Nested primers are regular primers they are just chosen within a DNA sequence that has been previously amplified. Nested primers are used to increase the specificity of an amplification or to reamplify the product of a PCR reaction that did not yield enough material. They are called nested because they are chosen within the target sequence of another set of primers.

Nested primers are regular primers they are just chosen within a DNA sequence that has been previously amplified. Nested primers are used to increase the specificity of an amplification or to reamplify the product of a PCR reaction that did not yield enough material. They are called nested because they are chosen within the target sequence of another set of primers.

How can we know the flanking sequence of the DNA if we use nested primers with more specificity?

(I suppose that TAIL PCR is used to know the flanking sequences. Is it true? Please explain. )

it is true that TAIL-PCR is used to discover flanking sequences. For amplifying the flanking region you should have the sequence of your interest gene. based on your goal, you can amplify the upstream or downstream of the interest gene by TAIL-PCR. the remaining things are tree nested primers(usually) and some arbitrary primers.

for more information you can read this paper: "Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR"

Nested primers add an extra element of selectivity in addition to the thermal selectivity. The method promotes a multitude of nonspecific products during the primary reactions. So, you could imagine just running the same primers without nesting would yield a lot of false positives due to the low annealing temperature cycles. By utilizing nested primer sequences toward the border region of a T-DNA insert that you know, ideally only the true TAIL-PCR products would be produced.

If this is something you're going to pursue in your research, I do recommend the more recent method introduced, "High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences". The revised method accommodates for the poor success rate of the original method.

I do understand that nested primers add an extra element of selectivity from your answer. If we use normal primers we get to amplify the required region of DNA (that's what is done by PCR) but how are nested primers different from the normal primers and how do they help in amplifying the flanking sequences?

Please explain the process.

Nested primers differ in their design, but they are no different than the ordinary oligonucleotides utilized in routine PCR with regard to their synthesis. They help in the return true flanking inserts as the low annealing cycles for degenerate primers used in the primary reaction (in the original TAIL-PCR method or preamplification in hi-TAIL-PCR) allow for a lot of mismatch annealing of the specific primer to occur in areas that aren't from the T-DNA insert(s) in question.

The nested primer design accommodates for these erroneous extensions of DNA produced due to the low annealing temperature cycles by demanding the subsequent nested primer sequences be present in the products to be amplified in reactions to follow, else no amplification would occur. Typically, nested primers are designed to go toward the right border of the T-DNA insert out to the unknown region of the genome. These known sequences from your insert used for the nested primers are the sequences that will be conditions necessary for amplification in high stringency cycles in each reaction. Keep in mind that the high stringency cycles are the composition of most of the time in the last two reactions.

In summary, nested primer design accommodates for the specific primer mismatch annealing in the low temperature stringency cycles needed for the degenerate primers to bind to unknown flanking genomic DNA.