Saturday, September 25, 2010

Multiplex STR

One of the more commonly encountered STR test designs in forensic testing is called, Multiplex STR.  There are multiplex, PCR reagent kits sold by both:  PE Applied Biosystems and by  Promega.  Such systems combine three or more different PCRs in one reaction that targets distinct STR loci at the same time.  Three of the commonly used loci are called, CSF1PO, TPOX and THO1.  Again, the names of the loci have historical significance, but are of little importance as names. 
 Profiler Plus and CofilerTM (PE Applied Biosystems) combines 13 different STR loci.  PowerplexTM (Promega) uses the same 13 loci but the primers used are different. The Promega kit incorporates published primer sequences, a significant scientific advantage, since without the primer sequences, it is unclear which STRs at some loci are targeted.  A newer typing kit, IdentifilerTM (PE Applied Biosystems) incorporates the original 13 loci but adds 2 additional loci.  By design (meaning where the primers were placed on the DNA by the designers) multiplex STR loci have different, non-overlapping size ranges so that DNA fragments from the different loci will have different, non-overlapping size ranges.  Or, if the sizes overlap, they are tagged with differing dyes to help distinguish the 13 loci.  These test systems have boldly ambitious designs and should be considered fairly experimental, especially for samples whose quantity and/or quality is outside tested limits.   
There have been some discrepancies in profiles obtained with test kits from the two manufactures when the same samples were analyzed.  These discrepancies are not extremely common but are noticeable and fairly dramatic when they occur.  Any base within DNA can mutate (ie. change).  For example, an A base at a particular position can change to a G. Such mutations usually first appear in a sperm or egg cell.  Each mutation then appears throughout the body of the person who results from such a sperm or egg.   Discrepancies in test kit results are thought to be due to mutations in the sites that the primers bind.  These events are called, primer binding site mutations or PBS mutations.
Multiplex STRs are often combined with PCR for another locus called, amelogenin (pronunciation varies, but usually it is AM'-EEL-O-GEN-IN).  Amelogenin adds little to the discriminating power of the test.  Its purpose is to help distinguish male and female sources of DNA by detecting the X and Y chromosomes.  The amelogenin products have sizes that place them outside the size ranges of the other loci.  
Compared to PCR-based systems originally introduced, such as PM plus DQA1 (PE Applied Biosystems) multiplex STRs are technically more simple and direct at the allele detection stage.  On the other hand, multiplex STR are slightly more vulnerable to missing alleles.  There are two reasons for this.  1)Larger DNA fragments are degraded before smaller ones.  This is simply due to the fact that larger DNA molecules are bigger targets for degradative enzymes than smaller DNA molecules.  2)PCR itself favors (will produce more of) smaller DNA targets compared to larger ones that take more time to copy. The copying is done by a protein called an enzyme.  It can finish copying smaller DNA fragments more rapidly than larger ones.  
Both of these factors result in a tendency for small DNA fragments to be seen more readily than larger ones.  This is not an overwhelming tendency but certainly should be considered when amounts of input DNA are low, when DNA degradation is suspected, and particularly when a single small STR allele is weakly observed at a given STR locus.  
STRs are prone to an artifact called, "stutter bands" or "shadow bands."  These are thought to be due to the DNA repeats slipping out of register during the PCR process.  These are spurious PCR products that are usually one repeat length smaller than the main band.  The main problem that these pose is that it may be difficult to impossible to determine whether light intensity bands are due to stutter or due to presence of a mixture.  Although the stutter bands are predictably below (shorter than) the main band, the stutter bands do often align with common alleles. 

Most forensic laboratories are aware of stutter artifacts and many take extremely careful and appropriate countermeasures.  However stutter artifacts conceivably could play a role if inappropriate attempts are made to interpret minor components of a mixture.  
Some of the current STR detection/typing schemes use thin tubes called capillaries, instead of flat gels.  When a capillary is used, the results are often displayed  as tracings on a graph, instead of the image display shown above.  On such tracings, each main STR product will appear as a large peak while stutter bands appear as smaller  peaks (to the left).  The tracings are called, electropherograms (ELECTRO-FERO-GRAMS).  The tracing data should be accompanied by 

 numeric data that reveals:  the measured size of each PCR product, the intensity (peak height) and the estimated allele size.  The numeric data can be important in determining the quality of the results. 

 The two figures above show some alternative ways in which STR results/data are presented.  Basically the peaks represent tracings of bands that have come off the end of a gel, or may represent tracings of the gel itself, depending on the equipment used.  Larger DNA fragments are on the right and smaller ones on the left.  There are recommended standards, called thresholds for how high or low the peaks may be.
All technology has limitations.  For multiplex PCRs, the most serious limitations are in the areas of samples that are minimal, degraded, mixed, over-interpreted, contaminated or even potential combinations of these.  Some current practices lack support by the established literature.  Over-interpretation can also occur when there are partial profiles.[1]  The scientific system recognizes the human tendency toward over-interpretation and offers the countermeasures:  independent review, independent verification, scientific controls and demonstrations of reproducibility.  These reviews and controls are considered integral parts of the scientific process.
PCR-based testing is potentially useful since it is currently the only quick method of amplifying really minuscule amounts of DNA.  However, it is important to recognize that PCR based methods are exquisitely sensitive to contamination and need to be interpreted with extreme caution.  Match probabilities generated with some STR typing systems may involve extreme numbers perhaps giving the impression of an infallible result.  Scientific rigor often requires that extreme numbers be placed in a context that considers all aspects of testing including laboratory error rates and technical limitations.

No comments:

Post a Comment