Nested+PCR

Nested polymerase chain reaction is a modification of polymerase chain reaction intended to reduce non-specific binding in products due to the amplification of unexpected primer binding sites.

Nested polymerase chain reaction involves two sets of primers, used in two successive runs of polymerase chain reaction, the second set intended to amplify a secondary target within the first run product.

People often perform nested PCR by doing pcr with one set of primers. They then use some volume from this first pcr reaction in a second pcr reaction. " The second-round PCR is performed with two new primers that anneal to sequences internal to the product of the first-round PCR. In this way, only specific first-round PCR products (and not nonspecific products) will be amplified in the second round. Alternatively, it is possible to perform semi-nested PCR by using one internal and one first-round primer in the second PCR" -Some people dilute the first reaction 1/100 and 1 uL of this reaction -- http://www.wantprogress.com/download10P35F3/PCR_guide016.pdf -Some people use 1 uL of the 1st reaction -- http://openwetware.org/wiki/Griffin:Nested_RT-PCR

-- some references for nested pcr

-One-sided polymerase chain reaction: The amplification of cDNA Osamu Ohara 1989 (PMID: 2788276) -http://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=4&cad=rja&ved=0CEYQFjAD&url=http%3A%2F%2Fwww.csub.edu%2F~kszick_miranda%2FNested%2520PCR.doc&ei=Abn2UdLcF4e5igLgmoGQAw&usg=AFQjCNGaINUGDvgm0S-6O-4rfabLFEqEjA&sig2=nMPhw5XLAuLiRKNEQX-z2w&bvm=bv.49967636,d.cGE