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August 10, 2003
 
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LA PCR Protocol (Revised from Takara Shuzo Co., Ltd.) [back]  
Principle of LA PCR

The key to LA PCR is an enzyme, TaKaRa LA TaqTM. It is a thermostable DNA polymerase, which possesses 3’ to 5’ exonuclease activity, or proofreading activity. The efficiency drastically declines when incorrect bases are incorporated. The 3’ to 5’ exonuclease activity removes these misincorporated bases and makes the further reaction proceed smoothly. Therefore, the amplification of long DNA fragments can be achieved.

Points to Consider

1. Template DNA: When using PCR to amplify DNA fragments longer than 10 kbp, the preparation of intact (free of nicks) and fully purified template samples through repeated extraction/purification is essential.

2. Primer concentration: The optimal concentration will range from 0.1 to 1.0 mM. At a lower than optimum concentration, amplification yield may be poor. On the other hand, at a higher concentration, non-specific reactions may outperform primer-specific amplifications. In ordinary practice, primer concentrations can be set depending on the characteristics and amounts of template DNA. Low concentrations are recommended either for highly complex DNA, such as human genomic DNA, or for high concentrations of template DNA. While high concentrations are preferred for low complexity templates such as plasmid DNA, or for limiting amounts of input template DNA.

3. Magnesium concentration: The optimal magnesium concentration may be affected by characteristics and properties of the reaction mix, including concentrations of dNTPs, template-primer combinations, and chelating agents carried over from template DNA. Determine the optimal level of Mg2+ by varying the concentration in 0.5 mM increments throughout a range of 1.5 to 4 mM. Excess Mg2+ tends to cause non-specific reactions and results in smeared electrophoresis bands, while scarce Mg2+ may generate less or no amplified products.

4. Enzyme amount: The recommended amount of enzyme in a 50 ml reaction is 2.5 units of TaKaRa LA TaqTM, but the amount can be changed for the optimum reaction condition. The quantity or complexity of template DNA and the length of amplified DNA fragment should be considered. Excess enzyme can cause non-specific reactions may occur and result in smear upon electrophoresis. The efficiency of amplification may be diminished when the enzyme concentration is low.

5. Cycle numbers: The optimum cycle number is around 25 to 30 cycles considering the quantity or complexity of template DNA, and the length of amplified DNA fragments. Less cycling may not generate enough amplified product, while over cycling may produce a diffuse smear upon electrophoresis.

6. Denaturation conditions: Denaturing conditions are recommended to be either 20 seconds at 98°C or 30 seconds at 94°C. A denaturation time that is too short or a denaturing temperature that is too low may cause either diffuse smearing upon electrophoresis or poor amplification efficiency. A denaturation time that is too long or a denaturation temperature that is too high may generate no identifiable product.

7. Conditions for annealing and extension: Determine the optimum annealing temperature experimentally over a range of 45-68°C. To carry out the combined anneal-extension at 68°C (two step PCR), the recommended time setting is 30 seconds to 1 minute per one kilobase. When the temperature is set below 68°C, a longer time period is required. An annealing temperature that is too high generates no amplification products, while a temperature that is too low enhances non-specific reactions. An extension time that is too short generates no amplification products or dominantly non-specific, short products. While too long extension time causes diffusely smeared electrophoresis bands. TaKaRa LA TaqTM shows sufficient activity at 60 to 68°C. Shuttle PCR (two temperature PCR) can be conducted by the anneal-extension temperature within this range. When long PCR amplification is required, a significant increase in amplification efficiency may be expected by employing an Autosegment Extension Method.

8. Hot start: This method is helpful in eliminating non-specific reactions with TaKaRa TaqTM. It suppresses adverse effects of the 3’ to 5’ exonuclease activity on the primers.

Standard Method

Reagent Volume Final concentration
10x LA PCR Buffer II (Mg2+ free) 5.0 ml [1x]
MgCl2 (25 mM) 5.0 ml 2.5 mM
DNTP Mixture (2.5 mM each) 8.0 ml 400 mM each
Primer 1 (20 pmol/ml) 0.5 ml 0.2 mM
Primer 2(20 pmol/ml) 0.5 ml 0.2 mM
TaKaRa LA TaqTM 0.5 ml 2.5 U/50 ml PCR
Purified genomic DNA 1.0 ml 500 ng/50 ml PCR
dH2O 29.5 ml  
Total 50.0 ml  


PCR Conditions

(1) Simple cycling

Cycles Temperature Time
1 94°C 1 min.
30 98°C 10 sec.
68°C 15 min.
1 72°C 10 min.

(2) Autosegment extension (Touchdown PCR)

Cycles Temperature Time
1 94°C 1 min.
14 98°C 10 sec. *
68°C 15 min.
16 98°C 10 sec.
68°C 15 min. + 15 sec./cycle **
1 72°C 10 min.

* When normal type of PCR tubes are used, denaturation time per cycle should be 20 seconds.
** Autosegment extension: At the 15th cycle and following, the extension time should be extended by 15 seconds each cycle. This extension procedure may increase yield of amplification products.

     

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