GENERAL CONSIDERATIONS

Native or natural DNA usually has two complementary strands.  The G residues on one strand bind C residues on the complementary strand and A residues bind T's.

 

 

Notice that the G-C pairs are depicted with three lines, or bonds between them while A-T base pairs have only 2 bonds.  This property of the DNA has been recognized since 1953.  The bonds between G-C base pairs and A-T base pairs are called, hydrogen bonds.  It is well known that the G-C base pairs are stronger than A-T pairings because of the extra hydrogen bond for G-C base pairs.  This means that the stability of the DNA can be predicted based on the % G+C content.  For example, the sequence shown above has 12 G-C base pairs and a total of 25 base pairs, for a G+C content of 50%.  The two strands of this sequence are held together more tightly than a similar length sequence with a 40% G +C content for example.  Such considerations are fairly important for DNA testing since any use of PCR or probe hybridization involves the disruption and reformation of the two strands.

For example, each cycle of PCR involves heating the DNA to separate the strands followed by cooling to the appropriate temperature to allow the primer DNAs to bind accurately to their complementary sequences.  This process is also important in hybridizations involving dot strips or Southern blots where the single-stranded probes must bind accurately to their complementary target DNA sequences. 

If the temperature of the cooling step is too warm (warmer than optimal) the probe may not bind to its target sequence.  If the temperature of the cooling step is too cool, the probe may bind incorrect targets as well as correct ones.  The latter effect is called cross-hybridization and has been documented for some of the probes of PM plus DQA1.  Incorrect binding can also happen to the primers of STR based PCR tests if conditions are improper.

With regard to accuracy of hybridization, the binding of a DNA probe to its complementary DNA target for example, there may be an important difference comparing the common research use of probes and primers and systems like PM plus DQA1 and even multiplex STRs.  The common use is to target a single sequence in each PCR using two primers to flank the sequence.  This is followed by some form of analysis of the sole PCR product.  Analysis may involve a Southern blot to size and probe the PCR product, or DNA sequencing of the product to determine the precise sequence of bases. 

In contrast, multiplex PCRs begin with the simultaneous binding of many different primers (two for each of the loci).  If 14 loci are targeted, there are at least 28 different primers involved.  For Polymarker, this is followed by simultaneous probings of the PCR products.  The PM typing strip alone has 14 different probes (one of the 13 dots has two probes) while the DQA1 strip has 11 probes.  Thus these systems are far more complex than usual applications of PCR.  The complexity was added to speed the analysis.  All of the loci of PM plus DQA1 could be analyzed one at a time.  

One would think that the sequences of the probes for PM plus DQA1 would have been chosen to all have roughly the same G + C content so that they could all be used at the same temperature with the same relative accuracy of each probe.  However, sequence inspection reveals that these in fact were not designed that way.  Based on empirically tested formulas for predicting best temperatures of probe binding, the S and C dot probes in particular appear to be as much as 20  C away from their temperature optima.  It is possible that this observation may account for some of the known artifacts that have been observed.  There is some evidence that PM plus DQA1 can function consistently when provided with relatively undegraded, unmixed DNA samples available in ample amounts.  However, there is evidence that this system can be fooled by aged or degraded DNA, mixtures and low input amounts of DNA.  For multiplex systems with unpublished primers, it is difficult for the scientific community to evaluate the general, thermal equality of the primers. 

Multiplex systems have the limitations of any PCR system in terms of the influence of contamination.  Stray DNA molecules can contribute alleles or complete DNA profiles.  PCR is a replication process similar to the replication of an infectious agent.  Contamination of a PCR can occur as easily as the spread of the common cold virus.  In fact, it may be easier to contaminate a PCR than it is to catch a cold since PCRs have no immune system to ward of the contaminating DNA.   

PCR is potentially useful since it is the only method of amplifying really minuscule amounts of DNA.  However, it is important to recognize that PCR methods are sometimes problematic, exquisitely sensitive to contamination and need to be interpreted with extreme caution. 

Analysis of Separated Sperm and Non-Sperm Fractions. 

In order to perform DNA typing on sperm DNA, it is desirable to separate the sperm DNA from any other DNA that may be present.  For example, in swabbed materials from a rape evidence kit, the swabs may contain non-sperm cells from the victim as well as sperm and non-sperm cells from the rapist.  To accomplish separation of the sperm cells, a process known as differential extraction is often performed.  This involves lysing (breaking open) the non-sperm cells followed by spinning (centrifugation) the mixture to remove the still unbroken sperm cells.  To do this, chemicals, usually an enzyme called proteinase K (PROTEIN-ACE-K) (breaks down most proteins), and a mild detergent (breaks down cellular membranes) are added to the original mixture of sperm and non-sperm cells.  The enzyme and the mild detergent can lyse most cell types but not sperm because the sperm cell membranes have cross-linking chemical bonds called disulfides (pronounced DI-SUL-FIDES).  Actually, the illustration below is slightly incorrect because the proteinase K does remove most of the sperm tails.  These were left in the illustration to assist in following what happens to the sperm.