Automated capillary sequencers are also capable of performing fragment analysis applications. The process is similar to sequencing because fluorescent dyes are used to label products amplified by PCR. Fragment analysis is often performed manually loading amplified products on agarose gel. It is a relatively simple and cost effective method for conducting fragment analysis. However, there are also limitations. First, there is no method for accurately calculating fragment sizes even with a size ladder. Second, multiplexing is not possible when product sizes from multiple primer pairs (markers) are similar in size. Finally, agarose gels does not have the resolution capabilities that capillary electrophoresis has. Capillary electrophoresis, used in forensics fingerprinting, provides remarkable accuracy to fragment analysis applications.
Automated Fragment Analysis Uses Color Fluorescent Dyes
The Polymerase Chain Reaction (PCR) first amplifies samples that will be compared by fragment analysis. For manual applications, the forward and reverse primer are simply unlabeled oligonucleotides. For automated capillary equipment the forward primer contains a fluorescent label on the 5’end. The amplified product will also be labeled following PCR because the product includes both primers (figure 1). The forward and reverse primers (markers) determine the region and base pair size of the resulting amplification.
There are advantages to using fluorescent labels in the amplification process. Capillary analyzers recognize fluorescent emission wavelengths (different colors). This adds the capacity to multiplex more than one set of primers in an amplification reaction. For example, forensic science currently runs 16 separate markers in one multiplexed reaction. Markers are labeled with four different fluorescent dyes. Fragment sizes add a second parameter because fragments with the same dye, but amplified with different markers, never overlap.
A Standard is Added to Every Sample
Another parameter that increases accuracy and resolution in automated fragment analysis applications is the addition of a given standard to every sample. The base pair sizes of test samples are calculated using this standard. Motility variance that could occur from one capillary to the next is eliminated because a standard curve is determined for each capillary. Each fragment in the standard would need to be labeled with fluorescent dye. However, the standard uses a unique label not used for any of the samples, typically ROX or TAMRA.
Resolution of Automated Capillary Fragment Analysis is 1 Base Pair
The resolution capability of capillary fragment analysis is 1 base pair. The same technology that separates each base in sequencing is also applied to fragment analysis. A single base pair separation is nearly impossible using an agarose gel (figure 2). Fragment size accuracy is also limited by manual methods because there is no way to view the standard and sample fragments loaded in the same lane.
The introduction of slab gel sequencers improved capacity to perform fragment analysis over manual agarose methods. Development of capillary analyzers has brought greater sensitivity and speed to this well-established process. Very little genomic DNA is now required to perform automated fragment analysis. Although science has developed new methods for sequencing, fragment analysis still remains an efficient and cost effective method to analyze genomic variability; particularly related to genetic disease and forensics.