can any one tell that is it possible to differentiate between the three sequences (having same size) and obtain their individual sequence in the same sequencing reaction and if yes then what is the principle..????
You have to clone the pool of PCR products into plasmids, select many plasmids, and then sequence them individually. If you try to sequence all of them at the same time, you will get overlapping reads and not know which peak goes with which molecule. Or use nano pore sequencing if money is no object.
Katie is correct that you cannot sequence a mixture of sequences when you do not know the sequence but in a special case if you know the sequences and they differ at polymorphic sites in different positions then it may be possible to say which sequences are derived from different species in a pcr of mixed amplimers. So for instance if you had a common species and assumed only one other sequence present then the polymorphic bases will show the second sequence but this makes the unjustified assumption that there is only one other species contributing the other polymorphic bases
thanks Paul your answer was so helpful.....actually I have to sequence the fragment of two different viruses that differ slightly...but the pcr product of both are obtained by using same primers and they have same size...but their sequences have already been reported so can their sequencing be performed in same reaction??
I am unsure what you are trying to do here Mufarrah. If the sequences vary only at SNPs then the existence of both bases shows that both sequences are present but Sanger sequencing will only give an approximate estimate of the relative abundance of each. Pyrosequencing would be more accurate for this. If the changes are small indels then the overlapping sequences will be messy and complex but this will also demonstrate the existence of 2 ( or more) sequences present. The danger is that you will never be entirely sure that a mixed sequence is cause by your expected sequences or two other sequences that give you the same mixed sequence effect. For example if your sequences have changes represented by AB and CD producing a mixed sequence of A+B+C+D the same effect could arrive from 2 sequences AC and BD or from 3 sequences AB AC and BD Katie's method is unequivocal and will tell you exactly which sequences are present
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