Protocol for Enhancing PCR of Very Difficult Regions

Ziyun Yao, Shaoping Lin, HongMin Wu and B.A. Roe



Target DNA (~5 ng/ul)                                                                                                            3ul

GeneAmp 10x PCR buffer*                                                                                                  10ul

25 mM MgCl2 (Applied Biosystems 58002032-01)                                                           10ul

7-deaza dGTP (Roche part No.92284126), dATP, dCTP, dTTP mix**                             10ul

Primer pair (from mermade)(5 ul, i.e. 500 pmoles of each primer)                                       10ul

Polyoxyethylenesorbitan monolaurate (1% v/v with sterile double distilled water)***       10ul

Igepal CA-630 (1% v/v with sterile double distilled water)***                                            10ul

Taq polymerase (~10U/ml)                                                                                                     1ul

Sterile double distilled water                                                                                                  36ul

Final volume                                                                                                                        100ul


Thermocycling reaction conditions:

95 degrees C for 5 minutes, followed by:

35 cycles of:

95 degrees C for 1 minutes

50 degrees C for 2 minutes

72 degrees C for 3 minutes

72 degrees C for 10 minutes

  4 degrees C hold


Then, clean the PCR product containing 7-deaza-dG by adding 5 ul of SAP and 5 ul of ExoI, incubating at 37 deg C for 30 minutes followed by 80 deg C for 15 minutes.  Store frozen and use 4 ul of this SAP-ExoI cleaned product in each subsequent sequencing reaction.


*(500 mM KCl, 100 mM Tris-HCl, pH 8.5 in sterile double distilled water or Applied Biosystems 58002026-01)


**The 7-deaza dGTP (Roche part No.92284126), dATP, dCTP, dTTP (the latter three are from Amersham-Pharmacia) mix has each dNTP at a final concentration of 2.0 mM and contains 5 mM Tris-HCl, pH 8.0 and 0.1 mM EDTA in sterile double distilled water.


*** Polyoxyethylenesorbitan monolaurate (1% v/v Tween 20, Sigma-Aldrich # P-9416 with sterile double distilled water), Igepal CA-630 (1% v/v Octylphenoxy)polyethoxyethanol [which is identical to NonidetP-40, a nonionic detergent], Sigma-Aldrich # I-8896 with sterile double distilled water) (both detergents are added to a final concentration of 0.1%), proline (Sigma-Aldrich # P-5607) and/or MMNO (4-Methylmorpholine N-oxide) (Sigma-Aldrich # 22428-6) (where proline and MMNO are at a final concentration of from 0.4M to 0.8M) may facilitate reading through G/C rich regions both in PCR and sequencing reactions.  See Life Technologies European Patent Number WO09946400.

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Bruce Roe,