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Ch 8.Immunohistoch / immunology
Ch 10.GC/MS, NMR and Proteomics
Molecular Biology / PCR / Variants of PCR / 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 Production
HOT ASYMMETRIC PCR (Mullins Lab)
Direct sequencing by thermal asymmetric
PCR
Detection
of asymmetric PCR products in homogeneous
solution by fluorescence correlation spectroscopy
Asymmetric PCR Using the Primers Anchored
on the surface of magnetic nanoparticles.