Assuming that your primers are for DNA and not cDNA, what checks have you done? Have you tried other human DNA primers eg b-actin etc ? Are you using the same PCR mixes and conditions as in the paper? Have you varied these -time, primer concentration, MgCl2, alternative PCR mixes? Different DNA ? . Have you tested for PCR inhibitors? Have you BLASTED your primer sequences to make sure they are what they are supposed to be. I have come across an instance where both primers were on the same strand.

Until we know what checks you have done it is impossible to tell you what is most likely not working.

Ian

What are you using as a positive and negative control? If your controls are okay then there's something wrong with the sample. I always take these steps when troubleshooting my PCR:

1: check quality of DNA in a nanodrop - just to make sure there is DNA to start with in your sample!

2: start new reagents. open new pack of buffer, taq, nucleotides and make up fresh primer dilutions. If this works something was contaminated.

3: If it's still not working you need to consider that the primers arn't correct and you're not getting amplification of the target sequence.

You really need a good positive and negative controls, HLA-B27 depending on the are of the world your patient is from is only expressed 8% - 24% of people. There is always the possibility that your patient is HLA-B27 negative.

Maybe you had wrong PCR conditions, I would suggest you to change the annealing temperature(or make a gradient for annealing temperature).

Another possible reason of "no amplification" could be low quality of DNA. Did you try to amplify some other sequences with this DNA sample?

Dear Saurabh, frequency of HLA-B27 positive individuals between patients with different forms of arthritis is not so much (~20-40%). Much more frequently this antigen can be detected in patients with ankylosing spondylitis (~95%). So, the most probable reason for inability to amplify is that the patient does not express HLA-B27.

Hello Ian, Its mentioned in literature that primers are for DNA, i run DNA samples with Endogenous gene beta globin that got amplified, so its sure that pcr is working. No as my annealing temp. was different so i used different Tm, and also have run gradient, with other conditions same as in paper and alsowith changed one, but i have not blasted those sequences, so i will do that.

Hello DMITRY, I agree with you, i took the sample form arthritis patient, so i have to look for AS positive patient. Thanks

Thanks Samantha and ANNA, i worked with all those possibilites you guys have mentioned. Thanks

Hello Saurabh, I now have a better idea of what your issues may be.

As Dimitry mentioned there may just be an issue of patients not expressing HLA-B27. Whilst a high % of AS patient have HLA-B27 only a small % of HLA-B27 patients have AS

From Wikipedia :

The prevalence of HLA-B27 varies markedly in the general population. For example, about 8% of Caucasians, 4% of North Africans, 2-9% of Chinese, and 0.1-0.5% of persons of Japanese descent possess this gene.[2] In northern Scandinavia (Lapland), 24% of people are HLA-B27 positive, while 1.8% have associated ankylosing spondylitis. The incidence for the Indian population is ~ 5% varying between North and S.India. (http://www.bjbs-online.org/article.asp?id=125)

Are your primers allele specific?

Ian

Hello Ian, These are specific primers for HLA b27 except HLA B2707. Thanks

From what you have said it seems that your issue may not be a PCR failure as you have done the obvious checks, but a choice of target. Have you only looked at 1 (reactive?) arthritis patient? As Dmitry suggested an AS control is a good idea. You really need to do more arthritis samples before you can conclude that the assay is not working.

Ian

Hello Ian, from last two months i myself was suffering from spine problem, so i took my own DNA and it got amplified with those primers. Thanks

Glad to hear you resolved your PCR problems. Sorry to hear you had to get spinal problems to resolve it !!!

Ian