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Genotyping RNA and DNA using padlock probes
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
2001 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

Novel techniques are needed to investigate the genetic variation revealed in the first draft of the human genome sequence. Padlock probes are recently developed reagents, suitable for detecting single-nucleotide variations of DNA and RNA in situ or in solution. The probes are oligonucleotides of about 70-140 nucleotides that can be circularized by ligation in the presence of a correct target sequence. Standard chemical synthesis of padlock probes is difficult due to the requirement for intact 5' and 3' ends of these long oligonucleotides.

A novel PCR-based method is presented in this thesis, whereby longer, densely labeled padlock probes can be made as compared to conventional chemical synthesis. PCR-generated padlock probes produced a stronger signal and a more resolved staining pattern, compared to chemically synthesized probes in fluorescence in situ analysis of an alpha-satellite sequence variant present in human chromosomes 13 and 21. Padlock probes used for in situ analysis of metaphase chromosomes had an optimal length of 140 nucleotides. They were used to identify individual chromosomes 7 and 15, and to follow the transmission of chromosome homologues for two consecutive generations. The specificity of the padlock probes to detect single copy genes in genomic DNA samples was demonstrated by detecting a single-nucleotide mutation in the ATP7B gene.

It has not previously been known if T4 DNA ligase can be used for RNA sequence analysis. In this thesis, it is demonstrated that T4 DNA ligase can be used for distinguishing single-nucleotide RNA sequence variants. Reaction conditions were defined where most mismatches could be discriminated by a factor of 80 and all mismatches by a factor of at least 20. Under these conditions padlock probes could detect and distinguish RNA sequence variants with ligation efficiency almost as high as on the corresponding DNA sequence.

A detailed study of the parameters influencing RNA-templated DNA ligation revealed that DNA ligation on RNA templates proceeds at a much slower rate compared to the same reaction on DNA, and that a molar excess of enzyme is required. Furthermore, the ligation reaction is inhibited by high concentrations of the cofactor ATP and NaCl.

The work presented in this thesis demonstrates that PCR-generated padlock probes can detect and distinguish single-nucleotide variation in both RNA and DNA.

sted, utgiver, år, opplag, sider
Uppsala: Acta Universitatis Upsaliensis , 2001. , s. 40
Serie
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 1050
Emneord [en]
Genetics, Padlock probe, enzymatic synthesis, PCR, in situ hybridization, RNA-templated DNA ligation
Emneord [sv]
Genetik
HSV kategori
Forskningsprogram
medicinsk genetik
Identifikatorer
URN: urn:nbn:se:uu:diva-687ISBN: 91-554-5057-1 (tryckt)OAI: oai:DiVA.org:uu-687DiVA, id: diva2:168331
Disputas
2001-05-23, Rudbeck salen, Rudbeck Laboratoriet, Dag Hammarskjölds väg 20, 751 85 Uppsala, Sverige, Uppsala, 13:15 (engelsk)
Tilgjengelig fra: 2001-05-02 Laget: 2001-05-02 Sist oppdatert: 2018-01-13bibliografisk kontrollert

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