Introduction
Forensic science is often referred to the use of science in criminal and civil law. Due to recent advances in microbiology, the resulting technological progress has been quite handy for parental tests, which falls into the category of forensics. There are important regulations within paternity test runs in the UK which will be discussed alongside the tests used in the past such as serological tests. The discovery of short tandem repeats has revolutionised these paternity test run which will be later explained. However, as with most biological tests, short tandem repeats and serological tests have their limitations. Both will be compared and contrasted between each other.
Legal aspects of paternity tests within the UK (in relation to the Mother)
In order to take a paternity test, the laboratory, which is responsible to carry out the test must be accredited by the Ministry of Justice.[1] The Human Tissue Act of 2004 covers England, Wales and Northern Ireland and states that it is an offense to use human tissue for analysis of DNA, without the consent of the person.[2] There are exemptions to this rule such as the use for clinical trials or if the person whose DNA is being analysed is not competent. If the alleged father is not competent, it has to be carried out in accordance with paragraph 4 or 7 of part 1 in schedule 1 of this act and is carried out by a person who believes this to be in the person’s best interest.[3] However, this is unlikely to apply to the mother and most likely she would require the consent of the male whose DNA is to be analysed. In hindsight, there seems to be a lot of legal barriers and a mother cannot simply conduct a DNA analysis without permission.
Serological tests
Serological tests involve the study of serum (Blood plasma, contains all proteins except from clotting factors). [5]
In early the 1920s, 4 different blood groups were discovered – A, B, AB and O. Each of these erythrocytes (red blood cells) have different antigens present on their cell membranes, this is known as the blood typing system (ABO system). [7]
• Type A has ‘A antigen’ and Anti-B antibodies
• Type B has ‘B antigen’ and Anti-A antibodies
• Type AB has ‘A and B antigen’ and have neither Anti-A or Anti-B antibodies
• Type O has no antigens on its surface and both Anti-A and Anti-B antibodies. [4]
These blood groups are inherited by a child from their parents. In reference to this case, serology tests could be used to identify the blood type of the parents and the child. It is done through a process known as agglutination.
The test is carried out in two steps:
1. A blood sample is taken from both parents. The samples are then mixed with antibodies for blood type A and B, then the blood sample is checked to see whether or not the red blood cells stick t¬¬ogether. If they stick together then that means the blood has reacted with one of the antibodies.
2. Serum is then mixed with blood (collected beforehand) containing both blood type A and B. This step is called back typing. The serum contains the antibodies and the sample of blood contains red blood cells with the antigens. [6] Agglutination reaction occurs between similar antigens and antibodies – such as when antibody A agglutinates with antigen A.
These steps can be used to distinguish what blood type a person has and then this analysis can be used to find out whose blood type matches the child’s. [8]
Another type of test can be taken out known as HLA testing – Human leukocyte antigen which is found in all body cells except erythrocytes. [9] They are highly polymorphic, this allows for a high rate of exclusion. This is because there are many different types so there are less people with identical antigens so matching with parents is more efficient than eukaryote (red blood cell) type matching. Finding HLA types in a person is known as HLA typing. This is done by adding tiny samples of lymphocytes to terasakti plates (serological cytoxicity). Terasakti plates are made of high-clarity polystyrene resin – they are used for serological determinartion of HLA antigens.
• Peripheral blood lymphocytes express HLA class I antigens are used for serological testing of HLA-A, HLA-B and HLA-C.
• HLA class 2 antigen have DP, DM, DO, DQ and DR antigens. [9][10]
The terasakti plates hold wells containing different specific antibodies (usually monoclonal antibodies made in the laboratory). The best cells for class 2 typing are B lymphocytes and for class 1 typing leucocytes are used.
When a HLA antigen and a specific antibody bind together, the complement is added which leads to the cells in the specific well to be killed. [9] This leaves a pattern in the well; this pattern can be used to find out what types of antigens were present on the HLA.
Short tandem repeats and polymorphisms
DNA profiling was first discovered in 1984 by Alec Jeffreys. Portions of DNA contained repeated DNA sequences, which all vary in size. DNA polymorphisms are DNA sequence variations which do not associate with an observable phenotype. They can exist anywhere on the genome of a human. [11]
There are many different classes of polymorphisms, the most common being SNPs – single nucleotide polymorphisms. This is where a single nucleotide is altered in a DNA sequence. They normally occur on average of 1 in every 300 nucleotides. They don’t always cause a change in expression in a human also known as are silent. Silent polymorphism means they don’t change the amino acid coded by the 3 nucleotides (codon). SNPs can act as biological markers – which help scientists locate genes that are associated with disease. [12][13]
Another type is variable number of tandem repeats (VNTRs) or minisatellites. This is where on a genome a short nucleotide sequence (typically 10-60 base pairs) is organised as a tandem repeat (repeat around 5-50 times) – they vary in size of how many repeats are made. [19] These VNTRs can be shown in RFLP where specific DNA sequences are cut and then analysed.
RFLP or Restriction fragment length polymorphism is where a sample of DNA is cut by a restriction endonuclease, which can detect and cut DNA at specific sequences. An RFLP probe is a labelled DNA sequence that hybridizes with one or more fragments of the digested fragments, it is used so it can be seen for analysis as these probes are usually labelled. Then the fragments are separated according to their length by agarose gel electrophoresis by southern blot procedures. These probes can be radioactive and so stain x-ray films to produce a pattern on x-ray film as shown in figure 1.[22] They vary in length – as described before VNTRs vary in length and are repeats. Depending on the length that these hybridized DNA sequences travel, the pattern can be used to see similarities in DNA sequences between people. [16][19]
Figure 1 [22]
STRs (short tandem repeats or microsatellites) are repeating regions in the DNA. They are 2-6 base pairs in length. The number of STRs in humans are highly variable from person to person, making them extremely useful for identification purposes. [13][14]
This makes them useful for paternal identification. The process which this done by is explained below. [21]
1. First step is to extract the DNA. Blood samples are taken and are treated with SDS and proteinase K. Other samples can be taken, and treatment depends on what type of tissue is taken.
2. Then the DNA is then amplified by a process known as PCR (polymerase chain reaction). Primers (short strands of DNA complementary to starting points of DNA strand) bind to either side of section of DNA that is to be amplified. This allows them to act as starting point. DNA deoxynucleotides (adenine, guanine, thymine, cytosine), TAQ polymerase and a buffer is needed in addition to the original DNA strand to amplify the STRs.
3. Denaturation – where the double strand in original DNA is split into two by breaking hydrogen bonds only, this is done at around 94-95°C
4. Annealing – takes place where temperature is lowered to 50-56°C to allow primers to attach to each strand as a template for new strands.
5. Extension – where temperature is raised to 72°C TAQ polymerase adds new nucleotides to ends of primers to make new DNA strands. This is repeated several times doubling DNA copies each time. [18]
6. Electrophoresis – DNA molecules are placed in a gel. DNA molecules are charged negatively, so can be pulled through gel by electric field (which can be supplied by an electric current). Small molecules move more quickly than larger DNA. The DNA fragments are then separated to form a band on the gel which can be seen if the primers are ‘labelled’ with something such as fluorescent agents. This creates a DNA ladder which can be used as an analysis tool to see where similarities lie on these ladders to the mother’s, child’s and alleged father. Usually nowadays a band of gel is not used, bioanalyzers are used as a machine for this. [18][20]
Accuracy of tests
Using erythrocytes to determine paternity isn’t quite powerful in comparison to other tests that have been discussed. The frequency of the genes that code for the antigens on red blood cells is quite high, so there is a large area for overlap thus suggesting that there will be likelihood that you can mistake someone as a father. As it is much more likely another male having a similar blood group to the original father. It is said that the accuracy of blood test grouping is about 30%. As explained the exclusion rate is quite low. This test can be deemed as not very accurate.
A more accurate test would be the HLA or white blood cell antigen analysis, which has about 95% exclusion rate. This is because there is much more variety of genes responsible for coding HLA antigens. As discussed before, if the frequency for different variety of genes is low there is less likelihood that people will have similar HLA antigens and so chances of overlap between two people is lower. This makes it much easier to exclude people who are not the father.
At the current moment DNA paternity testing can be called the most accurate form of testing. If the DNA patterns between the mother, alleged father and child analysed is matched, the likelihood of the alleged father being the actual father is 99.9%. If the alleged father does not match on two or more DNA probes the alleged father can disregarded. However there may be limitations with this technique as contamination can be amplified in results and thus make results unreliable.
Conclusion
In conclusion, we all have a similar genome, but a tiny portion is different in all of us. Offspring has a combination of maternal and paternal DNA. In earlier times, RBC analysis was used since that’s what was available at the time. It makes sense that blood types match up with offspring since they are derived from a person’s genome. Over time technology has advanced to where HLA analysis was available. This varies more from person to person – increasing exclusion rate. But the deal breaker was easily the discovery of the human genome. This directly allowed for analysis of what tiny portions of DNA that would match up with the child. As we look closer microscopically we have many more variations so finding specific DNA sequences in people are much easier this way. Mutations occurring within these STRs may cause by chance a result which may not show the correct father simply because the strands are different due to the mutation. Serological tests cannot be used to determine fatherhood alone, but they can be used to see the possibility that the alleged father is the actual father. A combination of these tests can be used to determine fatherhood efficiently.