Optimization of convenient real-time PCR machines

If it is real-time fluorescence quantitative PCR for virus quantification and SNP genotyping, we need a PCR machine with the highest possible specificity; if it is pathogen and mRNA detection, we need high sensitivity PCR machine, so how should we optimize this PCR machine?

Here is the content list:

l Improving PCR machine amplification efficiency

l Using real-time PCR machine primers for general PCR

Improving PCR machine amplification efficiency

To improve the PCR machine amplification efficiency, specificity, and sensitivity, we can optimize the real-time fluorescence quantitative PCR machine for the experiment through a series of measures. The first is the selection of the target sequence.

1, the choice of convenient real-time PCR machine target sequence appropriate length between 50 to 150bp. Shorter sequences take less time to synthesize and have shorter amplification times, so the possibility of contaminating DNA being amplified is reduced.

2, When selecting the target sequence, use BLAST search to analyze the target sequence for polymorphisms and sequencing errors and avoid them. If there are duplicate sequences in the target sequence, it will reduce the detection sensitivity of this PCR machine and should also be avoided.

3, control the GC content ≤ 60% in the target sequence. High GC content, will affect the denaturation of the target sequence in the thermal cycle, but also easy to produce this PCR machine non-specific amplification.

4, avoid generating secondary structure. Target sequences with reverse repetitive sequences are easy to produce secondary structure, which affects the hybridization of convenient real-time PCR machine primers and probes.

In addition to this, we also need to ensure that the quality of the template will not affect the amplification, the results of the convenient real-time PCR machine have reliability. For example, damaged DNA cannot be amplified adequately in the PCR machine, or not amplified at all. Also, the presence of DNA polymerase inhibiting reagents (DMSO, etc.) and contaminants (SDS, etc.) in the template can affect the reliability of the PCR machine amplification results.

Using real-time PCR machine primers for general PCR

Get convenient real-time PCR machine primers, this method can also be used for ordinary PCR.

1, The length should be 18-30bp to ensure the specificity of binding.

2, The melting temperature (Tm value) should be 55-60°C, and the Tm values of the two primers should differ by 2-3°C.

3, Each primer should have 1 to 3 guanines or cytosines at the 3' end, which can conveniently reduce the non-specific amplification during the PCR machine in real-time. More than 3 will backfire and cause a "sliding effect" leading to the wrong extension.

4. The GC content of the primers should be around 50%, too high GC content will increase the Tm value.

5, the PCR machine primers should avoid reverse repetitive sequences, because it will form a secondary structure, affecting the primer binding on the template.

6, If it is RT-PCR, you also need to avoid designing primers to bind on exon sequences, which will lead to false-positive PCR machine experimental results. The correct approach should be designed on the long intron flank, or short introns, or across the exon-exon junction region.

7. Desalting and purifying the primers.

Note that sometimes, when you have done all the points, the primers may not amplify, so you should redesign the primers.

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