Paternity and Parentage Testing

Although modern paternity testing is continues to gain wide acceptance across the Unite States, as of 2003 there were still some states where sixteenth century ancient English Common Law concepts remain dominate in determinations of parentage. Under these concepts, unless a husband who challenges the legitimacy of an offspring can prove impotency, or that he was out of the country at the time of conception, responsibility of parentage (including the financial responsibilities of contributing to the care and raising of the child) are assumed as a matter of law.

Since early in the twentieth century, several methods have been developed and utilized to establish paternity. The utilization of these techniques has followed the developing knowledge about the characteristics of the human genotype as well as having, in the last two decades, incorporated the development of new techniques in molecular biology. With the knowledge that each human is genetically unique as result of genetic polymorphism, blood group antigen typing and other immunological techniques including the human histocompatibility leukocyte antigen (HLA) typing, became widely used for paternity testing and forensic science application until the end of the 1970s.

In the mid-1980s, these techniques were replaced by direct analysis of the DNA polymorphisms. The first of such techniques, developed by Alec Jeffries utilized multilocus DNA probes. This technique is known as the restriction fragment length polymorphism (RFLP) testing. RFLP techniques are based on variable number of tandem repeats (VNTR), which are sequences of 10 to 60 bp (base pairs) of length, that lie adjacent to each other in the same chromosomal orientation (minisatellites).

Subsequently, several other laboratories developed similar methodology and in the late 1980s, the FBI adopted this technique. A second variation of this strategy was then introduced. It used a combination of single locus probes (SLP) to achieve a similar exclusion power, instead of the two MLP probes previously used.

Methodologically, both techniques are based on the digestion of the genomic DNA with restriction endonucleases, separation of the fragments by electrophoresis, followed by X transfer to a nylon membrane and finally, detection by hybridization with either a radioactive or chemiluminescent probe. This technique is known as Southern blot analysis.

A further significant improvement in the analysis of the RFLP took place through the use of the PCR (Polymerase Chain Reaction) technique, where a certain region of DNA is amplified, producing millions of copies of the fragment of interest. This reaction is carried on in a thermocycler machine, and the products of amplification are separated by electrophoresis and may be visualized and documented on a UV light box. Briefly, the main advantages of such techniques include the great discriminatory power of each loci, the ability to process mixed samples and the rich experience that was developed in the last decades through the utilization of these techniques. By the other hand, 50 ng or more of DNA material is required to obtain clear results, and degraded DNA samples can pose a significant limitation to the process. Furthermore, the process is time consuming, taking up to a number of weeks to be completed.

The next major change in the analysis of the DNA for paternity (and forensic) analysis incorporated the PCR amplification of microsatellites instead of minisatellites. Microsatellites are also formed by tandem repeats but consist of two to five nucleotides per repeat units. This means that the amplification requires less DNA (less than 1 ng) and the quality of the material may be less than ideal. This capability permits the analysis of some degraded DNAs. The potential number of loci is very large and the process is rapid; it may be completed in a day or two. This system also has the benefit of lending itself to multiplexing and automation. In addition, several kits are available, and for some multiplexes inexpensive silver stain materials may be employed without expensive equipment.