Rapid microfluidic analysis of a Y-STR multiplex for screening of forensic samples

Abstract

In this paper, we demonstrate a rapid analysis procedure for use with a small set of rapidly mutating Y chromosomal short tandem repeat (Y-STR) loci that combines both rapid polymerase chain reaction (PCR) and microfluidic separation elements. The procedure involves a high-speed polymerase and a rapid cycling protocol to permit PCR amplification in 16 min. The resultant amplified sample is next analysed using a short 1.8-cm microfluidic electrophoresis system that permits a four-locus Y-STR genotype to be produced in 80 s. The entire procedure takes less than 25 min from sample collection to result. This paper describes the rapid amplification protocol as well as studies of the reproducibility and sensitivity of the procedure and its optimisation. The amplification process utilises a small high-speed thermocycler, microfluidic device and compact laptop, making it portable and potentially useful for rapid, inexpensive on-site genotyping. The four loci used for the multiplex were selected due to their rapid mutation rates and should proved useful in preliminary screening of samples and suspects. Overall, this technique provides a method for rapid sample screening of suspect and crime scene samples in forensic casework. Graphical abstract ᅟ.

Forensic DNA Analysis

“…Increasing the speed of the PCR amplification step can be done in a variety of ways. Some groups modify commercially available kits and their protocols, while others create entirely new multiplexes specially designed for fast amplification. , Gibson-Daw et al used a 7-locus multiplex on a high speed thermocycler and a rapid polymerase to achieve a multiplex amplification in 6.5 min . Lower total volumes have been shown to help reduce the time of heating and cooling of the sample, reducing amplification time by 56–73% .…”

“…Rapid Y-STR typing for both screening samples and identification purposes was developed. ,, Screening methods are useful because much of the biological evidence received by laboratories often contains body fluids from a number of different contributors. Rapidly mutating Y-STR markers (RM Y-STRs) have been selected for inclusion in multiplexes.…”

The Future of Forensic Biology

“…The use of handheld 3-D laser scanners [60,61]and Near-infrared (Near IR) light scanners [62] can also help scientist/police to identify a potential suspect during an investigation. The microfluidic chips on the other hand allow scientists to reduce contamination and build DNA profile at the scene of crime [5][6][7]63]. Portable forensics labs could also be equipped with devices capable of transmitting data obtained through the use of next generation facial recognition software and fingerprint scans to government databases for comparison with stored files of such information.…”

“…Following refinement and rigorous testing, many methods/technologies have been adopted by forensic laboratories, including polymerase chain reaction (PCR) [1,2], capillary electrophoretic instrumentation (Genetic Analyzers) [3,4], automated liquid handling (Microfluidic devices) [5][6][7], and expert software systems [8][9][10][11]. Introduction of robust validation processes has a long term impact to test evidentiary samples to be presented to the court of law.…”

Ultrahigh Speed Direct PCR: A Method for Obtaining Y-STR and STR Based Genotypes in Under 20 Minutes

“…examined the effect of polymerase, buffer conditions and cycle time in an effort to increase the speed of commercially available forensic kits [Vallone 2008, Vallone 2009, Romsos 2015. And while many of these studies have successfully reduced amplification time to ~60 minutes [ Budowle 1990, Gibson-Daw 2017, only a few manage to reduce this below 30 minutes [Foster 2012 from Puritan Medical Products (Guildford, ME, USA). These samples were lysed, digested and the DNA extracted and purified using standard phenol chloroform isoamyl alcohol (PCIA) methods [Budowle 1990, Comey 1994 This method of extraction usually yields from 3-50 ng/ μL of DNA but for reasons of developing a rapid method the samples in this study were not quantified after extraction.…”