Binary sequencing for companion diagnostics
Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesisAlternative title
Binär DNA sekvensering för diagnostik (Swedish)
DNA sequencing is an invaluable tool for studying medical and biological systems. The genetic components of many diseases have been discovered, which holds great promise for new treatment and drug discovery for treat diseases such as cancer. However, the sequencing instruments of today are mainly research instruments and few have been developed for clinical diagnosis at a decentralized clinic. Therefore there is a need of a complementary small-scale sequencing platform that does not require trained personnel and expensive lab equipment. This project aims to contribute to that development by investigating and finding the optimal enzymatic reaction conditions for small-scale single nucleotide sequencing. The first part was to investigate the optimal conditions for on-slie extension and detection. Probes were printed on a CodeLink array followed by hybridisation to a primer-target complex. Extension was done on slide by the polymerase Klenow exo- with specific biotinylated nucleotides. The signals were captured by streptavidin-coated beads and investigated under a light microscope. The study found that an addition of apyrase to a concentration of 0.25 mU/ul during the extension produced a satisfactory signal. However, there were undesirable extensions that may have occurred as a result of cross hybridisation. Further experiments are needed to assess the extension accuracy. The second part was an investigation of the slippage activity properties of Type IIs restriction enzyme using high throughput sequencing. Synthetic oligonucleotides were cleaved in the conditions recommended by the manufacturers, in general 10 U per 1 µg substrate. The cleaved products were then ligated with enzyme specific adapters and amplified by PCR. Seven enzymes (BbvI, BmpI BsmFI, FauI, MmeI, GsuI and BpuEI) were tsted, and two enzymes were selected for sequencing in the Illumina MiSeq, BbvI and FauI. BbvI have been reported with low percentage of slippage and the sequencing data supported this claim. Moreover, the slippage analysis by sequences logos of the slippage area supports previous results. In contrast, FauI, which was repoted with high percentage slippage, was found to benefit from our conditions and slippage was slightly decreased wit different sequence logo patterns. From this study, it is suggest that the slippage activity and enzyme behaviour depends on reaction condition and may also depend on the sequence around the cognate site.
Place, publisher, year, edition, pages
DNA sequencing diagnostics binary
Engineering and Technology
IdentifiersURN: urn:nbn:se:kth:diva-150560OAI: oai:DiVA.org:kth-150560DiVA: diva2:744724