A novel method for whole blood PCR without pretreatment

Previous studies have shown that nylon flocked swabs outperform traditional fiber swabs in DNA recovery due to their innovative design and lack of internal absorbent core to entrap cellular materials. The microFLOQ Direct swab, a miniaturized version of the 4N6 FLOQSwab, has a small swab head that is treated with a lysing agent which allows for direct amplification and DNA profiling from sample collection to final result in less than two hours. Additionally, the microFLOQ system subsamples only a minute portion of a stain and preserves the vast majority of the sample for subsequent testing or re-analysis, if desired. The efficacy of direct amplification of DNA from dilute bloodstains, saliva stains, and touch samples was evaluated using microFLOQ Direct swabs and the GlobalFiler™ Express system. Comparisons were made to traditional methods to assess the robustness of this alternate workflow. Controlled studies with 1:19 and 1:99 dilutions of bloodstains and saliva stains consistently yielded higher STR peak heights than standard methods with 1ng input DNA from the same samples. Touch samples from common items yielded single source and mixed profiles that were consistent with primary users of the objects. With this novel methodology/workflow, no sample loss occurs and therefore more template DNA is available during amplification. This approach may have important implications for analysis of low quantity and/or degraded samples that plague forensic casework.

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“…Several studies have documented challenges with PCR amplification of blood samples without first performing DNA isolation [26][27][28][29][30].…”

Section: Resultsmentioning

confidence: 99%

Direct PCR amplification of DNA from human bloodstains, saliva, and touch samples collected with microFLOQ ® swabs

Ambers

Wiley

Novroski

et al. 2018

Forensic Science International: Genetics

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“…The specificity of the amplification was confirmed by melting temperature analysis which gave a very sharp and narrow melting point peak. An advantage of the SPR-assisted detection system is the ability to detect amplification products in nontransparent media which, for example, could be potentially useful in monitoring direct PCR amplification from blood [93,94]. A similar SPR detection technique has been utilized for isothermal amplification kinetic monitoring [74,95,96].…”

Section: Surface Plasmon Resonance Spectroscopymentioning

confidence: 99%

Recent developments in nucleic acid identification using solid-phase enzymatic assays

Khodakov

Ellis

2014

Microchim Acta

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“…DNA extraction is usually the limiting step for reducing the cost and time needed in molecular assays [31,32]. PCR without DNA purification cannot be done because of the chlorophyll, polysaccharides, and starches in the plant cells; which can inhibit the enzyme reaction [31,32,33,34,35,36,37]. Therefore, many efforts have been made towards the development of new methods for DNA analysis without DNA purification.…”

Section: Introductionmentioning

confidence: 99%

“…Therefore, many efforts have been made towards the development of new methods for DNA analysis without DNA purification. Several studies on DNA analysis by PCR without DNA purification have been reported using animal cells [33,34,35,36]. A method has also been developed to directly detect viruses infecting the Phalaenopsis orchid, using fresh leaves in RT-LAMP analysis [38].…”

Section: Introductionmentioning

confidence: 99%

Direct LAMP Assay without Prior DNA Purification for Sex Determination of Papaya

Tsai

Shih

et al. 2016

IJMS

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Papaya (Carica papaya L.) is an economically important tropical fruit tree with hermaphrodite, male and female sex types. Hermaphroditic plants are the major type used for papaya production because their fruits have more commercial advantages than those of female plants. Sex determination of the seedlings, or during the early growth stages, is very important for the papaya seedling industry. Thus far, the only method for determining the sex type of a papaya at the seedling stage has been DNA analysis. In this study, a molecular technique—based on DNA analysis—was developed for detecting male-hermaphrodite-specific markers to examine the papaya’s sex type. This method is based on the loop-mediated isothermal amplification (LAMP) and does not require prior DNA purification. The results show that the method is an easy, efficient, and inexpensive way to determine a papaya’s sex. This is the first report on the LAMP assay, using intact plant materials-without DNA purification-as samples for the analysis of sex determination of papaya. We found that using high-efficiency DNA polymerase was essential for successful DNA amplification, using trace intact plant material as a template DNA source.

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A novel method for whole blood PCR without pretreatment

Cited by 15 publications

References 12 publications

Direct PCR amplification of DNA from human bloodstains, saliva, and touch samples collected with microFLOQ ® swabs

Direct PCR amplification of DNA from human bloodstains, saliva, and touch samples collected with microFLOQ ® swabs

Recent developments in nucleic acid identification using solid-phase enzymatic assays

Direct LAMP Assay without Prior DNA Purification for Sex Determination of Papaya

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