Haematology and Cell Biology

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Browsing Haematology and Cell Biology by Author "Asfaw, Estifanos Kebede"

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    DNA characterization of the FGA locus in the human genome
    (University of the Free State, 2002-11) Asfaw, Estifanos Kebede; De Kock, André; Pretorious, G. H. J.
    English: The human alpha fibrinogen (FGA) short tandem repeat locus is found in the long arm of chromosome 4. It is located in the third intron of the alpha fibrinogen gene. This complex highly polymorphic tetranucleotide repeat locus together with other STR DNA markers is extensively used in personal identification in medical and forensic sciences. STRs are also used to study genetic variation in distinct ethnic groups and in disease diagnosis. More than 80 alleles have been reported for this locus from various population frequency studies. A few sequence studies have also reported 11 sequence variants to date. The FGA locus was found to have high heterozygosity and power of discrimination. The aim of this study was to characterise the sequence of microvariant and off-ladder complete and microvariant alleles of the FGA locus that were observed during routine paternity analyses. The characterization of the sequence of as many as possible of alleles observed in our study population would also be attempted. A total of 62 DNA specimens were selected and sequence characterized either for one or both alleles. The DNA specimens were 52 from Negroid, 5 mixed ancestry, 4 Caucasian and 1 SAN origin. The PCR reaction was used to amplify the selected alleles. The band of interest was cut from the gel and purified in consecutive PCR and purification steps till separate single bands were obtained. The purified single bands were sequenced using a BigDye terminator ready reaction kit in both forward and reverse reactions separately. These products were precipitated with ethanol acetate and subjected to capillary electrophoresis on an ABIPrism 310 Genetic Analyser using POP6 polymer. The results were analysed using the "Sequence Analysis Software version 2.1". The data obtained were checked, printed and compared with STR analysis results and FGA sequence reports. From the selected 62 specimens a total of 76 complete and microvariant alleles, the size of which ranged between 16.1 (224bp) to 44.2 (337bp) were found. These represent 27 different alleles (13 complete and 14 interalleles). In this study 2 novel (previously undescribed) alleles (40.2 and 41.2) were found. Three sequence variants (26, 28 and 43.2) with two variants each were observed. Two alleles 43.2 and 44.2 that had reported sequence variants were found to have different sequence structures from the published sequences. Forty-nine of the 76 sequenced alleles were within ladder and the remaining 29 were off-ladder. Only 8.41% (4/49) of the within ladder alleles had been wrongly assigned allelic numbers with routine STR analysis. The difference between the routine assignment and the sequencing of these alleles was only 1 or 2 bp. In contrast, all of the 29 off-ladder alleles were wrongly assigned. In this instance the difference was 2 or more base pairs. Although this study was conducted on conveniently selected DNA samples, it had significant results. Three sequence variants, 2 newalleles and, 1 allele, which had been reported, but sequence had not been described was found. Additionally, two other alleles with reported sequences were found, but their sequence structure differed from the published sequences. The samples in this investigation were not representative of the population groups that are found in the Free State province and we suggest further population-based studies of STR loci that are commonly used in paternity and forensic investigation. The information obtained from such studies will disclose the frequency of sequence variant alleles.