What is Direct PCR?
Direct PCR (dPCR) is a method of DNA amplification directly from an animal or plant tissue sample without performing DNA isolation and purification steps. This technique greatly reduces experimental time, and cost in genotyping and high-volume projects. It also provides a better option when faced with the challenges of amplifying a very small quantity of sample where the purification step could potentially lead to sample loss.
In many ways, dPCR works like conventional PCR. Both techniques depend on template DNA, primers and a master mix or PCR reagents, and both use a thermal cycler for amplification. The primary difference between dPCR and conventional PCR is the tailored buffers used in dPCR. The modified elements of dPCR enable robust amplification despite the presence of PCR inhibitors often found in crude samples.
Several papers have been published describing dPCR techniques and modifications of those techniques given special circumstances. “A rapid and inexpensive method for the direct PCR amplification of DNA from plants” provides excellent detail about experimental setup under usual conditions.
Kits are another avenue to dPCR. Direct PCR kits have the advantage of really simplifying the process and shaving off a lot of time. This is because kits do not involve the amount of preparation dPCR requires. Many techniques for dPCR (dPCR performed without kits) involve buffer and media preparation, mechanical disruption, and reagent variety. Kits, on the other hand, have all necessary reagents prepared in a ready-to-use format. GoldBio’s InstantAmp Plant Direct Kit, for example, is as easy as adding your plant sample (young leaf, root or seed) to the master mix with primers in PCR tubes and running it through a thermal cycler. Kit efficiency is ideal for large-scale sample analysis where saving time is extremely important.
What Value Does Direct PCR Offer a Lab Compared to Traditional PCR?
When dPCR aligns with your project goals, it offers several important benefits:
- Save Time: In situations where analyses of massive amounts of individual samples is needed, the omission of the purification step can dramatically reduce experimental time.
There is another aspect to timesaving, and that has to do with downstream methods that can only be performed after the upstream process is complete. By omitting a step and reducing process time, a researcher can move forward faster.
- Process Small Sample Quantities: Researchers often work with a tiny amount of precious sample. Classical DNA extraction can impose greater risk to smaller sample quantities. In forensics and anthropology, preventing sample loss can be especially important. One study reported a proportional loss of DNA collected from a variety of swabbing techniques to be between 20-76% during the extraction step. This loss is due to the series of wash steps involved in classical DNA purification. Elimination of the DNA purification step allows better conservation of material.
- Cut Costs: The amount of reagents used in DNA extraction can start to impact the budget, especially in large-scale situations. Extraction usually involves many buffers and reagents for optimization, detergents, phenol + chloroform, ethanol, digestion reagents, etc. When extraction is no longer factored into the equation, the reduced list of supplies can dramatically cut project expenses. There are also other costs involved when the purification step is involved such as cross contamination, prep time and sample loss.
Applications best suited for dPCR include genotyping, transgene detection, knockout analysis, sequencing and forensic detection.
Abstract
DNA evidence sample processing typically involves DNA extraction, quantification, and STR amplification; however, DNA loss can occur at both the DNA extraction and quantification steps, which is not ideal for forensic evidence containing low levels of DNA. Direct PCR amplification of forensic unknown samples has been suggested as a means to circumvent extraction and quantification, thereby retaining the DNA typically lost during those procedures.
Direct PCR amplification is a method in which a sample is added directly to an amplification reaction without being subjected to prior DNA extraction, purification, or quantification. It allows for maximum quantities of DNA to be targeted, minimizes opportunities for error and contamination, and reduces the time and monetary resources required to process samples, although data analysis may take longer as the increased DNA detection sensitivity of direct PCR may lead to more instances of complex mixtures. ISO 17025 accredited laboratories have successfully implemented direct PCR for limited purposes (e.g., high-throughput databanking analysis), and recent studies indicate that direct PCR can be an effective method for processing low-yield evidence samples.
Despite its benefits, direct PCR has yet to be widely implemented across laboratories for the processing of evidentiary items. While forensic DNA laboratories are always interested in new methods that will maximize the quantity and quality of genetic information obtained from evidentiary items, there is often a lag between the advent of useful methodologies and their integration into laboratories.
Delayed implementation of direct PCR of evidentiary items can be attributed to a variety of factors, including regulatory guidelines that prevent laboratories from omitting the quantification step when processing forensic unknown samples, as is the case in the United States, and, more broadly, a reluctance to validate a technique that is not widely used for evidence samples. The advantages of direct PCR of forensic evidentiary samples justify a re-examination of the factors that have delayed widespread implementation of this method and of the evidence supporting its use. In this review, the current and potential future uses of direct PCR in forensic DNA laboratories are summarized.
Direct Blood PCR Kit |
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| MBPCR171-100R | EWC Diagnostics | 1 unit | EUR 267.52 |
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Description: Direct Blood PCR Kit |
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Direct Blood PCR Kit |
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| MBPCR171-10R | EWC Diagnostics | 1 unit | EUR 71.73 |
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Description: Direct Blood PCR Kit |
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Direct Blood PCR Kit |
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| E0950-01 | EURx | 100reactions | EUR 69.76 |
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Description: Enables to perform PCR reactions directly from whole blood with no prior DNA extraction or purification |
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Direct Blood PCR Kit |
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| E0950-02 | EURx | 500reactions | EUR 277.95 |
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Description: Enables to perform PCR reactions directly from whole blood with no prior DNA extraction or purification |
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Direct Plant PCR Kit |
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| E0960-01 | EURx | 100reactions | EUR 55.59 |
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Description: Enables to perform PCR reactions directly from plants with no prior DNA extraction or purification |
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Direct Plant PCR Kit |
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| E0960-02 | EURx | 500reactions | EUR 251.79 |
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Description: Enables to perform PCR reactions directly from plants with no prior DNA extraction or purification |
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Direct Tissue PCR Kit |
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| E0940-01 | EURx | 100reactions | EUR 55.59 |
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Description: Direct Tissue PCR Kit enables to perform PCR reactions directly from tissue samples with no priorDNA purification |
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Direct Tissue PCR Kit |
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| E0940-02 | EURx | 500reactions | EUR 251.79 |
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Description: Direct Tissue PCR Kit enables to perform PCR reactions directly from tissue samples with no priorDNA purification |
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Blood Direct PCR Kit V2 |
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| PD103-01 | Vazyme | 50 rxn | EUR 51.41 |
Blood Direct PCR Kit V2 |
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| PD103-02 | Vazyme | 200 rxn | EUR 188.5 |
Mouse tissue direct PCR kit |
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| EP033-200T | ELK Biotech | 200T | EUR 125 |
Mouse tissue direct PCR kit |
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| EP033-50T | ELK Biotech | 50T | EUR 40 |
Mouse tissue direct PCR kit |
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| EP033 | ELK Biotech | 50T | EUR 125 |
Mouse Tissue Direct PCR Kit |
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| RK20736 | Abclonal | 100RXN | EUR 150 |
amfiSure Direct PCR Lysis Buffer |
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| D0300-010 | GenDepot | 100ml | EUR 165.6 |
Mouse Direct PCR Kit (For Genotyping) |
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| B40013 | Bimake | 200 ractions | EUR 250.8 |
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Description: The Mouse Direct PCR Kit provides a fast preparation and PCR amplIFication that is specIFically designed for mouse genotyping. Buffer L and Protease Plus rapidly digests mouse tissue to release intact genomic DNA that can be used directly as the template for PCR amplIFication. |
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Mouse Direct PCR Kit (For Genotyping) |
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| B40015 | Bimake | 500 ractions | EUR 490.8 |
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Description: The Mouse Direct PCR Kit provides a fast preparation and PCR amplIFication that is specIFically designed for mouse genotyping. Buffer L and Protease Plus rapidly digests mouse tissue to release intact genomic DNA that can be used directly as the template for PCR amplIFication. |
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Mouse Direct PCR Kit (For Genotyping) |
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| B45012 | Bimake | 1ml*5 (500 ractions) | EUR 336 |
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Description: The Mouse Direct PCR Kit provides a fast preparation and PCR amplIFication that is specIFically designed for mouse genotyping. Buffer L and Protease Plus rapidly digests mouse tissue to release intact genomic DNA that can be used directly as the template for PCR amplIFication. |
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Mouse Direct PCR Kit (For Genotyping) |
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| B45013 | Bimake | 1ml*20 (2000 ractions) | EUR 1038 |
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Description: The Mouse Direct PCR Kit provides a fast preparation and PCR amplIFication that is specIFically designed for mouse genotyping. Buffer L and Protease Plus rapidly digests mouse tissue to release intact genomic DNA that can be used directly as the template for PCR amplIFication. |
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BioLit Animal Tissue Direct PCR Kit (Research) |
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| 91210 | Sisco Laboratories | 50 Preps | EUR 222.28 |
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Description: Part E |
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Allele-In-One Mouse Tail Direct PCR Kit |
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| ABP-PP-MT02100 | Allele Biotech | 100 RXNS | Ask for price |
Allele-In-One Mouse Tail Direct PCR Kit |
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| ABP-PP-MT02500 | Allele Biotech | 500 RXNS | Ask for price |
Allele-In-One Human Blood Direct PCR Kit |
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| ABP-PP-BD02100 | Allele Biotech | 100 RXNS | Ask for price |
Allele-In-One Human Blood Direct PCR Kit |
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| ABP-PP-BD02500 | Allele Biotech | 500 RXNS | Ask for price |
FastAmp™ Plant Direct PCR Kit - 1,250 Reactions |
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| 4615 | Intact Genomics | 1/EA | EUR 495 |
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Description: Intact Genomics FastAmpTM Plant Direct PCR kit is an easy and robust method to amplify DNA fragments from plant tissues with high specificity and high sensitivity without the need of complicated DNA purification steps. The advanced formulation of this kit also allows fast PCR cycling conditions without compromising PCR sensitivity, specificity and yield. The FastAmpTM Plant Direct PCR kit is an ideal and powerful tool for high-throughput genotyping, DNA amplification, and plant genome analysis.Highlights:Direct PCR- no need to purify DNASpecially engineered Taq DNA polymerase with highest sensitivity and specificityExtremely short PCR protocol timesMaster mix format with premixed gel loading dye to reduce cross-contamination and sample handlingerrors5x magic enhancer for high GC containing DNA amplificationProduct Includes:FastAmp™ plant direct PCR master mix (2x)Dilution bufferControl primer mix (25 µM each)5x magic enhancerNuclease- free water |
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