高GC含量PCR Buffer配方

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The recommendation is: Betain 1M, DMSO 5%


ADDITIVE  Glycerol (5-10%), formamide (1-5%), or DMSO (2-10%) can be added to the PCR for template DNA with high GC content (these change the Tm of primer-template hybridization reaction and the thermostability of polymerase enzyme). Betain (0.5-2 M) is also useful in PCR of high GC content and long DNA. Perform a titration to determine optimum concentration. When using betain, reduce melting temperature (92-93°C) and annealing temperature (1-2°C lower). BSA (up to 0.8 ug/ul) may also improve efficiency of PCR reaction. PCRx Enhancer Solution (Cat. No. 11495-017) is a novel PCR cosolvent that facilitates efficient amplification of GC-rich sequences and remedies difficulties associated with PCR of problematic templates.

ANNEALING Use appropriate temperatures based on the calculated Tm of primers.

CYCLING Denaturation time can be increased if template GC content is high. Extension time should be increased for larger PCR products, but this might lead to damage of the enzyme. The number of cycles may be increased if the amount of template DNA is very low, and decreased if template DNA is abundant.

DENATURING Use a temperature appropriate for polymerase of choice.

DNTP The concentrations of dNTPs used in a reaction are determined by the affinity of the enzyme for substrate (the Km of the enzyme). dNTPs on the order of 10uM are generally appropriate. Concentration of dNTPs decreases as the reaction proceeds. Excessive dNTP concentration reduces polymerase accuracy and requires higher Mg2+ in the reaction mix. Up to 1.5 mM dNTP may be used. dNTPs chelate Mg2+. Excessive dNTP concentration may increase the error rate. Lowering the dNTP (10-50 uM) may reduce error rate. Large PCR fragments require more dNTPs than small fragments.

EXTENSION Typically 72°C. At 70-72°C the activity is optimal, and primer extension occurs at up to 100 bases/sec. One minute is sufficient for reliable amplification of 2 kb sequences. Longer products require longer times: 1 minute per 1 kb. Longer times may also be helpful in later cycles when product concentration exceeds enzyme concentration and when dNTP and/or primer depletion may become limiting.

MAGNESIUM Buffers often contain Mg2+ (from MgCl2 or MgSO4) as a necessary cofactor for enzyme activity. Taq polymerase is particularly sensitive to Mg2+ concentration. At low Mg2+, Taq polymerase activity (correct complementary base pairing) is high, but polymerization rate is low. Conversely, at high Mg2+, the polymerization rate is high but the accuracy is low. Reduce concentration to prevent non-specific and undesirable PCR products. Increase to attain more product. EDTA chelates Mg2+ and can change the Mg2+ concentration.

PCR BUFFER Higher concentration of PCR buffer may be used to improve efficiency.

pH The 10X buffer contains a buffering agent (usually Tris-HCl) to maintain constant pH. The ideal pH for PCR is 8.4. For long templates, a higher pH (pH 9.0) is suggested. The pH of the Tris buffer in the reaction mix will decrease in high temperatures. The lower pH may cause depurination of the template, resulting in a lower yield of amplicons.

POLYMERASES Taq DNA polymerase has a higher error rate (no proofreading 3' to 5' exonuclease activity) than Pfx. Use Pfx if high fidelity is needed. Taq tends to add non-templated A at the 3' end. Extra enzyme may be added to improve efficiency (since Taq may be damaged in repeated cycling), but this may increase non-specific PCR products.

PRIMER CONCENTRATION Primer concentration in a common PCR reaction is about 100-500 nM per primer. Increasing primer concentration may improve the outcome of the PCR reaction and should be considered as a way to optimize PCR reactions. High primer concentrations may inhibit the reaction. Example: How do I make a 10uM stock solution starting with 24 nmol of oligo? - Start by converting uM into nmol/ml: 10uM = 10 umol/L or 10 nmol/ml - Then figure out what volume you need to resuspend your oligo in based on your starting amount: 10 nmol/ml = 24 nmol/ X ml, solving for X = 2.4 ml or 2400 ul - To make a 10 uM stock solution, you would resuspend 24 nmol of oligo in 2400 ul. If you use 10 ul of a 10 uM stock solution in a 100-ul reaction, the final oligo concentration will be 1uM. Use the same amount for BOTH primers in your PCR. Up to 3 uM primers may be used. High primer to template ratio may result in non-specific amplification and primer-dimer formation. Store primers in small aliquots to avoid multiple freeze-thaw cycles.

PRIMER SEQUENCE Generally, primers used are 18-28 nt in length. This provides for practical annealing temperatures. Primers should avoid stretches of polybase sequences (e.g., poly dG) or repeating motifs -- these can hybridize inappropriately on the template. Aim for 50% GC content. High GC content results in the formation of stable imperfect hybrids while high AT content depresses the Tm of perfectly matched hybrids. If possible, the 3' end of the primer should be rich in GC bases (GC clamp) to enhance annealing of the end which will be extended. Inverted repeat sequences should be avoided to prevent formation of secondary structure in the primer which may prevent hybridization to template. Sequences complementary to other primers used in the PCR should be avoided so as to prevent hybridization between primers (primer dimers). Primer pairs should have compatible Tm (within 5 degrees). When adding sequences to the 5' end of the primer to create a restriction site, it is important to include a few extra bases (2-6 bases) to serve as a clamp to keep the 5' ends from breathing during digestion.

SALT Enzyme function and formation of primer-template hybrids require salt and buffer. Salt concentrations that maximize enzyme activity are generally sufficient to shield phosphate backbone repulsion. Buffers often contain 50mM KCl to provide the correct ionic strength.

TEMPLATE For most PCR reactions, template should be present at 10 to 1000 copies per reaction -- approximately 5-100 ng of tempate DNA. To reduce the likelihood of error by Taq DNA polymerase, a higher DNA concentration can be used. Too much template may increase the amount of contaminants and reduce efficiency