Molecular Techniques and Methods

Long Distance PCR

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

The ability to amplify fragments up to 20-50 kb would potentially enable the isolation of an entire gene from a cDNA, thereby obviating the time-consuming task of screening a genomic library for the target gene.




MATERIALS AND SOLUTIONS

10 x PCR Buffer (1 ml)
25 mM Tris-HCl (pH 8.9) ------------------------- 25 ul of 1 M Tris-HCl
100 mM KCl -------------------------------------- 100 ul of 1 M KCl
0.75 mM EGTA ----------------------------------- 15 ul of 50 mM EGTA
0.05% Tween-20 ---------------------------------- 5 ul of 10% Tween-20
0.01% Gelatin ------------------------------------- 10 ul of 1% Gelatin
Distilled H2O -------------------------------------- 845 ul

  • In 10 x PCR Buffer, the EGTA chelates manganese and inhibits any reverse transcriptase activity that might otherwise be exhibited by rTth DNA polymerase.




    PROCEDURES

    1. In a pre-PCR area, prepare the master mix of the followings and distribute to PCR tubes.

    Component
    Volume
    Final Concentration
    10 x PCR Buffer
    10 ul
    1 x Buffer
    50% Glycerol
    10 ul
    5%
    25 mM MgCl2
    10 ul
    2.5 mM MgCl2
    Primer 1 (Tm > 63oC)
    10 ul
    10 pmoles-200 nmoles
    Primer 2 (Tm > 63oC)
    10 ul
    10 pmoles-200 nmoles
    dNTPs Mix
    1-2 ul
    100-200 uM
    DNA Template
    -
    25-100 ng
    rTth DNA polymerase +
    Pfu (or, Vent) DNA polymerase (20:1 = v:v)
    -
    2.5 U
    Distilled H2O to make a final volume of
    -
    100 ul


    2. Do PCR reaction as follow:

    1 cycle
    Denaturation
    95oC
    2 min
    10 cycles
    Denaturation
    95oC
    10 sec
    Annealing
    Tm-5oC
    (>58oC)
    30 sec
    Extension
    72oC
    3 min
    20 cycles
    Denaturation
    95oC
    10 sec
    Annealing
    Tm-5oC
    (>58oC)
    10 sec
    Extension
    72oC
    3 min +
    20-40 sec automatic increment every cycle
    1 cycle
    Extension
    72oC
    10-15 min

  • Tm = [2 x (A+T)] + [4 x (G+C)]


    3. Analyze PCR products in agarose gel.




    NOTES

  • The majority of DNA in long distance PCR should be a high-quality template, because high-integrity DNA provides a greater concentration of intact initial template available.

  • Primer length is optimal at 18-25 nucleotides for long distance PCR. When designing primers for amplification of regions from complex genomes, or regions known to be particularly G+C-rich, increased length provides additional stability. The addition of each incremental nucleotide to the primer confers approximately four times more specificity. Complementarity at the 3' ends of the primers as well as secondary structure should be avoided.

  • High primer concentrations decrease the time necessary for sufficient annealing and therefore may increase specificity.

  • The nucleotide composition of primers should reflect the nucleotide composition of the region from which the desired target is to be amplified (e.g., if the target region is 50% G+C, then the primers should also be 50% G+C).

  • DNA polymerases with 5' to 3' exonuclease (nick translation) activity have yielded consistently superior performance in long-distance PCR as compared with those enzymes exhibiting 3' to 5' exonuclease ("proofreading") activity, both 3' to 5' and 5' to 3' exonuclease activities, or neither exonuclease activity.

  • Taq DNA polymerase extends at a rate of 0.25 nucleotides per second at 22oC, 1.5 nucleotides per second at 37oC, 24 nucleotides per second at 55oC, greater than 60 nucleotides per second at 70oC, and 150 nucleotides per second at 75-80oC. Thus, at 70-72oC, Taq DNA polymerase would extend at a rate of greater than 3.5 kb per minute. As a general rule, extension times of 1 minute per kilobase are more than sufficient to generate the expected PCR product. The same rule applies when using rTth DNA polymerase, because this enzyme has an extension rate that is very similar to that of Taq DNA polymerase.

  • As the PCR product begins to accumulate in the later cycles, the ability to extend a significant proportion of primers over a long distance in a given unit of time might be limited by the relative decrease in enzyme molecules per template. Thus, increasing the extension time in each of the later PCR cycles could increase the likelihood of synthesizing long PCR products.




    KIT INFORMATION




    REFERENCES

  • Barnes, WM (1994) PCR amplification of up to 55-kb DNA with high fidelity and high yield from lambda bacteriophage templates. PNAS 95: 2216-2220.

  • Foord, O, Rose, EA (1994) Long-distance PCR. PCR Methods Appl. 5: 5149-5161.

  • Kainz, P, Schrniedlechner, A, Strack, B (1992) In vitro amplification of DNA >10 kb. Anal. Biochem 202: 46-49.

  • Maga, EA, Richardson, T (1991) Amplification of a 9.0 kb fragment using PCR. BioTechniques 11: 185-186.

  • Ponce, MR, Nicol, L (1991) PCR amplification of long DNA fragments. Nucleic Acids Res. 20: 623.



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