by K Chico, Jessica Piczon

In traditional polymerase chain reaction (PCR), extraction is a necessary step used to purify DNA within a biological sample. Extraction steps effectively release trapped DNA from within cellular material such as saliva, blood, or even fingernails, hair, and bone. Additionally, extraction steps help remove contaminating inhibitors that may otherwise negatively affect PCR amplification. Although DNA extraction and isolation has its advantages, such steps may lead to a significant loss of DNA. Extraction steps may even reduce the potential for generating a reliable DNA profile downstream, especially in cases where the amount of available DNA within the starting sample is limited.

Direct PCR has been utilized for sample amplification since the 1990s, notably for colony PCR, as a rapid screening method for large numbers of bacterial cells for a gene of interest, and also for human leukocyte antigen (HLA) testing on whole blood. Currently, direct PCR is routinely used for the detection of viral and bacterial pathogens in the clinical setting, as well as for DNA barcoding purposes of taxa for biodiversity and species studies. Most recently, direct PCR has found valued use in forensic science. Because of which, a number of specialized direct PCR multiplex kits are commercially available which have reduced data analysis time, lowered personnel and material costs, and have shortened the number of steps required in experimentation.

Alternatively, direct PCR is a method of DNA and RNA amplification that does not require an extraction step. The methodology behind this technique is simple; a sample is first collected, then placed directly into the amplification reaction without preliminary treatment and subject to PCR. By bypassing the extraction processes, maximum quantities of DNA can be targeted, and personnel error or exogenous DNA contamination may be reduced. Direct PCR also circumvents the need for certain steps in the procedure, like isolation, purification and DNA quantification before PCR. This reduces overall sample processing time, removes the need for specialized equipment, and cuts costs associated with materials, including columns, beads, and various reagents. Moreso, direct PCR workflows can also be incorporated into high throughput automation techniques.

Direct PCR, however, does not come without consideration. One requirement of any robust, reliable direct PCR assay is the ability to overcome sources of PCR inhibition. These can exist in many forms, including excessive protein within a sample, particulates of the sample collection device (e.g., from a plastic swab), or even contaminants from the material storage solution (e.g., guanidine).

Typically, most direct PCR methods are less sensitive compared to a technique that requires extraction, so experimental optimization is often necessary. This means that primer and probe annealing should be highly specific to the intended target and the detection of the amplification signal should be high enough to avoid background signals. Due to the complexity of starting samples, data analysis is typically a lengthy process and requires experienced personnel.