PCR基本实验方法（三）

"Hot Start" PCR: In certain circumstances one wishes to avoid mixing primers and target DNA at low temperatures in the presence of Taq polymerase: Taq pol is almost as efficient as Klenow pol at 37oC; consequently, if primers mis-anneal at low temperature prior to initial template denaturation, "non-specific" amplification may occur. This may be avoided by only adding enzyme after the initial denaturation, before the reaction cools to the chosen annealing temperature. This is most conveniently done by putting wax "gems"TM into the reaction tube after addition of everything except enzyme, then putting enzyme on top of the gem: the wax melts when the temperature reaches +/-80oC, and the enzyme mixes with the rest of the reaction mix while the molten wax floats on top and seals the mix, taking the place of mineral oil. Information is that "gems" may be substituted by VaselineTM.

Asymmetric PCR for ssDNA Production:

Simply use a 100:1 molar ratio of the two primers (eg: primer 1 at 0.5uM, primer 2 at 0.005uM). This allows production of mainly ssDNA of the sense of the more abundant primer, which is useful for sequencing purposes or making ssDNA probes.

Detecting Products:

Take 1/10th - 1/3rd of the reaction mix CAREFULLY from under the oil or from under the Vaseline or solidified wax, using a micropipette with plugged tip, IN AN AREA AWAY FROM YOUR PCR PREPARATION AREA! 

Mix this with some gel loading buffer(1:1 - 1:5 mix:loading buffer): this is TBE containing 10 - 20% glycerol or sucrose and a dash of bromophenol blue (BPB) tracking dye. 

Load 5 - 30ul of sample into wells of 0.8 - 3.0% submarine agarose gel made up in TBE, preferably containing 50ng/ml ethidium bromide. 

Run at 80 -120 volts (not too slow or small products diffuse; not too fast or bands smear) until BPB reaches end of gel (large products) or 2/3 down gel (small products). Use DNA markers going from 2kb down to 100 bp or less (recommend BM PCR markers). 

View on UV light box at 254 - 300 nm, photo 1 - 5 sec.

NOTE:

Small products are best seen on 3% agarose gels that have been run fast (eg: 100 volts), with BPB run to ½ - 2/3 down the gel. It is best to include EthBr in the gel AND in the gel buffer, as post-electrophoresis staining can result in band smearing due to diffusion, and if there is no EthBr in the buffer the dye runs backwards out of the gel, and smaller bands are stripped of dye and are not visible.

NUSIEVE TM gel (FMC Corp) can also be used for small products - better resolution than agarose.

Polyacrylamide gels can be silver stained by simple protocols for extreme sensitivity of detection.

Gels can be blotted directly after soaking in 0.5M NaOH / 1.5M NaCl for 10-20 min: "dry blotting" works well (eg: gel is over- and under-layered with paper towel stacks and pressed; bands transfer up and down), as does classic "Southern" blotting. Bands blotted in this way are already covalently fixed onto nylon membranes, and simply need a rinse in 5xSSPE before prehybridisation.

The example shown is of detection of Human papillomavirus type 16 (HPV-16) DNA amplified from cervical biopsy samples (Williamson A-L, Rybicki EP (1991) Detection of genital human papillomaviruses by polymerase chain reaction amplification with degenerate nested primers. J Med Virol 33: 165-171).  The left panel is a photo of an EthBR-stained 2% agarose gel; the right is an autoradiograph of a Southern blot probed with 32P-labelled HPV-16 DNA.  Note how much more sensitive blotting is, and how much more specific the detection is.