What are the critical factors for amplification of GC-rich templates?

1. PCR conditions -Use higher denaturation temperatures (e.g., 98°C as opposed to 94°C or 95°C) to allow complete denaturation of the template. -Keep annealing times for GC-rich templates as short as possible. -Use primers with a higher Tm (>68°C), because annealing can occur at a higher temperature. 2. PCR polymerases Use a polymerase optimized for amplification of GC-rich sequences. To [...]

How do I determine if a template is GC rich?

The GC ratio varies across the genome. Templates with >65% GC content are considered GC rich. GC-rich regions of the genome are mostly concentrated in regulatory regions, including promoters, enhancers, and cis-regulatory elements. GC-rich tracts tend to form inverted repeats, or hairpin structures, that may not melt during the annealing step of PCR. Therefore, amplification of GC-rich [...]

What are the critical factors for amplification of long genomic targets?

1. Template quality DNA integrity is critical for amplification of long targets. DNA damage—such as DNA breakage during DNA isolation or DNA depurination at elevated temperatures and low pH—results in a greater amount of partial products and decreased overall yield. DNA damage can also occur in acidic conditions; therefore, avoid using water for resuspending DNA templates. DNA is most stable at [...]

What is the optimal amount of DNA template that should be used for PCR?

The optimal amount of template required depends on the complexity of the template and the copy number of the target sequence. Approximately 104 copies of the target DNA sequence are required to detect the amplification product in 25–30 PCR cycles. -Typically, 1 µg of human genomic DNA contains 3.04 x 105 molecules of DNA. For most PCR applications, 30–100 ng of human genomic DNA is sufficient. [...]

Which extension temperature should I use, 68°C or 72°C?

A 68°C extension temperature is preferred for two-step PCR and when amplifying longer templates (>4 kb). This lower extension temperature dramatically improves yields of longer amplification products by reducing the depurination rate that influences amplification. 72°C should be used as the extension temperature when performing three-step standard PCR and for amplification of short fragments [...]