On thing you could do is pick up the bands, purify the bands and send both for a Sanger sequencing assay. The sequencing never lie on what you amplified if it is only an unspecific band or better a new (or even known) length polymorphism. 

On thing you could do is pick up the bands, purify the bands and send both for a Sanger sequencing assay. The sequencing never lie on what you amplified if it is only an unspecific band or better a new (or even known) length polymorphism. 

Are your primers of same length for positive and test controls? If yes, then sequencing can sort out your problem. 

If not, there could be many possible reasons for extended nucleotides.

I am with Paolo in this one in that sequencing will give you the answer. The only thing I might add as you dont give amplimer size is that 1000 could be the size of your outer primer amplimer and in your samples one of the inner primers has a polymorphism at its 3' end so cannot anneal or work and there is enough outer primer in your second amplification to give more of the outer primer product. I might have tried second round amplification of outerf to inner r and outer r to inner f  just to make sure that all of your primers do anneal and can give the correct size product on both control and your sample. You did not make it clear but do your samples give only the larger band or are they giving the correct band and a larger band as well?

Thanks all

Paul, the samples gave only the 1000bp while the control gave only 650bp. 

Gaurav, I used the same primers for both tests and positive control

ok I am struggling a bit with this one.

possibilities are

the control is not a proper control  eg wrong species or your population has a common insertion not found in the control.

You are using too much first round pcr product and over amplification is allowing self annealing of product in the nested pcr reaction to produce a longmer but in this case I would expect some of the 650 product to be present as well.

I would blast all of the primers in case ,for instance, there is a pseudogene and for some reason ( polymorphisms under the primers perhaps) you are not amplifying the correct sequence in your samples but you can amplify a similar sequence which gives the wrong size product

i am still with sequencing the products but as a guide choose a restriction enzyme that cuts the 650 and see the fragment sizes and compare with fragments in your 1000 fragment just to see if they are similar or completely different in composition

is there a possibility of gene rearrangements..are you working on cell lines or paients

Paul, I can not really figure out what is happening, i did a nested PCR on the both control and test which are patients samples, behold, the test  samples are still showing 1000bp bands while the control produced a band of 350bp. Can the problem be from the samples?

Reasons may range in both physiological and experimentally-induced fields. Not only this but the same template at high concentration of himself or salts may run abundantly faster rather than the same sample in other conditions. I saw for example 5 months ago a shift of a expected 460 bp bands at ladder level of 200 bp, you should be sure first to load and run both at the same conc and conditions.

by the way nobody can say what happend in a DNA sample unless you have a sequencing file ready to align or blast or something other.

your samples are probably ok but when you produce the wrong size band in the control I worry most about the primers or the pcr conditions. You need to blat or blast the primer sequences to see how many hits they find on the genome and check the annealing temperatures of all of them . It might be that one of your primers is similar to a common repeat sequence and its commonness is being rescued to a single amplimer by the high quality of one of the other primers. Then if the good primer does not work because of too high an annealing temperature or because one primer has degraded you might get the wrong and wrong size product.. Blast the primers and make sure that they are all in the same area of the genome and do not have huge numbers of hits

you could try lowering the annealing temperature 2 degrees and see if the 650 correct band reappears if you are sure that 650 really is the correct size

I agree with Paul Rutland. I also had the same problem some time ago. Check the size of your primers, it could be the reason.  And be also sure that you are using both paire of primers which should be store seperatelly anytime.

try going back to your 1st round amplimer of both control and sample and rune all of the primer sets that can work eg outer f and inner r, outer r and inner f innerf and inner r on both samples at the annealing temperature of the lowest annealing primer and see which primer sets give an amplimer of the correct size. This expt may indicate if one of the primers is not working properly

Hi.

Your positive control is from the same organism than you are working with?  Maybe the organism you are working with, have diferences in the gen you are studing.

there may be insertion mutation in yours target gene of the sample. so sequence it to find out the actual status.

Dear,

Anyway, I think you have sequenced your PCR product to know about the sequence of your DNA target, right?.. So, have you tried to align your DNA target together with some complete genome of related species?.. If yes, how about that?.. If not yet, I suggest you to try it.. After that, we will know what happen with your DNA sequence..

Best regards,

try to check your primer .I mean's your specific primer to amplify internal fragment .it's maybe couldn't amplify your specific gene.

Before discussion other option you should sequence the samples. This will probably give you an answer.

Thanks all,I will try all the suggestions

Hi,

Sequence PCR amplified product. Possibility insertion, dubication etc etc. 

Perform electrophoresis carefully.