% will destroy the Taq but you should see an improvement unless the mastermix already contains DMSO but its worth a try. It is better to get a good clean amplimer but there is so much material in the band that a little non specific material may not matter very much but you will need to confirm the sequence by sequencing after cloning. Do not worry too much about less amplimer…it is still a lot of material and cleaner is better…you can always amplify 2 tubes if you want more amplimer. I would always take the cleaner amplimer for sequencing as the smear probably does contain the primer so is not entirely random for sequencing( Sanger)

I would not mix the amplimers for purification as structure may effect the amount of material purified in a sequence dependent way and you will have no idea how much of each amplimer you have purified. Purify them all individually and you will have good quality material to do whatever you want in the future for example ligating them together would be easier knowing each sample identity

best wishes,

Paul

Paul Rutland ,thank you very much. I'll give it a try with DMSO.

Hi Laith Al-Ani

Would it be possible to know where the table regarding the LR PCR amplification is from that you shared?

Many Thanks

Patricia