Reprints
PubMed
CAVEAT LECTOR
Carlos A. Muro-Cacho, M.D., Ph.D.
Department of Pathology, H. Lee Moffitt Cancer Center and Research Institute and University of South Florida College of Medicine, Tampa, Florida, USA
Received 1/10/97 Accepted 7/15/97
4. POLYMERASE CHAIN REACTION (PCR)
Priorto attempting In situ PCR protocols, it is important to have an appropriate level of knowledge and expertise in solution PCR methodologies. This is due to the fact that the conceptual background is the same for both techniques. It is also highly desirable to test reagents and conditions by PCR in solution (in vitro) as part of the necessary preliminary control process. It is of no use to spend time and resources optimizing In situ PCR if one has not confirmed that primers and amplification conditions work as expected, preferably on the nucleic acids extracted from the sample.
4.1 Polymerases (appendix 5)
A variety of polymerases with different properties are commercially available. Ideally, the selected enzyme should work effectively at a given temperature and retain its enzymatic activity at temperatures higher than 95o C . It is important to follow the conditions suggested by the supplier, since, depending on the vendor, the same enzyme may have different assay requirements. In general, 0.5-2.5 units of DNA Taq polymerase in a 100 microliter reaction is appropriate (21-24). However, based on the type of targets or primers, the concentration of the enzyme may vary. It is recommended to test concentrations ranging from 0.5 to 5 units/100 microliter.
4.2 Deoxynucleotide triphosphates (dNTPs) (appendix 5)
Equivalent concentrations of all four dNTPs in the range of 20-200 microM are recommended (25-30). Mispriming may be minimized, and sensitivity maximized, by reducing the concentration of the dNTPs (26-30). Typically, in a 100 microliter reaction, a dNTP concentration of 20 microM, will allow the synthesis of 2.6 micrograms of DNA or 10 pmol of a 400 bp sequence (25-27).
4.3 Buffer conditions (appendix 5)
It is important to include the appropriate metallic ion in the amplification buffer and to use the enzyme at optimal pH. In general, as compared with solution PCR, increased concentrations of DNA polymerase and Mg2+ ions are required. This is probably related to the sequestration of reagents on slides or due to the presence of inhibitors in the tissue sample (24). The concentration of ionsis one of the critical factors in PCR experiments and it has to be optimized to the particular assay conditions. MgCl2 is commonly used at a concentration of approximately 2-5mM.
Primers are usually designed to be 18-28 nucleotides in length, with a balanced G/C and A/T ratio, no complementarity between their 3' ends (to avoid primer-dimer formation) and with a low probability of internal secondary structure (23-26). Concentrations between 0.1 and 1.5 microM are generally optimal. The melting temperature (Tm) of oligonucleotides ranging in length from 14-70 bases can be calculated acording to the following formula: Tm in o C = 2oC (#A+#T) + 4oC (#G+#C) (24).
4.5 Cycling Profile (appendix 5)
For optimal binding of the primers to the DNA, separation (denaturation) of the two strands has to be complete. Incomplete "denaturation" is a common cause of PCR failure (25-30). Typically, denaturation is achieved at 95o C for 30 seconds or at 97o C for 15 seconds, although higher temperatures may be necessary for G+C rich targets. Higher temperatures and longer times may lead to the loss of enzyme activity since the half-life of Taq polymerase is approximately 5 minutes at 97o C, and 40 minutes at 95o C (24, 28-30). Annealing temperature and annealing time depend upon the base composition and length and concentration of the primers (26-30). It is generally agreed that an annealing temperature of 5o C below the true Tm of the amplification primers is appropriate and temperatures in the range of 55o C to 72o C yield the best results. The primer extension step (elongation) is usually performed at 72o C for 20 seconds to one minute. Using typical concentrations of primers (0.2 microM), annealing will require a few seconds. However, a longer extension time may be required at the beginning of the cycling when the substrate concentration is low or at later cycles when substrate concentration exceeds the enzyme concentration. Some investigators report that longer extension times (e.g., 2 min) are necessary (30) while others omit the extension step since annealing seems to be the rate limiting factor (2, 31-33). The reaction is maintained at 72o C for 5-15 minutes to allow completion of partial extension and annealing (25-29). It is believed, however, that In situ DNA amplification occurs at a low efficiency. It is generally estimated that in 30 cycles, even under the best conditions, amplification of DNA does not exceed 50-100 fold, in suspended cells, and it may be even lower on tissue sections and cytospins (20-23). The reasons are not clearly understood although cross-linking of histones to DNA, single-stranded DNA breaks, and sequestration of DNA polymerases and other reagents on the surface of slide, have all been proposed as potential causes (24). When all parameters are optimized, the number of cycles will primarily depend upon the starting concentration of DNA (26-30). It has been estimated that if the starting number of target molecules is 300,000, then 25-30 cycles of amplification would be appropriate. On the other hand, if only 50 target molecules are present, 40-45 amplification cycles may be necessary (30-32). Some investigators, however, reported poor results, with more than 20 cycles, in In situ PCR experiments (4).