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Quantitative Investigation of the Modular Primer Effect for DNA and Peptide Nucleic Acid Hexamers
KTH, Tidigare Institutioner                               , Bioteknologi.ORCID-id: 0000-0002-4657-8532
KTH, Tidigare Institutioner                               , Bioteknologi.
KTH, Tidigare Institutioner                               , Bioteknologi.
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1999 (Engelska)Ingår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 269, nr 1, s. 155-161Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

The effect on oligonucleotide–template duplex stability upon cohybridization of adjacently annealing oligonucleotides, the modular primer effect, was studied with biosensor technology. DNA and peptide nucleic acid (PNA) hexamer modules and sensor chip-immobilized template DNA strands were designed for analysis of nick, overlap, and gap modular hybridization situations. The fast hybridization kinetics for such hexamer modules allowed for the determination of apparent duplex affinities from equilibrium responses. The results showed that the hybridizational stability of modular hexamer pairs is strongly dependent on the positioning, concentration, and inherent affinity of the adjacently annealing hexamer module. Up to 80-fold increases in apparent affinities could be observed for adjacent modular oligonucleotide pairs compared to affinities determined for single hexamer oligonucleotide hybridizations. Interestingly, also for coinjections of different module combinations where DNA hexamer modules were replaced by their PNA counterparts, a modular primer effect was observed. The introduction of a single base gap between two hexamer modules significantly reduced the stabilization effect, whereas a gap of two bases resulted in a complete loss of the effect. The results suggest that the described biosensor-based methodology should be useful for the selection of appropriate modules and working concentrations for use in different modular hybridization applications.

Ort, förlag, år, upplaga, sidor
1999. Vol. 269, nr 1, s. 155-161
Nationell ämneskategori
Industriell bioteknik
Identifikatorer
URN: urn:nbn:se:kth:diva-13322DOI: 10.1006/abio.1999.4000OAI: oai:DiVA.org:kth-13322DiVA, id: diva2:323610
Anmärkning
QC 20100611Tillgänglig från: 2010-06-11 Skapad: 2010-06-11 Senast uppdaterad: 2020-01-10Bibliografiskt granskad
Ingår i avhandling
1. Biosensor technology applied to hybridization analysis and mutation detection
Öppna denna publikation i ny flik eller fönster >>Biosensor technology applied to hybridization analysis and mutation detection
1998 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

This thesis demonstrates the application of biosensor technology for molecular biology investigations, utilizing a surface plasmon resonance based optical device for mass sensitive detection of biomolecular interactions at a chipsurface. Oligonucleotide model systems were designed for analysis of the action of DNA manipulating enzymes. DNA ligation, DNA cleavage and DNA synthesis could be quantitatively monitored in real-time. A protocol for DNA minisequencing was also established based on prevention of chain elongation by incorporation of chain-terminators. Determinations of affinities for short oligonucleotides hybridizing to an immobilized target were performed with various sequence content, length, temperature and degree of complementarity. The decrease in affinity for hybridizations involving mismatch situations was found to be strongly dependent on the relative position of the mismatch. Interestingly, also end-mismatches were clearly detectable. The stabilization effect achieved upon co-hybridization of two adjacently annealing short oligonucleotide modules (modular primer effect) was also investigated for different module combinations and hybridization situations. The modular concept of hybridizations was subsequently demonstrated to result in enhanced Capture of single stranded PCR products. The sequence based DNA analysis, first introduced with oligonucleotide modelsystems, was extended to the scanning and screening formutations in PCR amplified DNA from clinically relevant samples. Several different formats were investigated, eitherwith the PCR products immobilized on the chip and oligonucleotides injected or vice versa. Again, mismatch discrimination could be observed for wild type and mutant specific oligonucleotides hybridizing to the targets. The experimental set-up for mutation detection was further developed by the introduction of a subtractive mismatch sensitive hybridization outside the instrument and a subsequent determination of the relative amounts of remain ingoligonucleotides with analytical biosensor monitoring of hybridizations between fully complementary oligonucleotides. In conclusion, the applied technology was found to be a suitable tool for a wide range of molecular biology applications, with emphasis on hybridization analysis and mutation detection.

Ort, förlag, år, upplaga, sidor
Stockholm: KTH, 1998. s. 56
Nyckelord
biosensor, genosensor, surface plasmon resonance, hybridization, nucleic acids, oligonucleotide, mutation detection, mismatch discrimination, modular
Nationell ämneskategori
Industriell bioteknik
Identifikatorer
urn:nbn:se:kth:diva-2703 (URN)91-7170-322-5 (ISBN)
Disputation
1998-11-06, 00:00
Anmärkning
QC 20100611Tillgänglig från: 2000-01-01 Skapad: 2000-01-01 Senast uppdaterad: 2010-06-11Bibliografiskt granskad

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Uhlén, MattiasNygren, Per-Åke

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Nilsson, PeterO'Meara, DeirdreEdebratt, FredrikUhlén, MattiasLundeberg, JoakimNygren, Per-Åke
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