Capillary Electrophoresis: Overcoming the Hurdles of Current Technologies

Gabriela Saldanha and Ann MacPhetridge, Promega

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The vast majority of the human genome is identical among diverse individuals. However, there are small regions of variation, especially in areas with repetitive sequences. One example is short tandem repeats (STRs) that can vary in length among individuals, with each repeat consisting of three to five nucleotides of DNA. Analysis of these repeats reveals a characteristic pattern for each individual that is inherited from paternal and maternal DNA. Forensic DNA databases use these patterns of STRs for human identification, and each national database has requirements for specific STR loci that are included.

To characterize these STR patterns, or DNA profiles, human genomic DNA is amplified using the polymerase chain reaction (PCR) with sets of primers for each STR being tested. The primers are labeled with fluorescent dyes, and the amplified products are separated according to size by capillary electrophoresis (CE). Fluorescence imaging by the CE instrument results in a set of peaks for each dye channel, which together constitute an electropherogram that serves as a DNA “profile” for that individual. Statistical analysis of the DNA profile and comparison to reference databases can predict whether the individual matches a known DNA profile, with a high power of discrimination.

CE instruments therefore play a key role in forensics labs, as they help enable scientists to generate DNA profiles. With this information, casework labs can help investigators to confirm or challenge the accounts of those involved in a crime and rule suspects in or out. Casework labs work with extremely challenging samples due to the conditions under which the DNA is collected. These labs require very sensitive, robust STR kits that work in the presence of inhibitors. The DNA evidence they produce often plays a critical role in the conviction or acquittal of those who are put on trial, so the stakes could not be higher.

In the case of database labs, they process a high volume of samples taken from convicted felons, felony arrestees, and misdemeanor arrestees. Paternity laboratories process samples to determine a characteristic pattern inherited from maternal and paternal DNA, which requires speed and cost-effective analysis. All of these laboratories require sensitive, flexible and cost-effective CE instruments, but to date, the choice available to them has been limited.

Drawbacks of existing technologies

The existing CE instruments are costly to purchase and run – and are designed for high throughput environments (with 96 well plate capacity). Subsequently in many forensics labs, particularly smaller ones, scientists currently have to work around the availability of the CE instrument. Oftentimes they have to batch their samples together and wait until other analyses are completed, which causes challenges, particularly with turnaround times and scheduling. Scientists might even be forced to carry out partial plate runs, which has a negative impact on cost due to wastage of array, polymer, reagents etc. Due to the critical and time-pressured nature of their work, this situation is far from ideal.

In the case of Y-STR analysis or mitochondrial DNA sequencing, some labs currently partner with an outside organisation, making the testing protocol lengthier and more complex. Critically, this means the chain of custody of the sample can be more complicated, and it can add complexity in the courtroom too, with more than one lab potentially having to testify, which should be avoided wherever possible.

What is needed is a more flexible instrument that can fit more seamlessly into the workflow of the forensics lab and enable all analysis to be done on site in the lab, when it is needed.

Emerging technologies

More innovative, affordable personal CE instruments are coming into the market, that are specifically designed to overcome some of the challenges forensics labs are facing. They have a smaller footprint, fit into the workflows more seamlessly and allow greater flexibility in sample batch sizes. They provide a lower cost alternative, but with the capabilities of their bigger counterparts, making them an ideal alternative.

The technical capabilities of new CE instruments are also improving, with eight dye color options on the horizon, enabling amplification and detection of more loci from challenging casework samples for better analysis of inhibited and degraded DNA (see Figure 1). Ultimately, this level of analysis provides more information about the sample, allowing scientists to be more confident and make better decisions.

Figure 1: DNA amplification with eight dye colors using a CE System.

With the caseload on forensics labs ever increasing, more convenient and easier to use technologies are going to help to ease the burden and streamline analysis. The growing choice for forensics labs of CE instruments – whether they have a high or low throughput of samples – is going to bring about important efficiencies in the near future.

In the longer term, CE analysis is likely going to remain the “gold standard” for generating STR profiles in the forensics market for years to come. While massively parallel sequencing offers some key advantages, wide adoption by forensic laboratories isn’t expected in the near future given the challenges of bioinformatics, a lack of training resources, funding constraints and the cost per sample. In the meantime, CE instruments and their associated STR kits will continue to enable laboratories involved in human identification applications to get meaningful information from precious samples.

About Promega Corporation  

Promega Corporation is a leader in providing innovative solutions and technical support to the life sciences industry. Our product portfolio of over 4,000 products support a range of life science work across areas such as cell biology; DNA, RNA and protein analysis; drug development; human identification and molecular diagnostics. For over 40 years these tools and technologies have grown in their application and are used today by scientists and technicians in labs for academic and government research, forensics, pharmaceuticals, clinical diagnostics and agricultural and environmental testing. Promega is headquartered in Madison, WI, USA with branches in 16 countries and over 50 global distributors. For more information, visit