Genomic DNA

Guidelines for PCR

PCR is a powerful tool that allows amplification of specific DNA sequences. Prerequisites for successful PCR include the design of optimal primer pairs, the use of appropriate primer concentrations, and optimization of the PCR conditions.

Tip

Genomic DNA sized up to 50 kb, such as that isolated using DNeasy and QIAamp Kits, shows the highest amplification efficiency under normal PCR cycling conditions.

Primer design
The following points should be considered when designing primers for PCR. Length: GC content: 1830 nucleotides 4060%

Tm:

Simplified formula for estimating melting temperature (Tm ): Tm = 2C x (A+T) + 4C x (G+C) Whenever possible, design primer pairs with similar Tm values. Optimal annealing temperatures may be above or below the estimated Tm. As a starting point, use an annealing temperature 5C below Tm. Avoid complementarity of two or three bases at the 3' ends of primer pairs to reduce primerdimer formation. Avoid mismatches between the 3' end of the primer and the target-template sequence. Avoid runs of 3 or more G or C at the 3' end.

Sequence:

Avoid a 3'-end T. Primers with a T at the 3' end have a greater tolerance of mismatch. Commercially available computer software (e.g., Primer Designer 1.0, Scientific Software, 1990; Oligo, Rychlik and Rhoads, 1989) can be used for primer design. Spectrophotometric conversion for primers: 1 A260 unit = 2030 g/ml. See Spectrophotometric Measurement of Nucleic Acid Concentration, page 91 for an example of the calculation for determining nucleic acid concentration when using a spectrophotometer. Molar conversions: Primer length 18mer 20mer 25mer 30mer pmol/g 168 152 121 101 20 pmol 119 ng 132 ng 165 ng 198 ng

Concentration:

Storage:

Use a concentration of 0.10.5 M of each primer. For most applications, a primer concentration of 0.2 M will be sufficient.

Lyophilized primers should be dissolved in a small volume of distilled water or TE to make a concentrated stock solution. Prepare small aliquots of working solutions containing 10 pmol/l to avoid repeated thawing and freezing. Store all primer solutions at 20C. Primer quality can be checked on a denaturing polyacrylamide gel; a single band should be seen.

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PCR conditions
The primer and Mg2+ concentration in the PCR buffer and annealing temperature of the reaction may need to be optimized for each primer pair for efficient PCR. In addition, PCR efficiency can be improved by additives that promote DNA polymerase stability and processivity or increase hybridization stringency, and by using strategies that reduce nonspecific primertemplate interactions (1). Use of high-purity reagents is also essential for successful PCR, especially for amplification of rare templates, for example, single copy genes in genomic DNA or pathogenic viral DNA sequences in genomic DNA isolated from an infected organism.

Tip

Q-Solution and QIAGEN PCR buffer, together with QIAGEN Taq DNA Polymerase or HotStarTaq DNA Polymerase, provide efficient template amplification without the need for optimization, even for templates that have extensive secondary structure or that are GC- rich (6).

Inclusion of control reactions is essential for monitoring the success of PCR reactions. Wherever possible, a positive control should be included to check that the PCR conditions used can successfully amplify the target sequence. As PCR is extremely sensitive, requiring only a few copies of target template, a negative control containing no template DNA should always be included to ensure that the solutions used for PCR have not become contaminated with the template DNA.

Tip

PCR setup should be performed in a separate area from PCR analysis to ensure that reagents used for PCR do not become contaminated with PCR products. Similarly, pipets used for analysis of PCR products should never be used for setting up PCR.

QIAGEN offers a wide range of products for isolation of genomic DNA from various sources and for all throughput requirements, as well as products for PCR analysis. For further information about QIAGEN products and literature please refer to the QIAGEN Product Guide, visit us online at www.qiagen.com, or contact QIAGEN Technical Services or your local distributor.

References 1. Ausubel, F.M., et al. (1987) Current Protocols in Molecular Biology. New York: John Wiley and Sons. 2. Animal Genome Size Database www.genomesize.com 3. Systma, K.K., Givnish, T.J., Smith, J.F., and Hahn, W.J. (1993) Collection and storage of land plant samples for macromolecular comparisons. Methods Enzymol. 234, 23. 4. Wilfinger, W.W., Mackey, M., and Chomczynski, P. (1997) Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. BioTechniques 22, 474. 5. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. 6. Critical Factors for Successful PCR, QIAGEN

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