RFLP method: Characterise human DNA & paternity analysis
QUE: Discuss the use of method to characterise human DNA samples as applied to paternity testing.
Restriction Fragment Length Polymorphism (RFLP) is a difference in homologous DNA sequences that can be detected by the presence of fragments of different lengths after digestion of the DNA samples in question with specific restriction endonucleases. It differentiates minor nucleotide sequence variations in homologous fragments of DNA. The technique relies on the specificity of restriction endonucleases, which are highly sequence-specific and cut the double-stranded DNA only at their recognition sites. The action of restriction enzymes produces variable lengths of homologous DNA molecules containing minor sequence variations or polymorphisms and the cleaved fragments in the digested DNA can be separated by electrophoretic techniques. Two individuals cannot have the same RFLP unless they are identical twins hence this difference can be used to distinguish between two individuals. RFLP are inherited thus a child has RFLPs that are similar to that of the mother and father. Matching these pieces of DNA can establish either if a particular man is the biological father or not and this forms the basis of paternity testing using RFLPs. Polymorphisms are inherited differences found among the individuals in more than 1% of normal population. (Goodwill, et al,. 2007)
In RFLP high-quality DNA is isolated from the samples and digested with restriction enzymes. The fragments are segregated on Agarose Electrophoresis Gel, transferred to a nitrocellulose or nylon membrane and hybridized with a probe. The DNA bands are detected by autoradiography, chemiluminescence or fluorescence. An RFLP probe is a labelled DNA sequence that hybridizes with one or more fragments of the digested DNA sample after they were separated by gel electrophoresis, thus revealing a unique blotting pattern characteristic to a specific genotype at a specific locus. Restriction endonucleases are enzymes that cleave DNA molecules at specific nucleotide sequences depending on the particular enzyme used. Restriction enzymes are isolated from a wide variety of bacterial genera. Because analysis of RFLP need high quantity of DNA PCR for RFLP might be necessary. (Goodwill, et al,. 2007)
The restriction fragments have negative charge and can be separated by gel electrophoresis, which separates the pieces of DNA based on their size. Because of the negative net charge of DNA the fragments migrate toward the positive and smaller fragments move the fastest and furthest toward the positive terminal and the bigger ones move the least thus separating the DNA samples into distinct bands. These are then visualise under a florescence light. If a child is tested with his/her biological father the fragments of the father for that probe must tally with those of the child, otherwise if they do not tally it can be concluded that he is not the biological father. However, to prove paternity with this method several RFLP probes with have to be used so as to increase the confidence level of the results. Analysing many RFLPs will reduce the chances of including mutations in the final concluding statement.
The main advantage of RFLP analysis over PCR-based technique is that no prior sequence information, nor oligonucleotide synthesis, is required. Furthermore, in some cases, it may not be feasible to develop a PCR technique to detect a particular form of allelic variation. Also, RFLPs provide a convenient means for turning an uncharacterized DNA clone into a reagent for the detection of a genetic marker. Even though RFLP is a specific technique it also has its delimitations. Sample size, sample quality and the length of time for analysis are the major limitations of the RFLP paternity testing technique. DNA samples are often taken by swabbing the inside of the cheek (buccal swabs). “At one time, blood alone was used for RFLP DNA tests because larger DNA samples are needed to for the RFLP testing technique reports S.P.A.R.C,” FitzGerald (2016). The other limitation in RFLP paternity testing concerns the quality of the sample. The cells must be fresh and undamaged, the collection, storage and handling of DNA samples can comprise the quality of DNA (FitzGerald, 2016).
References.
FitzGerald, S. & FitzGerald, S. (2016). Limitations of the RFLP Paternity Testing Technique | eHow. eHow. Retrieved 16 November 2016, from http://www.ehow.com/list_7474181_limitations-rflp-paternity-testing-technique.html
Goodwin, W., et al. (2007) An introduction to forensic genetics. John Wiley & Sons Inc., 111 River Street, Hoboken, NJ 07030, USA.
Restriction fragment length polymorphism - ScienceDirect Topics. (2016). Sciencedirect.com. Retrieved 16 November 2016, from http://www.sciencedirect.com/topics/page/Restriction_fragment_length_polymorphism
