Asymmetric PCR

What is asymmetric PCR? A PCR in which the predominant product is a single-stranded DNA, as a result of unequal primer concentrations. As asymmetric PCR proceeds, the lower concentration primer is quantitatively incorporated into double-stranded DNA. The higher concentration primer continues to primer synthesis, but only of its strand.

Asymmetric PCR Protocol 1. Pick a phage plaque and place in 100 ul TE or scrape a fresh colony of a bacterial transformant of choice and place in 50 ul of TE/TX100 in a microcentrifuge tube. 2. Heat the tube for 10 min at 95C. 3. Centrifuge at maximum speed for several minutes in a microcentrifuge to pellet cell debris. Collect the supernatant. 4. Add the following components in a PCR tube: 5 ul of phage or bacterial extract (from Step #A3) 50 uM of dNTPs 50 pmol of Primer 1 1 pmol of Primer 2 in 1X PCR Reaction Buffer to give a final reaction volume of 50 to 100 ul 2.5 Units of Taq polymerase 5. Run 30 to 35 cycles in a thermocycler using the following PCR program (see Hint #1 and #2) 95C for 60 sec 60C for 30 sec 72C for 2 min 6. Run a small aliquot on an agarose gel to analyze for single-stranded DNA (see Protocol on Agarose Gel Electrophoresis of DNA). 7. Purify the PCR products and sequence, if desired.
Asymmetric PCR for ssDNA ProductionHOT ASYMMETRIC PCR (Mullins Lab)
Direct sequencing by thermal asymmetric PCR

Rapid sequencing of unpurified PCR products by thermal asymmetric PCR cycle sequencing using unlabeled sequencing primers.

Detection of asymmetric PCR products in homogeneoussolution by fluorescence correlation spectroscopy
Asymmetric PCR Using the Primers Anchored on the surface of magnetic nanoparticles.