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dc.contributor.advisorDe Kock, André
dc.contributor.advisorPretorious, G. H. J.
dc.contributor.authorAsfaw, Estifanos Kebede
dc.date.accessioned2017-03-22T06:56:34Z
dc.date.available2017-03-22T06:56:34Z
dc.date.issued2002-11
dc.identifier.urihttp://hdl.handle.net/11660/5949
dc.description.abstractEnglish: 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.en_ZA
dc.description.abstractAfrikaans: Die menslike alfa fibrinogeen (FGA) kort tandem herhalingslokus (STR) is geleë op die lang arm van chromosoom 4. Dit kom voor in die derde intron van die alfa fibrinogeen geen. Hierdie komplekse hoogs polimorfiese tetranukluotied herhalingslokus word saam met ander STR DNA merkers aangewend vir persoonlike identifikasie in die mediese en forensiese wetenskappe. STR lokusse kan ook aangewend word om genetiese variasie in etniese groepe te bestudeer asook in siekte assosiasie studies. Tydens veskeie populasie frekwesie studies is daar al meer as 80 allele in die FGA lokus gevind. Basispaaropeenvolgingstudies het ook al 11 variant allele aangetoon. Die FGA lokus is aangetoon om 'n hoë heterosigisiteit en krag van diskriminasie te vertoon. Die doel van hierdie studie was om die basispaaropeenvolging te bepaal van die mikrovariant- en buite alleliese leer allele wat tydens roetine vaderskapsondersoeke gevind is. Die basispaaropeenvolging van so veel al moontlik van die allele van die FGA lokus sou ook bepaal word. Twee-en-sestig DNA monsters is gekies en die basispaaropeenvolging van een of beide allele is bepaal. Die DNA monsters was afkomstig van swart (52), kleurling (5), blanke (4) en boesman (1) persone. Die geselekteerde allele is met behulp van PKR vermeerder. Die spesifieke alleelband is uit die gel gesny en gesuiwer in herhaalde PKR en suiwerings stappe totdat 'n suiwer enkel alleelband gevind is. Die suiwer allele se basispaaropeenvolging is daarna bepaal met behulp van die "BigDye terminator" reaksie. Voorwaartse- en tru-reaksies is apart uitgevoer. Die produkte van die reaksies is met etanolasetaat neergeslaan waana kapilêre elektoforese in POP-6 polimeer gevolg het op 'n ABIPrism 310 genetiese analiseerder. Die data is verwerk met "Sequence Analysis" sagteware. Die resultate is nagegaan, gedruk en vergelyk met STR analises en FGA basispaaropeenvolgings publikasies. Uit die 62 DNA monsters is 76 volledige- en mikrovariantallele gevind wat wissel vanaf 'n 16.1 (224bp) tot 'n 44.2 (337bp) alleel. Hierdie allele verteenwoordig 27 veskillende allele (13 volledig en 14 inter-allele). In hierdie studie is twee nuwe allele (40.2 en 41.2) beskryf. Daar is ook variasie in die basispaaropeenvolging gevind in allele 26, 28 en 43.2 met twee variante elk. Twee allele, 43.2 en 44.2, se basispaaropeenvolging het ook verskil van die gepubliseerde data. Van die 76 allele waarvan die basispaaropeenvolging bepaal is, was 49 binne die alleliese leer en 29 buite die alleliese leer. Slegs 8.41% (4/49) van die allele binne die alleliese leer is verkeerd aangetoon tydens roetine STR analise. Hierdie 4 allele het verskil met 1 of 2 basispare. In teenstelling hiermee is al 29 buite alleliese leer allele verkeerd aangetoon met 'n verskil van meer as twee basispare. Alhoewel hierdie studie uitgevoer is op geselekteerde DNA monsters is betekenisvolle resultate verkry. Drie variante in basispaaropeenvolging, twee nuwe allele sowel as een alleel waarvan die opeenvolging onbekend was, is beskryf. Daar is verder ook twee allele beskryf waarvan die struktuur van die opeenvolging verskil van dit wat in die literatuur beskryf is. Aangesien die DNA monsters wat in hierdie studie gebruik is, nie verteenwoordigend is van die bevolkingsverspreiding van die Vrystaat Provinsie nie, stelons verdere populasie studies voor om die STR verspreiding van lokusse, wat algemeen aangewend word in vaderskaps- en forensiese ondersoeke, deeglik te beskryf. Sodanige studies sal lig werp op die frekwensie van variasie in die alleel basispaaropeenvolging van STR lokusse.af
dc.description.sponsorshipIrish Aid Ethiopiaen_ZA
dc.language.isoenen_ZA
dc.publisherUniversity of the Free Stateen_ZA
dc.subjectPaternity testingen_ZA
dc.subjectForensicen_ZA
dc.subjectShort Tandem Repeats (STRs)en_ZA
dc.subjectDNA markersen_ZA
dc.subjectPolymerase chain reaction (PCR)en_ZA
dc.subjectDNA sequencingen_ZA
dc.subjectSequence variantsen_ZA
dc.subjectEletrophoresisen_ZA
dc.subjectFGAen_ZA
dc.subjectMicrovariant allelesen_ZA
dc.subjectTetranucleotide repeatsen_ZA
dc.subjectHuman chromosomesen_ZA
dc.subjectGenetic codeen_ZA
dc.subjectDissertation (M.Med.Sc. (Haematology and Cell Biology))--University of the Free State, 2002en_ZA
dc.titleDNA characterization of the FGA locus in the human genomeen_ZA
dc.typeDissertationen_ZA
dc.rights.holderUniversity of the Free Stateen_ZA


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