Dear Vikash,

I'm not quite sure about the reason for this phenomena but in my experience it commonly happens with PCR from PCR. I would suggest you sequence the amplicons to make sure if you have the right or a random target. In regards to the PCR-Reaction itself I would consider a gradient PCR with higher TM or a Touchdown.

Best regards

Dear Soner

These were specific right  PCR product.

Best regards

You have 2 possible cases for the size of the middle product (674): a) The second primers give additional size; b) Second PCR products often form heteroduplexes, and their migration is slower. 

713bp (right side) - is non specific reaction

This happens sometimes. Sometimes your PCR products contain repeated sequences that bind to different targets in the complementary strand. In that case,, you go, after annealing,  into the next round of synthesis with overhanging single strands. These are repaired by the Taq-polymerase and serve, of course, also as targets for your primers and are ampified to one or more longer products. You will see this by looking at the sequences. You may be able to find small duplications.

should product A theoretically be 600 bases or is it unknown size but runs at 600 bases? How many cycles of pcr are used to produce  the  A product and then how many cycles to produce the B and C product please. At the moment one idea is that product A is the wrong product but there is some proper product produced but too little to see in the gel, Second round amplification then is too much amplification so produces B which may be correct and a smear of over amplified mess in lane C, Every 10 cycles makes up to 1000 times more product so 30 cycles then 30 cycles would be a billion billion times more product so over amplification concatenation of product with product making random sizes may be happening. usually wither amplifications you can use 1/1000 ul of first round product and re amplify with 10-20 cycles for the second round pcr

Hi Vikash,

This seems to be a reoccurring problem, with may people that I have spoken to. I have had a similar experience in the past with a house keeping gene that I was amplifying for a human sample, I was curious to know what it was, so we sequenced it. The band turned out to be from algae, we eventually traced the source to our ultra pure water, which needed to be thoroughly decontaminated. For another colleague with similar problems, it turned out to be cross-contamination from PCR run in the same lab. If you have not done so, I would recommend running the second PCR with everything except the template, I also agree with Soner, start with a higher temperature and run a touchdown PCR. If this does not work, you may need to try different nested primers

Best regards  

Hi vikas,

PCR product as template , most of the times is problem.  it will have the original template molecules and primers also which is  enough to interfere in subsequent PCR. The nonspecific bands are all result of the earlier template and primers. You may have to dilute the PCR product a lot( depending on the concentration ) to use it as template.    if the PCR product is gel purified and used as template, it may be better.

• I think that prior answers are informative
• Like most people I have found that PCR on PCR can often result in non specific bands even when nested primers are used during secondary PCR
• In terms of amount of primary PCR I would recommend  maximum of 1:100 of your original PCR product; which of course you must column purify or treat with Shrimp alkaline phosphatase (SAP) and exonuclease III (exo) to inactivate excess dNTPs and primers (respectively)
• In fact try titrants from 1:100 to 1:1000 to reduce non specific background
• Furthermore, like prior commentators touch down PCR id DEFINITELY the way to go for secondary PCR:
• Commence PCR with very stringent conditions, cf Tm+3C for first annealing cycle then touch down in 0.5C to 1C intervals to Tm-3C
• An illustrative example of TD is provided below

95oC       3 min x1

95oC       15 sec

63oC*1    30 sec ( - 0.5oC/cycle)                  x14

72oC       3 min

94oC       15 sec

57oC*2    30 sec           x25

72oC       3 min

72oC       10 min   x1

4oC         ∞

*1: Initial Ta = Tm + 3oC

*2: Final Ta = Tm – 3oC

Andrey has given the most probable answer to your question.

I am thankful to all of you for giving your opinions. I wish to inform you that PCR product is Lana (A) ( First lane in pic 2) is actually Parallel stranded DNA which actually consist of 769+ bp.  By using this as a template I got specific PCR product of 674 bp and 713 bp. Now most important question is that why this 769+bp DNA migrates around 600 bp, if we compare with the marker used. I wish to share with you one study published in 1988 which says that Parallel stranded DNA migrates faster than antiparallel DNA (kindly check Figure no 3 and compare  Lane 4 and Lane 5 in that study available at  https://academic.oup.com/nar/article-lookup/doi/10.1093/nar/16.14.6659. ) There is another study published in 2000, also states that Parallel DNA migrates faster than antiparallel DNA (http://www.sciencedirect.com/science/article/pii/S0022283600940737)

Few years back I have published about a novel PD PCR technique which is available at https://f1000research.com/articles/3-320/v1.  I wish to inform you that we have done a lot of progress in molecular biology field by applying concept of antiparallel DNA but we have totally ignored formation of Parallel DNA (https://f1000research.com/articles/4-438/v1) . Worldwide there is an issue of irreproducibilty in research  and a study says 28 billion $ are wasted in USA alone (http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.1002165) . Till date Human Genome project is not yet complete  (https://www.ncbi.nlm.nih.gov/pubmed/25652027) and I also have a doubt whether Data collected from Human Genome project is 100% accurate. Is it a Natural genome sequence data or artificial? Many fundamental molecular techniques show errors and we are ignoring just by saying all those errors by saying Non-specific reactions.  We should found reasons behind non-specific reactions and should  make our tools free of errors if we want to find solutions of mankind’s problems at molecular level.

If you like to have more interaction, you may contact me at [email protected].   I look forward for your valuable thought,

With regards

Dr Vikash Bhardwaj

Is the MPR relevant here? - See e.g., the following articles:

Itsko M, Rabinovitch A, Zaritsky A. 2009. Kinetics of repeat propagation in the Microgene Polymerization Reaction. Biophys J 96: 1866-1874

Itsko M, Zaritsky A, Rabinovitch A, Ben-Dov E. 2011. Tandem DNA Repeats: Generation and Propagation in the Microgene Polymerization Reaction and in vivo. Chapter 6 (pp. 175-202) in, Applications of Thermodynamics to Biological and Material Science, ISBN 978-953-307-980-6 (Ed. Tadashi, M). InTech Publishers

Melnikov O, Baranes N, Einav M, Ben-Dov E, Manasherob R, Itsko M, Zaritsky A. 2011. Tandem repeats in a new toxin gene from Bacillus thuringiensis and in other cry-like genes. J molec Microbiol Biotechnol 20: 204-210