Effect of PCR conditions on the formation of heteroduplex and single-stranded DNA products in the amplification of bacterial ribosomal DNA spacer regions
| Autor: | Mark Anton Jensen, Neil Alexander Straus |
|---|---|
| Rok vydání: | 1993 |
| Předmět: |
DNA
Bacterial DNA clamp Base Sequence Base pair Circular bacterial chromosome DNA polymerase II Molecular Sequence Data Multiple displacement amplification Nucleic Acid Heteroduplexes DNA Single-Stranded Spacer DNA Biology Endonucleases Molecular biology DNA Ribosomal Polymerase Chain Reaction RNA Ribosomal 23S Reannealing RNA Ribosomal 16S Genetics biology.protein Electrophoresis Polyacrylamide Gel Genetics (clinical) Heteroduplex DNA Primers |
| Zdroj: | PCR methods and applications. 3(3) |
| ISSN: | 1054-9803 |
| Popis: | PCR amplifications of 16S/23S rDNA spacer regions were carried out from conserved 16S and 23S sequences for genomic DNA samples from strains representing 16 bacterial species (12 genera). Multiple products were produced containing conserved homologous sequences at the 3' and 5' ends, separated by highly variable internal spacer sequences. These products cross-hybridized forming heteroduplex DNA structures containing double-stranded ends surrounding an internal single-stranded loop. Single-stranded DNA was also produced in the amplification of rDNA spacer sequences. Fragments comprising the nonhomoduplex DNA components were identified by their susceptibility to removal by digestion with a single-stranded endonuclease. The relative formation of heteroduplex and single-stranded DNA was reduced by reaction conditions favoring primer/template annealing, for example, higher ionic strength, higher primer concentration, and lower annealing temperature, as well as by decreasing the number of amplification cycles. Heteroduplex and single-stranded DNA structures were also generated by denaturing and reannealing spacer amplification products in the absence of polymerase activity. Whereas heteroduplex and single-stranded DNA structures provide additional information that is helpful in distinguishing between species of bacteria that produce similar homoduplex products, the mobility of heteroduplex and single-stranded DNA structures DNA structures is extremely sensitive to electrophoretic conditions. |
| Databáze: | OpenAIRE |
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