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== Genotyping of RhD gene ==
The presence of the RhD gene in an individual’s genome is determined by [[genotyping]]. Firstly, the body fluid containing an individual’s DNA will be extracted. DNA will then be isolated from unwanted impurities. The isolated DNA will then be mixed with various reagents to prepare the [[Polymerase chain reaction|polymerase chain reactions]] (PCR) mixture. The PCR mixture usually contains [[Taq polymerase|Taq DNA polymerase]], [[DNA primer|DNA primers]], [[Deoxyribonucleotide|deoxyribonucleotides]] (dNTP) and [[buffer solution]]<ref name=":5">{{cite journal | vauthors = Lorenz TC | title = Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies | journal = Journal of Visualized Experiments | issue = 63 | pages = e3998 | date = May 2012 | pmid = 22664923 | pmc = 4846334 | doi = 10.3791/3998 }}</ref>. The DNA primers are specific for [[exon]] 7 and exon 10<ref>{{cite journal | vauthors = Hromadnikova I, Vechetova L, Vesela K, Benesova B, Doucha J, Kulovany E, Vlk R | title = Non-invasive fetal RHD exon 7 and exon 10 genotyping using real-time PCR testing of fetal DNA in maternal plasma | journal = Fetal Diagnosis and Therapy | volume = 20 | issue = 4 | pages = 275–80 | date = Jul 2005 | pmid = 15980640 | doi = 10.1159/000085085 }}</ref>. Under different circumstances, primers for other regions of the RhD gene, such as [[intron]] 4 and exon 5, may also be used<ref>{{cite journal | vauthors = Dovč-Drnovšek T, Klemenc P, Toplak N, Blejec T, Bricl I, Rožman P | title = Reliable Determination of Fetal RhD Status by RHD Genotyping from Maternal Plasma | journal = Transfusion Medicine and Hemotherapy | volume = 40 | issue = 1 | pages = 37–43 | date = February 2013 | pmid = 23637648 | pmc = 3636019 | doi = 10.1159/000345682 }}</ref>. The mixture will be subjected to a series of PCR which is performed by a [[thermal cycler]]<ref name=":5" />. By the end of the PCR, the amount of RhD gene will be amplified if it is present. The product of the PCR will be analysed by [[gel electrophoresis]]. Before gel electrophoresis, [[Molecular-weight size marker|DNA reference ladder]], [[positive control]]{{dn|date=May 2019}} containing DNA with RhD gene and the PCR product will be loaded onto the wells of the gel<ref name=":5" />. An [[Electric current|electrical current]] will be applied and the DNA fragments will migrate to the positive terminal as they are negative in charge. Since DNA fragments have different molecular sizes, the larger they are, the slower they migrate<ref name=":6">{{cite journal | vauthors = Lee PY, Costumbrado J, Hsu CY, Kim YH | title = Agarose gel electrophoresis for the separation of DNA fragments | journal = Journal of Visualized Experiments | issue = 62 | date = April 2012 | pmid = 22546956 | pmc = 4846332 | doi = 10.3791/3923 }}</ref>. Utilising this property, DNA fragments with different molecular masses can be segregated. With the help of gel staining and visualising devices such as [[Transillumination|UV trans-illuminators]], RhD gene DNA fragments, if present, will be visible as a band with its corresponding molecular mass<ref name=":6" />. Further DNA sequencing can be conducted to confirm that the sequence of product DNA fragments matches that of the RhD gene sequence.
== Clinical Applications ==
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