ABOUT PATERNITY TESTS

1. Sample Registration

As soon as the samples reach the laboratory they should be registered in the lab's database. Here, data such as the case number and the date of sample receipt are recorded.

For legal paternity tests, the samples are generally referenced by the date of birth of the sample donors and the identification number of the case. These two pieces of information allow the unique identification of a sample while keeping the donor anonymous to the laboratory personnel. Once the tests are carried out, a report is issued to the test requestor who can match the results back to the corresponding sample donors.

For curiosity paternity tests, the person responsible for test registration generally assigns an identification number to the case and a unique donor number to each of the samples as soon as they arrive at the laboratory. In most labs, all personal information is then kept in a secure place to ensure confidentiality. Samples are identified solely by case and donor number during the testing process, and the laboratory analysts have no access to any personal information of the sample donors. Once results are determined, the person in charge matches the identification numbers back to the sample donors and issues a report.

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2. DNA Extraction

The saliva or blood samples received by the laboratory contain the genetic material that will be analysed in the paternity test. The genetic material is called DNA (Deoxyribonucleic Acid) and it is found in the centre (or nucleus) of the cells in the sample.

Before the DNA can be analysed it must first be extracted and purified from the other components in the sample, a process that takes about 2-3 hours. For saliva and blood spot samples (see Collecting Samples section for information on different sample types), the laboratory analyst punches out a small circle from the filter paper on which the donor's sample was deposited during the sample collection process. The circle of paper is transferred to a sterile tube labelled with the donor's identification code. In the case of whole blood samples, an adequate volume of the whole blood is transferred to the labelled sterile tube.

The first step of the DNA extraction process is to break up (or lyse) the cells so that the DNA inside is released. This is achieved by adding a special solution to degrade certain components of the cell, leaving the DNA floating freely. The tubes are now inserted into a special centrifuge machine and spun at a high speed for several minutes. This causes the DNA to collect in the bottom of the tube in the form of a firm pellet, whereas the other cellular components float freely in the solution. This solution is then removed, and the remaining DNA is purified further by washing out the tube several times with an alcoholic solution. Finally, the DNA is dissolved in a liquid and is ready for the next step of the paternity test.

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3. DNA Amplification

Only relatively small sections of a person's DNA need to be analysed in order to perform a paternity test. These sections are referred to as microsatellite markers or loci (see The Paternity Testing Process section for more details). The amount of DNA extracted from a sample is small, so before the microsatellite markers can be analysed, they must first be copied (or amplified) millions of times so that there are enough copies present to allow detection. This amplification process is known as a Polymerase Chain Reaction (PCR) and it takes about 4 hours.

But how can the laboratory analyst control which fragments of DNA to amplify? The trick lies in knowing the sequence of the DNA bordering these fragments. Each bordering sequence is unique, meaning that the DNA pattern constituting it is not found anywhere else throughout the entire human DNA. On the other hand, this DNA pattern is identical amongst all humans. Thus, the pair of sequences bordering a microsatellite marker uniquely identifies the start and end of the marker.

For the PCR, specially designed molecules (called primers) are introduced into the purified DNA solution. These primers are single-stranded DNA molecules designed to bind specifically to the unique sequences bordering the microsatellite markers. Single nucleotides, which are the building blocks that constitute DNA, together with an amount of another special molecule, or enzyme, called Taq polymerase, are also added to the mixture.

In the PCR reaction, the double stranded DNA is first denatured by exposing it to a high temperature of around 94 Celsius. At this temperature, the DNA unwinds and the bonds between the two strands of the DNA molecule are broken, leaving two single stranded DNA molecules. The solution is then allowed to cool down, and as this happens, the primers bind to their targets at both ends of the DNA markers. The Taq polymerase recognises the site where the primer ends and starts inserting single nucleotides at this point, building the single strand DNA between the two primers back into double-stranded DNA. This constitutes the end of the first PCR cycle.

A PCR cycle is therefore made up of three steps:

Thus, at the end of each PCR cycle the targeted DNA fragments would have doubled in number. For paternity testing the PCR cycle is repeated about 40 times, so that by the end of the PCR reaction there are millions of copies of the required DNA fragments.

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4. DNA Analysis

Once the microsatellite markers have been amplified, they are placed into an instrument called an automatic DNA analyser, which separates the markers by size. The primers used during the PCR are fluorescently labelled. This allows the DNA analyser to detect the DNA markers and measure their size by means of a laser. The size information is fed into a computer, and a special software program analyses the data and assigns a repeat number to each fragment.

The detection and data analysis process takes between 5 and 7 hours and the final result is displayed in graphic form on the computer as show in Example 1 and Example 2. Each microsatellite marker appears as two peaks, one for each complementary copy. The position of a peak is determined by its size, so if the two genetic complements of a given marker are of equal size they appear superimposed as one peak.

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5. Statistical Analysis

Once results from all donors for all the DNA markers have been collected, they are inputted into a statistical software program for interpretation. For a given marker, if the number of repeats (or frequency) does not match between a child and alleged father, it is highly likely that the latter is not the real biological father. There is however, a slight chance that the discrepancy may be due to other causes, such as a mutation that may have occurred between father and child. So the conclusion cannot be made with certainty. For each additional locus in which the frequency differs between alleged father and child, there is an increase in the probability that the alleged father is not the biological father. In fact, if the frequencies differ in three or more loci, it is practically impossible that the alleged father is the real biological father.

On the other hand, a match in the frequencies between an alleged father and a child for a given marker may occur for two reasons. It may either be that the alleged father is the real biological father. However it could also be that the two donors happen to have identical frequencies purely by chance. The probability of the latter occurring depends to a large extent on the ethnic origin of the donors. For this reason, population databases defined by ethnic group and geographical region are generally employed to calculate probabilities of a match occurring if the two donors are not related. (This is the reason why laboratories generally request information on the ethnic origins of the donors.) The larger the number of matching markers between a child and an alleged father, the higher the likelihood that the latter is the biological father. For this reason it is better to select tests that employ 12 or more markers. In fact, if more than 10 markers show a match between child and alleged father it is practically certain that the latter is the biological father.

During a paternity test, the laboratory analysts examine the markers one by one. Whenever a match occurs between the child and alleged father, the frequency observed for the particular locus is looked up in a population database, and the probability value obtained is inputted in the statistical program. The program processes the information and outputs a result called a paternity index for this particular locus. When all loci are analysed and inputted, the program computes the combined paternity index and the probability of paternity for the overall test.

The combined paternity index is a likelihood ratio that expresses how many more times an individual is likely to be the biological parent as compared to an unrelated and untested person of the same ethnic group. An index of 100 or greater is considered strong genetic evidence of parentage.

The probability of paternity is defined as a percentage, and it compares the likelihood that the tested individual has passed on the particular marker frequencies to the likelihood that an untested random person of the same ethnic group may have passed on these frequencies. If an alleged father is not the biological father, the paternity test generates a probability of paternity of 0%. If the alleged father is the biological father, the test usually generates a probability of paternity on the order of 99.99% or more.

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6 . The Paternity Test Report

The final step in the laboratory procedure for a Paternity Test, is to issue a report and dispatch it to the person requesting the test. The format of the Paternity Test Report varies between laboratories, but all reports should include the following components:

• The case identification number.
• A listing of the samples, including identification information and pertinent data such as the date of sample collection and the date it was received at the laboratory.
• A description of the method used to perform the test.
• A presentation and interpretation of the results.

The person responsible for issuing the results must sign and date the report.
Typical examples of Paternity Test Reports may be viewed by clicking on the links below:

Report 1: Results indicate that the alleged father is in fact the biological father.
Report 2: Results exclude the alleged father from being the biological father.

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