Extraction, sequencing and bioinformatics analysis of DNA from dried blood spots.
Mjoli, Phiwokuhle Bulelwa.
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The Africa Centre for Health and Population Studies has a demographic surveillance site 200km north of Durban at the Umkhanyakude district. In this setting, blood samples are routinely collected and used for the diagnosis of HIV infection. In this setting and in many settings in Africa, samples are normally collected and stored on filter paper as dried blood spots (DBS) as those are easy to transport and store. DNA can be isolated/extracted from DBS and used for viral and/or host genomic analysis. The aim of this work was to extract DNA from DBS of sufficient quality and yield that could be used for subsequent analysis. Specifically, we aimed to perform host HLA genotyping from DBS as HLA type has an impact on HIV-1 replication levels. Dried blood spots were prepared from anonymous samples that are commonly used for the validation of new laboratory methods at the Africa Centre Virology Laboratory in Durban. The QIAamp DNA Mini Kit method was optimised to isolate DNA from DBS. DNA levels were quantified using the Qubit 2.0 Fluorometer (Qubit Assay). Polymerase chain reactions (PCR) were used to amplify the HLA Class 1A, 1B and 1C loci and the PCR products were purified using the Pure Link Purification Kit (Invitrogen Life Technologies). Sanger sequencing techniques were used to genotype the HLA‟s PCR products. AssignTM ATF Softwere v1.5 was used to detect HLA allele variations in the consensus sequences produced through Sanger sequencing. The DNA yield that could be extracted from the DBS was low. This was most probably due to the low quantity of blood that can be stored in one DBS. Despite the relatively low DNA yields sequencing of the target gene (HLA Class 1A, 1B and 1C) was successful using Sanger Sequencing and variations in the majority of the HLA alleles were detected. This MSc study shows that it is possible to sequence HLA loci directly from DBS.