Direct PCR

What is Direct PCR?

Direct polymerase chain reaction, (direct PCR) is a type of PCR that allows direct amplification from tissue sources (cultured cells, animal tissue, animal body fluids (including blood, semen, lymph fluid, cerebrospinal fluid, etc.), plant leaves, plant seeds, etc) without DNA purification (which is usually time-consuming and expensive). The premise of traditional PCR technology is to obtain a purified DNA / RNA template. Whether it is cells or animal and plant tissues, the purification of DNA / RNA templates is time-consuming and laborious, and plant tissues require liquid nitrogen grinding. In direct PCR, manual processing of the sample as well as the amount of buffers and consumables needed are limited to a minimum which helps to avoid the introduction of contamination. Since DNA extraction results in significant loss of DNA from the samples because of multiple tube changes and sometimes low DNA recovery rates. Furthermore, it saves time and demonstrates excellent sensitivity. Direct PCR is also effective for low amounts of DNA, simplifying experiments. The amplification can be achieved in the presence of PCR inhibitors, growth serum, and other source material components. This is a more rapid and simple approach because the untreated environmental sample is used directly as a template in PCR, eliminating the steps of cell recovery or DNA extraction. Direct PCR can be performed in two alternative ways, direct protocol and dilution protocol (Fig.1).

DNA polymerases used in direct PCR have some features superior to other PCR enzymes: they are highly robust and tolerant of many PCR inhibitors present in unpurified samples. These features guarantee reliably amplifying DNA in extremely challenging conditions-such as directly from crude samples.

Direct PCR workflow. Direct PCR can be performed using  two alternatives. In the direct protocol, a small amount of sample is added  directly to the PCR reaction. Hands-on time is minimized and carried on from  sample to PCR in one step. The dilution protocol uses a short pre-culture step  before PCR to release DNA from the sample material. It's for multiple PCR  reactions and long/difficult amplicons Fig. 1 Direct PCR workflow. Direct PCR can be performed using two alternatives. In the direct protocol, a small amount of sample is added directly to the PCR reaction. Hands-on time is minimized and carried on from sample to PCR in one step. The dilution protocol uses a short pre-culture step before PCR to release DNA from the sample material. It's for multiple PCR reactions and long/difficult amplicons. (Pak C, et al, 2012)

Process of Direct PCR Amplification

Direct PCR is conducted by placing the relevant sample into a 0.2 mL thin walled tube containing 10 µL of PCR master mix along with 5 µL of the primer mix and 1 µL (5 U) of additional DNA polymerase. Adding DNA polymerase is to increase the overall units of enzyme in the reaction to assist in overcoming inhibitors that may be present in the sample. An extra 9 µL of sterile H2O were added to make the final volume 25 µL. The amplification could be conducted in a thermal cycler in the conditions as manufacturer's recommend.

Application of Direct PCR

Direct PCR technique itself has been used since the 1990s. It is capable for DNA amplification from a wide range of materials, such as mouse tissues (ear, tail, hair, liver, spleen and brain), cultured mouse cells (fibroblasts), drosophila (wing and whole insect) Dog (hair), Human specimen (buccal swabs, fingernails, saliva, teeth, skin biopsies, hair, FFPE tissue). It can also analyze sexual assault samples which are common samples encountered in forensic analysis.

Direct PCR represents an innovative example of translational medicine as well as the application of pharmacogenetics in the clinical practice encouraging personalized, safer and cost-effective therapy. Current application of direct PCR includes genotype analysis to identify the roles of genes in development, physiology and disease.

Key Points in Direct PCR

This technology has extremely high resistance to stress and adaptability to PCR (including RT-PCR) reaction systems. Because the intracellular material composition of different cells and different tissues is extremely responsible, proteins, polysaccharides, and salt ions have a great impact on the PCR reaction, and it is difficult to have a system that can adapt to multiple samples. Plant tissues are more pronounced, and different plant tissues have significantly different inhibitory effects on PCR reactions due to the huge differences in secondary metabolites. Therefore, optimizing the PCR reaction system according to different species so that it has corresponding resistance and adaptability is the key to the realization of direct PCR assay.

References:

  1. Cascella R, Strafella C, Ragazzo M. et al. Direct PCR: A new pharmacogenetic approach for the inexpensive testing of HLA-B*57:01. Pharmacogenomics J, 2015, 15: 196–200.
  2. Tobe S S, Swaran Y C, Lynn Dennany, et al. A proof of principal study on the use of direct PCR of semen and spermatozoa and development of a differential isolation protocol for use in cases of alleged sexual assault[J]. International Journal of Legal Medicine, 2016, 131(1):1-8.
  3. Franzo G, Legnardi M, Centelleghe C, et al. Development and validation of direct PCR and quantitative PCR assays for the rapid, sensitive, and economical detection of porcine circovirus 3[J]. Journal of Veterinary Diagnostic Investigation, 2018, 30(4):538-544.
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